(Return to overview of bibliography)

AU - Cogniaux J
TI - Interaction between Sindbis virus RNA and ribosomes from chick fibroblasts.
SO - Biochem Biophys Res Commun 1971 Jul 2;44(1):22-8

AU - Nicoli J, Coulomb P, Meyer F
TI - [Structural proteins of Sindbis virus and translation of the viral genome]. [French]
SO - Ann Inst Pasteur (Paris) 1971 May;120(5):683-97

AU - Eaton BT, Faulkner P
TI - Heterogeneity in the poly(A) content of the genome of Sindbis virus.
SO - Virology 1972 Dec;50(3):865-73

AU - Johnston RE, Bose HR
TI - An adenylate-rich segment in the virion RNA of Sindbis virus.
SO - Biochem Biophys Res Commun 1972 Jan 31;46(2):712-8

AU - Eaton BT, Donaghue TP, Faulkner P
TI - Presence of poly (A) in the polyribosome-associated RNA of Sindbis-infected BHK cells.
SO - Nat New Biol 1972 Jul 26;238(82):109-11

AU - Mowshowitz D
TI - Identification of polysomal RNA in BHK cells infected by sindbis virus.
SO - J Virol 1973 Apr;11(4):535-43

AU - Donaghue TP, Faulkner P
TI - Characterisation of the 3'-terminus of Sindbis virion RNA.
SO - Nat New Biol 1973 Dec 12;246(154):168-70

AU - Hsu MT, Kung HJ, Davidson N
TI - An electron microscope study of Sindbis virus RNA.
SO - Cold Spring Harb Symp Quant Biol 1974;38:943-50

AU - Sreevalsan T, Rosemond-Hornbeak H
TI - Inhibition of Sindbis virus replication in HeLa cells by poliovirus.
SO - Antimicrob Agents Chemother 1974 Jan;5(1):55-62

AU - Simmons DT, Strauss JH
TI - Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes.
SO - J Mol Biol 1974 Jun 25;86(2):397-409

AU - Cancedda R, Schlesinger MJ
TI - Formation of Sindbis virus capsid protein in mammalian cell-free extracts programmed with viral messenger RNA.
SO - Proc Natl Acad Sci U S A 1974 May;71(5):1843-7

AU - Simmons DT, Strauss JH
TI - Replication of Sindbis virus. V. Polyribosomes and mRNA in infected cells.
SO - J Virol 1974 Sep;14(3):552-9

AU - Pisano MR, Poiree JC, Lanuc L, Buret M, Nicoli J
TI - [The length of polyadenylic acid tracts of Sindbis virus RNA: the possible existence of two synthetic mechanisms]. [French]
SO - C R Acad Sci Hebd Seances Acad Sci D 1975 Feb 24;280(8):1023-6
AB - The poly (A) tracts of RNA's from viral particles and from replicative complex belong to two classes of 180 and of 60 nucleotides in length. Those of the mRNA are homogenous and around 280 nucleotides in length. The results presented here strongly suggest the possibility of a copying mechanism in the first case and of a posttranscriptional addition in the latter.

AU - Hefti E, Bishop DH, Dubin DT, Stollar V
TI - 5' nucleotide sequence of sindbis viral RNA.
SO - J Virol 1975 Jan;17(1):149-59

AU - Schlesinger MJ
TI - Function of Sindbis virus 49S and 26S RNAs in in vitro protein synthesizing systems. A summary.
SO - Med Biol 1975 Oct;53(5):380-2

AU - Cancedda R, Villa-Komaroff L, Lodish HF, Schlesinger M
TI - Initiation sites for translation of sindbis virus 42S and 26S messenger RNAs.
SO - Cell 1975 Oct;6(2):215-22
AB - Sindbis virus 26S RNA is the principal species of virus-specific RNA found in the infected cell; it is derived from a one third segment of virion 42S RNA. When translated in cell-free extracts from mouse ascites cells or rabbit reticulocytes, 26S RNA directed the synthesis primarily of the 33,000 dalton virus capsid protein, and the protein products were in the form of free peptides rather than peptidyl-tRNA. In contrast, the polypeptides synthesized in either extract in response to Sindbis virus 42S RNA were heterogeneous, ranging in molecular weight from 33,000 to 190,000, and were largely in the form of peptidyl-tRNA. The number of independent initiation sites on the 26S and 42S RNAs was determined by analyzing a tryptic digest of reaction products labeled with yeast N-formyl-35S-methionyl-tRNAFmet. The 26S RNA appeared to contain a single initiation site, and this site could also be found in varying amounts in different preparations of 42S RNA. However, a second initiation site, distinct from that of 26S RNA, was the major site in 42S virion RNA. These results suggest that 42S virion RNA contains two potential sites for initiation of protein synthesis. Only one of these may be active, however, and it is postulated that the second site functions primarily, if not exclusively, in the subgenomic 26S RNA species. In this regard, Sindbis virus 42S RNA may represent a novel form of a eucaryotic messenger RNA.

AU - Margotat A, Laplane J, Pisano MR, Nicoli J
TI - [Polyuridylic sequences and negative strands of Sindbis virus-specific RNAs: study by affinity chromatography on poly (A)-sepharose columns (author's transl)]. [French]
SO - Ann Microbiol (Paris) 1976 Aug-Sep;127B(2):243-56
AB - The experimental conditions of affinity chromatography on poly (A)-Sepharose columns have been determined. This method makes obvious the existence of polyuridylic acid sequences on the negative strands of Sinbis virus-spedific RNAs. The isolated RNAs are negative and positive strands hybrids. By polyacrylamide gel electrophoresis, it has been shown that the negative strands have the same length as the 26 S interjacent RNA at the 6th hour, and as the 42 S virion RNA at the 9th hour postinfection. The polyadenylic acid sequences of the virion RNA and of the replicative intermediate are therefore probably genetically coded.

AU - Deborde DC, Leibowitz RD
TI - Polyadenylic acid size and position found in Sindbis virus genome and mRNA species.
SO - Virology 1976 Jul 1;72(1):80-8

AU - HsuChen CC, Dubin DT
TI - Di-and trimethylated congeners of 7-methylguanine in Sindbis virus mRNA.
SO - Nature 1976 Nov 11;264(5582):190-1

AU - Kennedy SI, Bruton CJ, Weiss B, Schlesinger S
TI - Defective interfering passages of Sindbis virus: nature of the defective virion RNA.
SO - J Virol 1976 Sep;19(3):1034-43
AB - Defective interfering particles of Sindbis virus contain 20S RNA identical to that found in BHK cells co-infected with standard and defective virions. We have characterized these RNAs by their oligonucleotide fingerprints. Most of the oligonucleotides were identical to those found in the mRNA (26S RNA) that codes for the virion structural proteins. Three oligonucleotides found in 20S RNA were absent from the 26S RNA pattern and may represent sequences from the 5' end of the virion RNA. Previous difficulties in describing the nature of the defective virion RNA were due to the aggregated state of the RNA. Nucleocapsids obtained from standard and defective virions were essentially the same size and had about the same density, suggesting that defective particles contain more than a single molecule of 20S RNA.

AU - Dubin DT, Stollar V, Hsuchen CC, Timko K, Guild GM
TI - Sindbis virus messenger RNA: the 5'-termini and methylated residues of 26 and 42 S RNA.
SO - Virology 1977 Apr;77(2):457-70

AU - Martire G, Bonatti S, ALIPERTI G, De Giuli C, Cancedda R
TI - Free and membrane-bound polyribosomes in BHK cells infected with Sindbis virus.
SO - J Virol 1977 Feb;21(2):610-8
AB - The data presented in the paper demonstrate that in BHK cells infected with Sindbis virus virtually all the 42S mRNA not in nucleocapsid is associated with free polyribosomes, whereas the 26S mRNA is distributed between free and membrane-bound polyribosomes. We suggest that the 26S RNA polyribosomes are bound to the membranes through the nascent chains of the B1 protein and that a large percentage of 26S RNA polyribosomes free in the cytoplasm may be due to the small amount of rough endoplasmic reticulum in BHK cells. In addition, we found that intracellular nucleocapsid is in the nonmembrane fraction of the cytoplasm of infected cells.

AU - Schlesinger RW, Stollar V, Guild GM, Igarashi A, Shenk TE, Peleg J
TI - The significance and nature of defective interfering viruses.
SO - Bull Schweiz Akad Med Wiss 1977 Sep;33(4-6):229-42
AB - Deletions in viral genomes appear to be a common occurrence in the replication of all DNA and RNA viruses which have been adequately studied. Such defective genomes can replicate in the presence in the same cell of a helper virus as long as the deletion does not involve the initiation site for genome replication. Coinfection of a cell with defective and "normal" infectious virus leads to reduction in the yield of the latter. The nature of DI viruses and genomes found in Sindbis virus-infected vertebrate cells during "undiluted passage" series is discussed. This procedure leads to the accumulation of progressively shorter viral RNA genomes with internal deletions. The enrichment is limited to genome lengths which are integral fractions (1/2, 1/3, 1/4, etc.) of the complete genome, and these are also found in viral particles released at the corresponding passage levels. It is believed that the selective accumulation of these fragments is governed by constraints of assembly which demand that one full genome equivalent be packaged in a released particle. In contrast to vertebrate cells, cultured mosquito cells do not seem to produce or "recognize" DI particles. Possible implications for the epidemiology of arthropod-transmitted alphaviruses are presented.

AU - Frey TK, Gard DL, Strauss JH
TI - Replication of Sindbis virus. VII. Location of 5-methyl cytidine residues in virus-specific RNA.
SO - Virology 1978 Sep;89(2):450-60

AU - Martin JD, Riggsby WS, Beck RW
TI - The effect of ribonuclease on the replicative forms of Sindbis virus RNA.
SO - Arch Virol 1979;60(2):131-46
AB - Three species of double-stranded RNA, designated RF I, RF II, and RF III in order of decreasing size (25), are produced by ribonuclease treatment of extracts of chicken embryo cells infected for 6 hours with Sindbis virus. Only one class of replicative form RNA is present in extracts not treated with ribonuclease; this class contains some molecules which can be enzymatically cleaved to produce the other two replicative forms. At a low level of enzyme (0.001 microgram/ml) the major species obtained was RF I, the replicative form of the genome. When the enzyme concentration was increased 10-, 100-, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concomitant decrease in the proportion of RF I. The generation of RF's II and III by nuclease resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. In preparations of isolated double-stranded RNA, only RF I and replicative intermediate RNA were present. Mild nuclease treatment of these preparations converted the replicative intermediates primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. Treatment of the isolated naturally occurring replicative form with 0.01 microgram of ribonuclease per ml cleaved some molecules migrating as RF I during gel electrophoresis into molecules which migrated as RF II and RF III.

AU - Dohner D, Monroe S, Weiss B, Schlesinger S
TI - Oligonucleotide mapping studies of standard and defective Sindbis virus RNA.
SO - J Virol 1979 Feb;29(2):794-8
AB - Oligonucleotide mapping studies of the RNA from standard and defective interfering particles of Sindbis virus demonstrate that 3'- and 5'-terminal regions of the genome are conserved in the defective RNAs. These studies also suggest that defective RNAs contain multiple deletions.

AU - Frey TK, Gard DL, Strauss JH
TI - Biophysical studies on circle formation by Sindbis virus 49 S RNA.
SO - J Mol Biol 1979 Jul 25;132(1):1-18

AU - Dubin DT, Timko K, Gillies S, Stollar V
TI - The extreme 5'-terminal sequences of sindbis virus 26 and 42 S RNA.
SO - Virology 1979 Oct 15;98(1):131-41

AU - Sonenberg N, Rupprecht KM, Hecht SM, Shatkin AJ
TI - Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.
SO - Proc Natl Acad Sci U S A 1979 Sep;76(9):4345-9
AB - A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.

AU - Czarniecki CW, Sreevalsan T
TI - Sindbis virus RNA replication. I. Properties of the 38s RNA species.
SO - J Gen Virol 1979 Sep;44(3):759-71
AB - Four species of single-stranded virus RNA (49S, 38S, 33S and 26S) were detected in chick embryo fibroblasts infected with Sindbis virus. The relative amounts of these RNAs were unaffected by the m.o.i. There was also no significant difference in the molar proportions of the four RNA species when purified virion RNA was used as the inoculum. These findings suggest that the 38S and 33S species represent products of the transcription of non-defective virion RNAs. Kinetic analyses of RNA synthesis indicated that during a 1 min pulse more radioactivity was associated with the 38S than with the 49S RNA and as the length of the pulse increased, the ratio of 38S/49S decreased, with the 49S appearing as the predominant species. Furthermore, addition of cycloheximide within the first 3 h p.i. resulted in detection of only the 49S species. Synthese of all four species was unaffected when the drug was added after this time period. These data suggest that the 38S species may represent newly synthesized 49S molecules and some protein(s) synthesized within the first 3 h p.i. is necessary for maintaining the 38S conformational form.

AU - Bonatti S, Sonenberg N, Shatkin AJ, Cancedda R
TI - Restricted initiation of protein synthesis on the potentially polycistronic Sindbis virus 42 S RNA.
SO - J Biol Chem 1980 Dec 10;255(23):11473-7
AB - Sindbis 42 S genome RNA was isolated from virions and translated in vitro before and after purification by oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation under denaturing conditions. In intact 42 S RNA, only the 5'-proximal initiation site for the synthesis of nonstructural proteins was used. The internally located start site for viral structural protein formation was active in broken genome RNA molecules where, as a consequence of fragmentation, it was closer to a 5' end. The results of several kinds of experiments indicate that the fragmentation-dependent synthesis of structural proteins directed by virion RNA was not due to the presence of 26 S subgenomic messenger RNA.

AU - Bonatti S, Cerasuolo A, Cancedda R, Borgese N, Meldolesi J
TI - Studies on the intracellular distribution of Sindbis messenger RNA in infected chick-embryo fibroblasts. 1. Presence of extrapolyribosomal 26-S RNA in the membrane fraction.
SO - Eur J Biochem 1980 Jan;103(1):53-64
AB - Four hours after infection with Sindbis virus, chick embryo fibroblasts were studied by electron microscopy and cell fractionation. Electron microscopy of infected and non-infected cells revealed that infection produced a disaggregation of polyribosomes into monomers. Apart from this observation most cells appeared well preserved, and no degranulation of the rough endoplasmic reticulum was visible. Analysis of postnuclear supernatants by sucrose density gradients showed that no change in the relative proportions of free and membrane-bound ribosomes was produced by infection. Approximately 30% of the ribosomes and 50% of the viral RNA were found to be associated with membranes. Of the membrane-associated viral RNA, 70% was recovered as 26-S RNA. Similar results were obtained with fibroblasts infected by the temperature-sensitive Sindbis mutant ts2, which is defective in the co-translational processing of the viral gene products at the nonpermissive temperature. Sucrose gradient analysis of membrane-bound polyribosomes solubilized by detergent indicated that as much as 50% of the membrane-associated viral 26-S RNA is not integrated into polyribosomes and that most of the ribosomes are present as monomers or ribosomal subunits. Treatment with puromycin of living cells or of isolated membrane fractions under a variety of ionic conditions revealed that the viral RNA-membrane linkage is insensitive to puromycin but sensitive to high concentrations of monovalent ions. The bulk of the membrane-bound ribosomes were detached by high salt and recovered as ribosomal subunits on sucrose gradients. These results are consistent with the idea that in chick embryo fibroblasts infected with Sindbis virus only a small percentage of the ribosomes are engaged in protein synthesis, and that the Sindbis messenger RNA may attach to endoplasmic reticulum membranes through a ribosome-independent, salt-sensitive link.

AU - Borgese N, Meldolesi J, Bonatti S, Cancedda R
TI - Studies on the intracellular distribution of Sindbis messenger RNA in infected chick embryo fibroblasts. 2. Non-parallel distribution of 26-S RNA and ribosomes within microsomal subfractions.
SO - Eur J Biochem 1980 Jan;103(1):65-73
AB - The submicrosomal distribution of membrane-associated viral 26-S RNA in chick embryo fibroblasts infected with Sindbis virus was studied. Infected chick embryo fibroblasts were homogenized in the presence of low amounts of EDTA and fractionated by differential centrifugation. Analysis of postmitochondrial supernatants by isopycnic flotation on continuous sucrose gradients showed that membrane-associated 26-S RNA and ribosomes were not distributed in parallel, with an enrichment in 26-S RNA in the light microsomal subfractions. This distribution could not be explained by adsorption artifacts. Analysis of the distribution of microsomal constituents on sucrose gradients after treatment with digitonin ruled out the possibility that the 26-S RNA might be associated with Golgi or plasma membranes. The attachment of viral RNAs to endoplasmic reticulum membranes is discussed in relation to its possible role in viral morphogenesis.

AU - Czarniecki CW, Sreevalsan T
TI - Sindbis virus RNA replication. II. Strand composition and metabolic fate of the multi-stranded RNA species.
SO - J Gen Virol 1980 May;48(1):75-85
AB - Double-stranded RNA from SB virus-infected cells was denatured and analysed on agarose-methylmercuric hydroxide gels. Equimolar amounts of three single-stranded species with mol. wt. of 4 x 10(6), 2.5 x 10(6) and 1.8 x 10(6) were found. Pulse and chase experiments in infected cells established a precursor-product relationship between the multi-stranded and single-stranded virus RNA species. The present results support the model in which the 49S and 26S species of virus RNA are synthesized in infected cells from two distinct replicating structures.

AU - Ou JH, Strauss EG, Strauss JH
TI - Comparative studies of the 3'-terminal sequences of several alpha virus RNAs.
SO - Virology 1981 Mar;109(2):281-9

AU - Rupprecht KM, Sonenberg N, Shatkin AJ, Hecht SM
TI - Design and preparation of affinity columns for the purification of eukaryotic messenger ribonucleic acid cap binding protein.
SO - Biochemistry 1981 Nov 10;20(23):6570-7
AB - 2',3'-O-[1-(2-Carboxyethyl) ethylidene]-7-methylguanosine 5'-diphosphate (5) and 7-(5-carboxypentyl) guanosine 5'-diphosphate (13) have been synthesized and immobilized on AH-Sepharose 4B to the extent of 17.4 and 36.6 mumol of ligand/g of gel, respectively. The affinity resins thus derives were employed in columns for the purificaton of 24K cap binding protein (CBP) from rabbit reticulocytes. Each resin was found to retain the protein of interest; elution of 24K CBP could then be effected by washing with 70 microM m7GDP. The 24K CBPs released from both columns were found to be active, both as judged by a cross-linking assay that utilized 10(4)-oxidized methyl-3H-labeled reovirus mRNA as a substrate for the protein and also by the ability of the isolated 24K CBP to stimulate the translocation of capped Sindbis virus mRNA in HeLa cell extracts.

AU - Ou JH, Trent DW, Strauss JH
TI - The 3'-non-coding regions of alphavirus RNAs contain repeating sequences.
SO - J. Mol. Biol. 1982 Apr 25;156(4):719-30

AU - Ou JH, Rice CM, Dalgarno L, Strauss EG, Strauss JH
TI - Sequence studies of several alphavirus genomic RNAs in the region containing the start of the subgenomic RNA.
SO - Proc. Natl. Acad. Sci. USA 1982 Sep;79(17):5235-9
AB - The alphaviruses produce two mRNAs after infection: the genomic (49S) RNA which is translated into the nonstructural (replicase) proteins and the subgenomic (26S) RNA which serves as the mRNA for the virion structural proteins. The sequence of the region of the genomic RNA that contains the 5' end of the subgenomic RNA and the 5' flanking sequences in the genomic RNA were determined for several alphaviruses. A highly conserved sequence of 21 nucleotides was found which includes the first two nucleotides of the subgenomic RNA and the 19 nucleotides preceding it. We propose that the complement of this sequence in the minus strand is the recognition site used by the viral transcriptase for initiation of transcription of 26S RNA and that, in general, such short recognition sequences are commonly used among the RNA viruses. The COOH-terminal sequence of the nonstructural polyprotein precursor has been deduced for each virus. These protein sequences are highly homologous and are followed by multiple in-phase termination codons clustered in the nontranslated region of the 26S RNA in each case. In contrast to the proposed transcriptase recognition site, the particular triplets used for a given conserved amino acid have diverged markedly during evolution of these viruses. The protein homology is sufficient, however, for deduction of the correct coding phase of the RNA and allows the alignment of the corresponding nucleic acid sequence data from different alphaviruses without knowledge of the sequence of the entire genomes.

AU - Wengler G, Wengler G, Gross HJ
TI - Terminal sequences of Sindbis virus-specific nucleic acids: identity in molecules synthesized in vertebrate and insect cells and characteristic properties of the replicative form RNA.
SO - Virology 1982 Dec;123(2):273-83

AU - Monroe SS, Ou JH, Rice CM, Schlesinger S, Strauss EG, Strauss JH
TI - Sequence analysis of cDNA's derived from the RNA of Sindbis virions and of defective interfering particles.
SO - J Virol 1982 Jan;41(1):153-62
AB - Sindbis virus generates defective interfering (DI) particles during serial high-multiplicity passage in cultured cells. These DI particles inhibit the replication of infectious virus and can be an important factor in the establishment and maintenance of persistent infection in BHK cells. In an effort to understand how these DI particles are generated and how they interfere with the replication of standard virus, we performed a partial sequence analysis of the RNA obtained from two independently isolated populations of DI particles and from two Sindbis virus variants and compared these with the RNA of the parental wild-type virus. The 3'-terminal regions of the RNAs were sequenced by the dideoxy chain terminating method. Internal regions of the RNA were examined by restriction endonuclease digestion of cDNA's made to the various RNAs and by direct chemical sequencing of 5' end-labeled restriction fragments from cDNA made to the DI RNAs. One of the variant viruses examined was originally derived from cells persistently infected with Sindbis virus for 16 months and is resistant to interference by the DI strains used. In the 3'-terminal region of the RNA from this variant, only two base changes were found; one of these occurs in the 20-nucleotide 3'-terminal sequence which is highly conserved among alphaviruses. The DI RNA sequences were found to have been produced not by a single deletional event, but by multiple deletion steps combined with sequence rearrangements; all sequences examined are derived from the plus strand of Sindbis virion RNA. Both DI RNAs had at least 50 nucleotides of wild-type sequence conserved at the 3' terminus; in addition, they both contained conserved and perhaps amplified sequences derived from the non-26S region of the genome which may be of importance in their replication and interference ability. MS - GENBANK/J02363, GENBANK/J02368, GENBANK/J02364, GENBANK/J02365, GENBANK/J02366, GENBANK/J02367, GENBANK/J02369, GENBANK/J02370, GENBANK/J02371, GENBANK/J02372, GENBANK/J02373, GENBANK/J02374, GENBANK/J02375, GENBANK/J02376, GENBANK/J02377, GENBANK/J02378, GENBANK/J02379, GENBANK/J02380, GENBANK/J02381, GENBANK/J02382, GENBANK/J02383, GENBANK/J02384, GENBANK/J02385, GENBANK/V00073

AU - Ou JH, Strauss EG, Strauss JH
TI - The 5'-terminal sequences of the genomic RNAs of several alphaviruses.
SO - J. Mol. Biol. 1983 Jul 25;168(1):1-15
AB - The 5'-terminal sequences of the genomic RNAs of several alphaviruses have been determined. The nucleotide sequences at the extreme 5' termini are not highly conserved among the alphaviruses, but a similar stem and loop structure, which begins at the 5' end and utilizes about the first 40 nucleotides, can be formed in each case. Downstream from this structure, beginning about 150 nucleotides from the 5' end, a conserved sequence of 51 nucleotides is found which can form two stable hairpin structures. Examination of the 5'-terminal and 3'-terminal sequences suggests that part of this conserved nucleotide sequence may be involved in cyclization of the RNA. A model is proposed for the function of the 5'-terminal sequences in RNA replication. In addition, sequence homologies among these RNAs strongly support the hypothesis that an AUG codon, which occurs at 60 to 80 nucleotides from the 5' end, depending on the virus, and which may or may not be the first AUG codon, is used for initiation of translation of the non-structural proteins and allows a comparison of the deduced amino acid sequences in the NH2-terminal regions.

AU - Dalgarno L, Rice CM, Strauss JH
TI - Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins.
SO - Virology 1983 Aug;129(1):170-87
AB - The complete sequence of the 26 S RNA of Ross River virus (T48 strain) has been obtained and from this the amino acid sequences of the encoded structural proteins have been deduced. These include a basic capsid protein and two envelope glycoproteins. The nucleotide sequence was obtained by chemical sequence analysis of both single-stranded and double-stranded cDNA made to RNA and the sequence data so obtained was rapidly aligned by making use of the protein homology found among the alphaviruses. The polyprotein precursor encoded by the 26 S RNA of Ross River virus is 75% homologous to that of Semliki Forest virus and 48% homologous to that of Sindbis virus. The extent of homology is not uniform within a protein or between proteins and this is discussed with respect to the possible function of the various polypeptide domains in the virus life cycle. In each case the putative attachment site of the amino proximal carbohydrate chains of the three glycoproteins is conserved, whereas the attachment site of a second chain, if present, is not conserved. The 3'-untranslated region of Ross River virus RNA is 524 nucleotides long. It contains a sequence of about 50 nucleotides in length which is present in four copies but which is not shared with other alphaviruses examined. MS - GENBANK/K00046, GENBANK/J02337

AU - Monroe SS, Schlesinger S
TI - RNAs from two independently isolated defective interfering particles of Sindbis virus contain a cellular tRNA sequence at their 5' ends.
SO - Proc Natl Acad Sci U S A 1983 Jun;80(11):3279-83
AB - Defective interfering (DI) particles are deletion mutants that interfere specifically with the replication of homologous standard virus. We have determined the 5'-terminal nucleotide sequences of two DI RNA populations by the following methods: (i) cloning of the cDNA from one of the DI RNA populations and sequencing a representative clone, and (ii) using both DI RNA populations as templates for preparing primer-directed cDNA transcripts and sequencing these transcripts. The 5' terminal sequences of the two DI RNA populations were not derived from standard Sindbis viral RNA but were almost identical to those of a cellular tRNAAsp. MS - GENBANK/J02368, GENBANK/J02369, GENBANK/J02370, GENBANK/J02371, GENBANK/J02372, GENBANK/J02373, GENBANK/J02374, GENBANK/J02375, GENBANK/J02376, GENBANK/J02377, GENBANK/J02378, GENBANK/J02379, GENBANK/J02380, GENBANK/J02381, GENBANK/J02382, GENBANK/J02383, GENBANK/J02384, GENBANK/J02385

AU - Strauss EG, Rice CM, Strauss JH
TI - Sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon.
SO - Proc Natl Acad Sci U S A 1983 Sep;80(17):5271-5
AB - We have obtained the nucleotide sequence of the genomic RNAs of two alphaviruses, Sindbis virus and Middelburg virus, over an extensive region encoding the nonstructural (replicase) proteins. In both viruses in an equivalent position an opal (UGA) termination codon punctuates a long otherwise open reading frame. The nonstructural proteins are translated as polyprotein precursors that are processed by posttranslational cleavage into four polypeptide chains; the sequence data presented here indicate that the COOH-terminal polypeptide, ns72, may be produced by read-through of this opal codon. The high degree of amino acid homology between the ns72 polypeptides of the two viruses, in contrast to the lack of conserved sequence upstream from the read-through site, suggests that ns72 plays an important role in viral replication, possibly modulating the action of other replicase components. MS - GENBANK/J02246, GENBANK/J02363, GENBANK/J02364, GENBANK/J02365, GENBANK/J02366, GENBANK/J02367, GENBANK/V00073

AU - Strauss EG, Rice CM, Strauss JH
TI - Complete nucleotide sequence of the genomic RNA of Sindbis virus.
SO - Virology 1984 Feb;133(1):92-110
AB - The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5' cap and the 3'-terminal poly(A) tract. After the 5'-terminal cap there are 59 nucleotides of 5' nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 untranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3' untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of post-translational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3' end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented. MS - GENBANK/J02363, GENBANK/J02364, GENBANK/J02365, GENBANK/J02366, GENBANK/J02367, GENBANK/V00073

AU - Barrett AD, Dimmock NJ
TI - Variation in homotypic and heterotypic interference by defective interfering viruses derived from different strains of Semliki Forest virus and from Sindbis virus.
SO - J Gen Virol 1984 Jun;65 ( Pt 6):1119-22
AB - There was strong interference between various virulent and avirulent strains of Semliki Forest virus (SFV) and their respective defective interfering (DI) viruses but in other combinations interference was variable: it could be equally strong, weak or could not be demonstrated. On passage, this spectrum of interfering activity changed, some combinations showing greater interference than before and others less. Heterotypic interference between DI SFV, DI Sindbis virus and standard viruses was clearly demonstrated although this was strongest between DI SFV preparations and Sindbis standard virus than in the reciprocal combinations. Variation in interference between DI SFVs and different SFV strains was similar in magnitude to that between DI SFVs and Sindbis virus, suggesting that a similar DI RNA sequence is recognized by both viruses.

AU - Monroe SS, Schlesinger S
TI - Common and distinct regions of defective-interfering RNAs of Sindbis virus.
SO - J Virol 1984 Mar;49(3):865-72
AB - Defective-interfering (DI) particles are helper-dependent deletion mutants which interfere specifically with the replication of the homologous standard virus. Serial passaging of alphaviruses in cultured cells leads to the accumulation of DI particles whose genomic RNAs are heterogeneous in size and sequence composition. In an effort to examine the sequence organization of an individual DI RNA species generated from Sindbis virus, we isolated and sequenced a representative cDNA clone derived from a Sindbis DI RNA population. Our data showed that: (i) the 3' end of the DI RNA template was identical to the 50 nucleotides at the 3' end of the standard RNA; (ii) the majority (75%) of the DI RNA template was derived from the 1,200 5'-terminal nucleotides of the standard RNA and included repeats of these sequences; and (iii) the 5' end of the DI RNA template was not derived from the standard RNA, but is nearly identical to a cellular tRNAAsp (S. S. Monroe and S. Schlesinger, Proc. Natl. Acad. Sci. U.S.A. 80:3279-3283, 1983). We have also utilized restriction fragments from cloned DNAs to probe by blot hybridization for the presence of conserved sequences in several independently derived DI RNA populations. These studies indicated that: (i) a 51-nucleotide conserved sequence located close to the 5' end of several alphavirus RNAs was most likely retained in the DI RNAs; (ii) the junction region containing the 5' end of the subgenomic 26S mRNA was deleted from the DI RNAs; and (iii) the presence of tRNAAsp sequences was a common occurrence in Sindbis virus DI RNAs derived by passaging in chicken embryo fibroblasts. MS - GENBANK/J02368, GENBANK/J02369, GENBANK/J02370, GENBANK/J02371, GENBANK/J02372, GENBANK/J02373, GENBANK/J02374, GENBANK/J02375, GENBANK/J02376, GENBANK/J02377, GENBANK/J02378, GENBANK/J02379, GENBANK/J02380, GENBANK/J02381, GENBANK/J02382, GENBANK/J02383, GENBANK/J02384, GENBANK/J02385

AU - Tsiang M, Monroe SS, Schlesinger S
TI - Studies of defective interfering RNAs of Sindbis virus with and without tRNAAsp sequences at their 5' termini.
SO - J Virol 1985 Apr;54(1):38-44
AB - Three of six independently derived defective interfering (DI) particles of Sindbis virus generated by high-multiplicity passaging in cultured cells have tRNAAsp sequences at the 5' terminus of their RNAs (Monroe and Schlesinger, J. Virol. 49:865-872, 1984). In the present work, we found that the 5'-terminal sequences of the three tRNAAsp-negative DI RNAs were all derived from viral genomic RNA. One DI RNA sample had the same 5'-terminal sequence as the standard genome. The DI RNAs from another DI particle preparation were heterogeneous at the 5' terminus, with the sequence being either that of the standard 5' end or rearrangements of regions near the 5' end. The sequence of the 5' terminus of the third DI RNA sample consisted of the 5' terminus of the subgenomic 26S mRNA with a deletion from nucleotides 24 to 67 of the 26S RNA sequence. These data showed that the 5'-terminal nucleotides can undergo extensive variations and that the RNA is still replicated by virus-specific enzymes. DI RNAs of Sindbis virus evolve from larger to smaller species. In the two cases in which we followed the evolution of DI RNAs, the appearance of tRNAAsp-positive molecules occurred at the same time as did the emergence of the smaller species of DI RNAs. In pairwise competition experiments, one of the tRNAAsp-positive DI RNAs proved to be the most effective DI RNA, but under identical conditions, a second tRNAAsp-positive DI RNA was unable to compete with the tRNAAsp-negative DIs. Therefore, the tRNAAsp sequence at the 5' terminus of a Sindbis DI RNA is not the primary factor in determining which DI RNA becomes the predominant species in a population of DI RNA molecules. MS - GENBANK/K02741

AU - Migliaccio G, Castagnola P, Leone A, Cerasuolo A, Bonatti S
TI - mRNA activity of a Sindbis virus defective-interfering RNA.
SO - J Virol 1985 Sep;55(3):877-80
AB - We obtained Sindbis defective-interfering particles by nine and undiluted passages of standard virus on chicken embryo fibroblasts. These particles contain a deleted 20S RNA molecule which has mRNA activity, as shown by translation in cell-free systems in vitro. In infected cells, this mRNA activity appeared to be totally inhibited except at very late times postinfection.

AU - Kuge S, Saito I, Nomoto A
TI - Primary structure of poliovirus defective-interfering particle genomes and possible generation mechanisms of the particles.
SO - J Mol Biol 1986 Dec 5;192(3):473-87
AB - The genomes of defective-interfering (DI) particles derived from the Sabin strain of type 1 poliovirus (PV1(Sab] were characterized by nuclease S1 mapping using complementary DNA (cDNA) copies of PV1(Sab) genome as probes. The results demonstrated variety in the size and location of the deletions, which were compatible with our previous prediction. The results further indicated that the locations of the deletions were limited within the internal genome region encoding viral capsid proteins and that the deletion sites were clustered in certain areas on the genome. Sequence analysis of a number of cloned cDNAs to the DI genomes revealed that every DI genome retained the correct reading frame for viral protein synthesis. These results strongly suggested that one or all of the viral non-structural proteins might be cis-acting at least at a certain stage in viral replication. A computer search for secondary structures with regard to the deletion sites provided a possible common structure from which, supported by sequences existing on the plus or minus RNA strand of PV1(Sab), deletion regions looped out from the remaining sequences. Replicase might, therefore, skip these transiently formed loop structures with certain frequencies, resulting in the generation of DI genomes. This model could also be considered as a model for genetic recombination in these RNA genomes. Possible "supporting sequences" were also found for every rearranged site on the RNAs of influenza virus and sindbis virus. Thus, we propose a new copy-choice model, designated the "supporting sequence-loop model", for the generation of rearrangements occurring on single-stranded RNA genomes.

AU - Levis R, Weiss BG, Tsiang M, Huang H, Schlesinger S
TI - Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging.
SO - Cell 1986 Jan 17;44(1):137-45
AB - Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging. They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome. To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase. The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus. After one to two passages the DI RNA became the major viral RNA species in infected cells. Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes. MS - GENBANK/M12563

AU - Takkinen K
TI - Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus.
SO - Nucleic Acids Res 1986 Jul 25;14(14):5667-82
AB - The nucleotide sequence coding for the nonstructural proteins of Semliki Forest virus has been determined from cDNA clones. The total length of this region is 7381 nucleotides, it contains an open reading frame starting at position 86 and ending at an UAA stop codon at position 7379-7381. This open reading frame codes for a 2431 amino acids long polyprotein, from which the individual nonstructural proteins are formed by proteolytic processing steps, so that nsPl is 537, nsP2 798, nsP3 482 and nsP4 614 amino acids. In the closely related Sindbis and Middelburg viruses there is an opal stop codon (UGA) between the genes for nsP3 and nsP4. Interestingly, no stop codon is found in frame in this region of the Semliki Forest virus 42S RNA. In other aspects the amino acid sequence homology between Sindbis, Middelburg and Semliki Forest virus nonstructural proteins is highly significant. MS - GENBANK/J02361, GENBANK/J02362, GENBANK/L00018, GENBANK/V01399, GENBANK/V01400, GENBANK/V01401, GENBANK/X04129

AU - Kinney RM, Johnson BJ, Brown VL, Trent DW
TI - Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins.
SO - Virology 1986 Jul 30;152(2):400-13
AB - A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3' terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alphaviruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5' and 3' noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%, 50 to 53%, and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses. MS - GENBANK/M14937

AU - McClure MA, Perrault J
TI - RNA virus genomes hybridize to cellular rRNAs and to each other.
SO - J Virol 1986 Mar;57(3):917-21
AB - In this communication we show that the RNA genomes of vesicular stomatitis, Sindbis, and reoviruses can specifically hybridize under stringent conditions to the large rRNAs present in HeLa cell cytoplasmic extracts. In addition, we show that some virus genome RNAs can also hybridize to each other. On the basis of our previous detailed studies identifying specific regions of hybridization between the poliovirus genome and 28S rRNA, we suggest that a similar phenomenon of "patchy complementary" may be responsible for the interactions described here (M. A. McClure and J. Perrault, Nucleic Acids Res. 13:6797-6816, 1985). The possible biological implications of these cross-reacting hybridizations and practical considerations in the use of viral probes for diagnosis are discussed.

AU - Kaariainen L, Takkinen K, Keranen S, Soderlund H
TI - Replication of the genome of alphaviruses.
SO - J Cell Sci Suppl 1987;7:231-50
AB - The genome of Semliki Forest virus (SFV) is 11,442 nucleotides with a 5' cap-structure and a 3' poly(A) tail of about 100 residues. The genome of the closely relate Sindbis virus (SIN) is slightly longer (11,703 nucleotides). The parental RNA is first translated from the 5' two thirds to yield; nsP1, nsP2, nsP3 and nsP4, which are cleaved from a polyprotein of 2431 amino acids (SFV). The parental genome is copied to a full-length minus strand with poly(U) at the 5' end. The minus strand is used as template for the synthesis of 42 S RNA in membrane-bound replicative-intermediate (RI) structures. In addition to 42 S RNA, a 3'-coterminal subgenomic 26 S mRNA, coding for the structural proteins, is synthesized by internal initiation at the minus strand. Capping and methylation of both plus-strand RNAs occur concomitantly with their synthesis. Analysis of Sindbis virus temperature-sensitive RNA-negative mutants have shown that one complementation group (B) is specifically associated with the synthesis of minus strands. Another, group F, is involved in the polymerization step of both minus- and plus-strand 42 S RNA, and of the 26 S mRNA. The synthesis of minus strands is normally dependent on protein synthesis. There is a shut off of the minus-strand RNA synthesis at about 3 h post-infection. This is apparently regulated by a virus-specific protein, represented by the complementation group A. The same protein is involved in the regulation of the initiation of 26 S RNA together with a component represented by group G mutants. Comparative analysis of SFV and SIN RNAs and DI RNAs of both viruses suggests that perhaps only 19 nucleotides from the 3' end and about 150 nucleotides from the 5' end are needed for replication of the alphavirus RNAs. In some SIN DI RNAs the proposed secondary structure at the 5' end is replaced by a cellular tRNA(ASP) suggesting that the secondary structure rather than nucleotide sequence is sufficient for the recognition by the viral polymerase. Even when the primary structure of the four non-structural proteins of both SFV and SIN is known, the correlation of the genetic data with the individual proteins has not yet been possible.

AU - Chang GJ, Trent DW
TI - Nucleotide sequence of the genome region encoding the 26S mRNA of eastern equine encephalomyelitis virus and the deduced amino acid sequence of the viral structural proteins.
SO - J Gen Virol 1987 Aug;68 ( Pt 8):2129-42
AB - The 26S mRNA and most of the nsP4 encoding regions of the eastern equine encephalomyelitis (EEE) viral genome have been cloned. Excluding the poly(A) tail, the 26S mRNA region was determined to be 4139 nucleotides long and to share the same general organization as that of other alphaviruses. A highly conserved region of 19 nucleotides, the putative transcriptase recognition site for 26S mRNA synthesis, was present at the 26S/42S junction region of the 42S genomic RNA. Translation of the 26S mRNA began at the first AUG (positions 59 to 61) initiation codon and continued with an open reading frame that coded for a polyprotein of 1258 amino acids ending at a UAA ochre termination codon (positions 3776 to 3778). All four putative posttranslational cleavage sites used to generate the capsid, E3, E2, 6K and E1 proteins were conserved. Transmembrane domains present in the EEE virus structural polyprotein have been identified and their functions discussed. Pairwise comparison of the deduced amino acid sequences of the polyproteins of five alphaviruses (EEE, Venezuelan equine encephalitis, Sindbis, Semliki Forest and Ross River viruses) revealed EEE virus to be more closely related to VEE virus than to the other three viruses. MS - GENBANK/D00145

AU - Rice CM, Levis R, Strauss JH, Huang HV
TI - Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.
SO - J Virol 1987 Dec;61(12):3809-19
AB - We constructed full-length cDNA clones of Sindbis virus that can be transcribed in vitro by SP6 RNA polymerase to produce infectious genome-length transcripts. Viruses produced from in vitro transcripts are identical to Sindbis virus and show strain-specific phenotypes reflecting the source of RNA used for cDNA synthesis. The cDNA clones were used to confirm the mapping of the causal mutation of ts2 to the capsid protein. A general strategy for mapping Sindbis virus mutations is described and was used to identify two lethal mutations in an original full-length construct which did not produce infectious transcripts. An XbaI linker was inserted in the cDNA clone near the transcriptional start of the subgenomic mRNA; the resulting virus retains the XbaI recognition sequence, thus providing formal evidence that viruses are derived from in vitro transcripts of cDNA clones. The potential applications of the cDNA clones are discussed.

AU - Faragher SG, Meek AD, Rice CM, Dalgarno L
TI - Genome sequences of a mouse-avirulent and a mouse-virulent strain of Ross River virus.
SO - Virology 1988 Apr;163(2):509-26
AB - The nucleotide sequence of the genomic RNA of a mouse-avirulent strain of Ross River virus, RRV NB5092 (isolated in 1969), has been determined and the corresponding sequence for the prototype mouse-virulent strain, RRV T48 (isolated in 1959), has been completed. The RRV NB5092 genome is approximately 11,674 nucleotides in length, compared with 11,853 nucleotides for RRV T48. RRV NB5092 and RRV T48 have the same genome organization. For both viruses an untranslated region of 80 nucleotides at the 5' end of the genome is followed by a 7440-nucleotide open reading frame which is interrupted after 5586 nucleotides by a single opal termination codon. By homology with other alphaviruses, the 5586-nucleotide open reading frame encodes the nonstructural proteins nsP1, nsP2, and nsP3; a fourth nonstructural protein, nsP4, is produced by read-through of the opal codon. The RRV nonstructural proteins show strong homology with the corresponding proteins of Sindbis virus and Semliki Forest virus in terms of size, net charge, and hydropathy characteristics. However, homology is not uniform between or within the proteins; nsP1, nsP2, and nsP4 contain extended domains which are highly conserved between alphaviruses, while the C-terminal region of nsP3 shows little conservation in sequence or length between alphaviruses. An untranslated "junction" region of 44 nucleotides (for RRV NB5092) or 47 nucleotides (for RRV T48) separates the nonstructural and structural protein coding regions. The structural proteins (capsid-E3-E2-6K-E1) are translated from an open reading frame of 3762 nucleotides which is followed by a 3'-untranslated region of approximately 348 nucleotides (for RRV NB5092) or 524 nucleotides (for RRV T48). Excluding deletions and insertions, the genomes of RRV NB5092 and RRV T48 differ at 284 nucleotides, representing a sequence divergence of 2.38%. Sequence deletions or insertions were found only in the noncoding regions and include a 173-nucleotide deletion in the 3'-untranslated region of RRV NB5092, compared with RRV T48. In the coding regions, most of the nucleotide differences are silent; there are 36 amino acid differences in the nonstructural proteins and 12 in the structural proteins. The distribution of amino acid differences between the two RRV strains correlates with the location of domains which are poorly conserved in sequence between alphaviruses. The possible role of amino acid differences in envelope glycoproteins E1 and E2 in determining the different antigenic and biological properties of RRV NB5092 and RRV T48 is discussed. MS - GENBANK/M20162

AU - Hahn CS, Lustig S, Strauss EG, Strauss JH
TI - Western equine encephalitis virus is a recombinant virus.
SO - Proc Natl Acad Sci U S A 1988 Aug;85(16):5997-6001
AB - The alphaviruses are a group of 26 mosquito-borne viruses that cause a variety of human diseases. Many of the New World alphaviruses cause encephalitis, whereas the Old World viruses more typically cause fever, rash, and arthralgia. The genome is a single-stranded nonsegmented RNA molecule of + polarity; it is about 11,700 nucleotides in length. Several alphavirus genomes have been sequenced in whole or in part, and these sequences demonstrate that alpha-viruses have descended from a common ancestor by divergent evolution. We have now obtained the sequence of the 3'-terminal 4288 nucleotides of the RNA of the New World Alphavirus western equine encephalitis virus (WEEV). Comparisons of the nucleotide and amino acid sequences of WEEV with those of other alphaviruses clearly show that WEEV is recombinant. The sequences of the capsid protein and of the (untranslated) 3'-terminal 80 nucleotides of WEEV are closely related to the corresponding sequences of the New World Alphavirus eastern equine encephalitis virus (EEEV), whereas the sequences of glycoproteins E2 and E1 of WEEV are more closely related to those of an Old World virus, Sindbis virus. Thus, WEEV appears to have arisen by recombination between an EEEV-like virus and a Sindbis-like virus to give rise to a new virus with the encephalogenic properties of EEEV but the antigenic specificity of Sindbis virus. There has been speculation that recombination might play an important role in the evolution of RNA viruses. The current finding that a widespread and successful RNA virus is recombinant provides support for such an hypothesis. MS - GENBANK/J03854

AU - Tsiang M, Weiss BG, Schlesinger S
TI - Effects of 5'-terminal modifications on the biological activity of defective interfering RNAs of Sindbis virus.
SO - J Virol 1988 Jan;62(1):47-53
AB - We have been studying defective interfering (DI) genomes of the RNA enveloped virus Sindbis virus. Deletion mapping of a DI cDNA demonstrated that only sequences at the 3' and 5' termini of the genome are required for the DI RNA to be biologically active. We constructed a series of cDNAs that transcribe DI RNAs differing only in 5'-terminal sequences. Two of the 5' termini identical to ones found in naturally occurring DI RNAs are the 5' terminus of the virion RNA (DI-549) and the first 142 nucleotides from the 5' terminus of the subgenomic 26S mRNA attached to the 5' terminus of the virion RNA (DI-15). The latter has a 42-nucleotide deletion from nucleotides 25 to 66 in the 26S RNA sequence. These DI RNA transcripts were biologically active, but one (DI-526) which did not have the 42-nucleotide deletion of DI-15 was not replicated. The DI RNA isolated after the presumed amplification of the DI-526 transcript had deleted the first 54 nucleotides of the 26S RNA sequences. The 5' terminus of Sindbis virion RNA contains a stem and loop region that is conserved among alphaviruses. An 11-nucleotide deletion in DI-549 that disrupted this stem and loop rendered this DI RNA inactive. In contrast, this same deletion in DI-15 and one that removed an additional 100 nucleotides of the virion 5' terminus did not prevent its amplification. We did not detect by computer analysis any common secondary structures among the biologically active DI RNAs that distinguished them from those RNAs that were not amplified. Our results support the conclusion that tertiary structure or the ability of the RNA to adapt its structure upon interaction with protein is important in the recognition process.

AU - Grakoui A, Levis R, Raju R, Huang HV, Rice CM
TI - A cis-acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis.
SO - J Virol 1989 Dec;63(12):5216-27
AB - The synthesis of Sindbis virus minus-strand and genomic and subgenomic RNAs is believed to require specific cis-acting sequences or structures in the template RNAs and a combination of virus-specific proteins and host components which act in trans. A conserved sequence of about 21 nucleotides in the junction region and encompassing the start site for the subgenomic RNA has been proposed to function as the promoter on the minus-strand template for synthesis of the subgenomic RNA (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982). We introduced a three-base insertion in this sequence, which also inserts a single amino acid near the COOH terminus of nsP4, in a cDNA clone of Sindbis virus from which infectious RNA transcripts can be generated. The phenotype of this mutant, called Toto1100CR4.1, was studied after RNA transfection of chicken embryo fibroblasts or BHK cells. The mutation leads to a drastic reduction in the level of the subgenomic RNA but does not alter the start site of the RNA. Probably as a consequence of depressed structural-protein synthesis, very few progeny virions are released and the mutant makes tiny or indistinct plaques even after prolonged incubation. The cis-acting effect of this mutation was demonstrated by incorporating either a wild-type or mutant junction region into a defective-interfering RNA and examining the relative synthesis of defective-interfering RNA-derived subgenomic RNA in vivo in the presence of wild-type helper virus. These results show that the junction region is recognized by yet unidentified viral trans-acting components for subgenomic RNA synthesis. When the Toto1100CR4.1 mutant was passaged in culture, plaque morphology variants readily arose. A total of 24 independent revertants were isolated, and 16 were characterized in detail. All revertants analyzed showed an increase in the level of subgenomic RNA synthesis. Sequence analysis of the junction region showed that all were pseudorevertants, with only two containing potentially compensating changes in the junction region. An assay was developed to identify revertants with second-site changes in trans-acting viral components involved in subgenomic RNA synthesis. At least two such revertants were identified. Mapping of these and other second-site compensating mutations may provide genetic clues as to which virus-specific protein(s) is responsible for interaction with the conserved junction region to promote subgenomic RNA synthesis.

AU - Weiss B, Nitschko H, Ghattas I, Wright R, Schlesinger S
TI - Evidence for specificity in the encapsidation of Sindbis virus RNAs.
SO - J Virol 1989 Dec;63(12):5310-8
AB - We investigated the interaction of the capsid protein of Sindbis virus with Sindbis viral RNAs and defined a region of the genome that is required for binding in vitro and for packaging in vivo. The binding studies were performed with purified capsid protein immobilized on nitrocellulose and 32P-labeled RNAs transcribed in vitro from viral and nonspecific cDNAs. Genomic and defective interfering (DI) RNAs bound capsid protein significantly better than either the subgenomic (26S) RNA or nonspecific RNAs. Transcripts prepared from either truncated or deleted cDNAs were used to define the segment required for binding. This segment, which is represented twice in DI RNA, lies between nucleotides 746 and 1226 of the genomic RNA and is within the coding region of the nonstructural protein nsP1. Insertion of a domain covering these sequences into a nonviral RNA was able to convert it from a background level of binding to an activity that was 80% that of the Sindbis virus DI RNA. We analyzed DI RNA transcripts in detail because they could be studied not only for the ability to bind capsid protein in vitro but also for the ability to be replicated and packaged in vivo in the presence of helper virion RNA. The results obtained with three DI RNAs are reported. One (CTS14), which has one copy of the binding domain, bound efficiently to capsid protein in vitro and was packaged in vivo as measured by amplification on passaging. In contrast, a DI RNA (CTS1) which lacked this region did not bind to capsid protein and was not detected on passaging. By using lipofectin (P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J.P. Northrop, G. M. Ringold, and M. Danielson, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987) to enhance RNA uptake, we were able to demonstrate that CTS1 RNA was replicated in the transfected cells. It was replicated to the same level as another DI RNA (CTS253) which has only the 3' 279 nucleotides of the binding domain and these are located near the 3' terminus of the RNA. CTS253 bound capsid protein to an intermediate level but was amplified on passaging. The binding studies and the in vivo packaging data, taken together, provide strong support for the conclusion that there is a specific capsid recognition domain in Sindbis virus RNA that plays a role in nucleocapsid assembly.

AU - Li GP, Rice CM
TI - Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication.
SO - J Virol 1989 Mar;63(3):1326-37
AB - Sindbis virus (SIN) contains an in-frame opal termination codon in the nonstructural protein-coding region separating nsP3 and nsP4 and provides a useful tool to study the readthrough phenomenon of the termination codon in host cells and its role in viral replication. We have changed the opal codon by site-directed mutagenesis of a full-length SIN cDNA clone to either sense amino acids (serine, tryptophan, or arginine) or the other two translation termination codons (amber or ochre). Transcripts from all of the mutant cDNA clones were infectious when used to transfect chicken embryo fibroblasts. The resulting progeny virus stocks were then used to study the effects of these mutations on viral protein and RNA synthesis, growth properties, host range, and fitness compared with the parental strain. None of the mutants showed temperature sensitivity in plaquing efficiency or plaque morphology on chicken embryo fibroblast monolayers. Relative to the wild-type parent, the mutants containing sense replacements overproduced nsP34 but not nsP4 and made slightly decreased levels of nsP3, with a delay in its appearance. This indicates that the cleavage separating nsP3 and nsP4 occurs in these mutants and also that the level of nsP4 is not regulated solely by readthrough of the opal codon. The amber and ochre mutants produced decreased levels of nsP34, and the ochre mutant grew significantly more slowly than the other mutants or wild-type virus. For all five mutants, RNA synthesis early in infection was inhibited compared with that of the parental virus. This effect was apparent at multiplicities of infection of 20 PFU per cell but not at 100 PFU per cell. Using in situ hybridization to distinguish between mutant and wild-type plaques, we have studied the behavior of the serine mutant in a high-multiplicity growth competition experiment with wild-type virus. The wild-type virus eventually outcompeted the mutant after several passages, and these results indicate that this mutation has resulted in effects that are at least partially cis acting. Furthermore, by studying the growth, plaque formation, and protein synthesis of the mutants in various cell types, we have observed host range effects of the mutations, especially in mosquito and human cells. In addition, we have demonstrated, at least indirectly, that opal, amber, and ochre termination codons in the SIN nucleotide context can be suppressed in cultured cells of chicken, human, hamster, and mosquito origin.

AU - Kinney RM, Johnson BJ, Welch JB, Tsuchiya KR, Trent DW
TI - The full-length nucleotide sequences of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and its attenuated vaccine derivative, strain TC-83.
SO - Virology 1989 May;170(1):19-30
AB - Nucleotide sequence analysis of cDNA clones covering the entire genomes of Trinidad donkey (TRD) Venezuelan equine encephalitis (VEE) virus and its vaccine derivative, TC-83, has revealed 11 differences between the genomes of TC-83 virus and its parent. One nucleotide substitution and a single nucleotide deletion occurred in the 5'- and 3'-noncoding regions of the TC-83 genome, respectively. The deduced amino acid sequences of the nonstructural polypeptides of the two viruses differed only in a conservative Ser(TRD) to Thr(TC-83) substitution in nonstructural protein (nsP) three at amino acid position 260. The two silent mutations (one each in E1 and E2), one amino acid substitution in the E1 glycoprotein, and five substitutions in the E2 envelope glycoprotein of TC-83 virus were reported previously (B.J.B. Johnson, R.M. Kinney, C.L. Kost, and D.W. Trent, 1986, J. Gen. Virol. 67, 1951-1960). The genome of TRD virus was 11,444 nucleotides long with a 5'-noncoding region of 44 nucleotides. The carboxyl terminal portion of VEE nsP3 contained two peptide segments (7 and 34 amino acids long) that were repeated with high fidelity. The open reading frame of the nonstructural polyprotein was interrupted by an in-frame opal termination codon between nsP3 and nsP4, as has been reported for Sindbis, Ross River, and Middelburg viruses. The deduced amino acid sequences of the VEE TRD nsP1, nsP2, nsP3, and nsP4 polypeptides showed 60-66%, 57-58%, 35-44%, and 73-71% identity with the aligned sequences of the cognate polypeptides of Sindbis and Semliki Forest viruses, respectively. The lack of homology in the nsP3 of the viruses is due to sequence variation in the carboxyl terminal half of this polypeptide. MS - GENBANK/J04332

AU - Levis R, Schlesinger S, Huang HV
TI - Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription.
SO - J Virol 1990 Apr;64(4):1726-33
AB - Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA.

AU - Niesters HG, Strauss JH
TI - Mutagenesis of the conserved 51-nucleotide region of Sindbis virus.
SO - J Virol 1990 Apr;64(4):1639-47
AB - We have constructed 25 site-specific mutations in a domain of 51 nucleotides in Sindbis virus that is highly conserved among all alphaviruses sequenced to date. These 51 nucleotides are capable of forming two hairpin structures and are found from nucleotides 155 to 205 in Sindbis virus within the region encoding nsP1. Of the mutations, 21 were silent and did not lead to a change in the amino acid sequence encoded. These silent mutations changed not only the linear sequence but also the stability of the hairpins in most cases. Two double mutants that were constructed led to the replacement of one base pair by another so that the linear sequence was altered but the nature of the hairpins was not. All of the mutants with silent mutations were viable, but 19 of the 21 mutants were severely impaired for growth in both chicken and mosquito cells. Compared with the parental virus, they grew slowly and produced virus at rates of 10(-1) to 10(-4) times the parental rate. Surprisingly, however, the plaques produced by these mutants were indistinguishable from those produced by the parental virus. Two of the silent mutations, found within the first hairpin structure, produced virus at a faster rate than the parental virus. It is clear that the exact sequence of this region is important for some aspect of virus replication. We suggest that one or more proteins, either virus encoded or cellular, bind to the hairpin structures in a sequence-specific fashion in a step that promotes replication of the viral RNA. Of the mutations that resulted in a change of coding, only one of four was viable, suggesting that the amino acid sequence encoded in this domain is essential for virus replication.

AU - Kuhn RJ, Hong Z, Strauss JH
TI - Mutagenesis of the 3' nontranslated region of Sindbis virus RNA.
SO - J Virol 1990 Apr;64(4):1465-76
AB - A cDNA clone from which infectious RNA can be transcribed was used to construct 42 site-specific mutations in the 3' nontranslated region of the Sindbis virus genome. The majority of these mutations were made in the 3'-terminal 19-nucleotide conserved sequence element and consisted of single nucleotide substitutions or of small (1 to 8) nucleotide deletions. An attempt was made to recover mutant viruses after transfection of SP6-transcribed RNA into chicken cells. In most cases, viable virus was recovered, but almost all mutants grew more poorly than wild-type virus when tested under a number of culture conditions. In the case of mutations having only a moderate effect, the virus grew as well as the wild type but was slightly delayed in growth. Mutations having a more severe effect led to lower virus yields. In many cases, virus growth was more severely impaired in mosquito cells than in chicken cells, but the opposite phenotype was also seen, in which the mutant grew as well as or better than the wild type in mosquito cells but more poorly in chicken cells. One substitution mutant, 3NT7C, was temperature sensitive for growth in chicken cells and severely crippled for growth in mosquito cells. Insertion mutations were also constructed which displaced the 19-nucleotide element by a few nucleotides relative to the poly(A) tail. These mutations had little effect on virus growth. Deletion of large regions (31 to 293 nucleotides long) of the 3' nontranslated region outside of the 19-nucleotide element resulted in viruses which were more severely crippled in mosquito cells than in chicken cells. From these results, the following principles emerge. (i) The entire 3' nontranslated region is important for efficient virus replication, although there is considerable plasticity in this region in that most nucleotide substitutions or deletions made resulted in viable virus and, in some cases, in virus that grew quite efficiently. Replication competence was particularly sensitive to changes involving the C at position 1, the A at position 7, and a stretch of 9 U residues punctuated by a G at position 14. (ii) The panel of mutants examined collectively deleted the entire 3' nontranslated region. Only mutants in which 8 nucleotides in the 3' terminal 19 nucleotides had been deleted or in which the 3' terminal C was deleted were nonviable. Although the 3' terminal C was essential for replication, it could be displaced by at least 7 nucleotides from its 3' terminal position adjacent to the poly(A) tract.(ABSTRACT TRUNCATED AT 400 WORDS)

AU - Levinson RS, Strauss JH, Strauss EG
TI - Complete sequence of the genomic RNA of O'nyong-nyong virus and its use in the construction of alphavirus phylogenetic trees.
SO - Virology 1990 Mar;175(1):110-23
AB - The alphaviruses are a group of about 25 positive-strand RNA viruses that are important human and veterinary pathogens and that are geographically dispersed. We report here the complete nucleotide sequence of the genomic RNA of the alphavirus, O'nyong-nyong virus. The RNA is 11,835 nucleotides in length and the organization of the genome is typical of alphaviruses. Phylogenetic trees were constructed from the protein sequences of O'nyong-nyong and six other alphaviruses. Trees were constructed for each nonstructural and structural viral protein individually in order to detect any possible recombination events, as well as to examine the differential divergence among the various proteins. The members of each tree can be divided into three subgroups: the Semliki Forest virus subgroup (Semliki Forest, O'nyong-nyong, and Ross River viruses), the eastern equine encephalitis virus subgroup (eastern equine encephalitis and Venezuelan equine encephalitis viruses), and the Sindbis virus subgroup. Sindbis virus, which is geographically restricted to the Old World, is more closely related to the eastern equine encephalitis subgroup, which are New World viruses, than it is to the Semliki Forest virus subgroup, which are mostly Old World viruses. Western equine encephalitis virus is a special case because it is a recombinant virus. Its nonstructural and capsid proteins are most closely related to those of eastern equine encephalitis virus while its glycoproteins are most closely related to those of Sindbis virus. All members of a given subgroup have diverged the same amount from their common node point. However, the structural proteins of the Semliki Forest virus subgroup are more closely related to one another than those of the eastern equine encephalitis virus subgroup. This difference probably indicates that the members of the eastern equine encephalitis virus subgroup diverged earlier than the members of the Semliki Forest virus subgroup, which suggests that the alphaviruses originated in the New World. MS - GENBANK/M20303

AU - Niesters HG, Strauss JH
TI - Defined mutations in the 5' nontranslated sequence of Sindbis virus RNA.
SO - J Virol 1990 Sep;64(9):4162-8
AB - We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant.

AU - Weiss BG, Schlesinger S
TI - Recombination between Sindbis virus RNAs.
SO - J Virol 1991 Aug;65(8):4017-25
AB - The genome (49S RNA) of Sindbis virus is a positive-strand RNA of 11.7 kb that consists of two domains. The 5' two-thirds of the RNA codes for the proteins required for replication and transcription of the RNA. The 3' one-third codes for the structural proteins. The latter are translated from a 26S subgenomic RNA identical in sequence to the 3' one-third of the genome. The 26S RNA is transcribed by initiation from an internal promoter that spans the junction between the nonstructural and structural genes. We have used Sindbis virus RNAs transcribed from cloned cDNAs to demonstrate recombination between Sindbis virus RNAs in cultured cells. Several different combinations of deleted or mutationally altered RNAs gave rise to infectious recombinants. In 7 of 10 different crosses, the infectious recombinant RNAs were larger than wild-type 49S RNA. We sequenced the recombinant RNAs in the region spanning the junction between the nonstructural and structural protein genes from five different crosses. In three of the crosses, this is the only region within which recombination could have taken place to produce an infectious 49S RNA. Recombination also occurred in this region in the other two crosses. The recombinant RNAs were distinct from wild-type RNA and from each other. All contained sequence insertions derived from the parental RNAs. One contained a deletion and a rearrangement, and one also contained a stretch of 11 nucleotides not found in the Sindbis virus genome. When each of the parental RNAs contained a functional subgenomic RNA promoter, both promoters were present and functional in the recombinant RNA. Those recombinants with large sequence insertions showed evidence of evolution toward the wild-type single-junction RNA.

AU - Durbin R, Kane A, Stollar V
TI - A mutant of Sindbis virus with altered plaque morphology and a decreased ratio of 26 S:49 S RNA synthesis in mosquito cells.
SO - Virology 1991 Jul;183(1):306-12
AB - When our stock of standard Sindbis virus (SVSTD) is assayed by plaque formation on Aedes albopictus mosquito cells, about 1-2% of the plaques appear much clearer and sharper than the majority of the plaques. One of these clear plaques was picked, grown into a viral stock (SVCP), and used to prepare viral cDNA. Making use of the infectious Sindbis virus plasmid, Toto 1101 (Rice et al., 1987), we mapped the causal mutation for the clear plaque phenotype to a region between nt 7334 and 7716, and by sequencing of the viral RNA identified a mutation at nucleotide 7592. This mutation lies in the junction region of the viral genome, specifically at nucleotide -6, with reference to the initiation site for 26 S RNA synthesis. In SVCP-infected mosquito cells, but not in SVCP-infected chick cells, the ratio of subgenomic 26 S to 49 S (genomic) RNA synthesis was decreased relative to that observed in SVSTD infected cells. In terms of amino acid coding, the SVCP mutation is silent.

AU - Kuhn RJ, Niesters HG, Hong Z, Strauss JH
TI - Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus.
SO - Virology 1991 Jun;182(2):430-41
AB - We have constructed a full-length cDNA clone of the virulent T48 strain of Ross River virus, a member of the alphavirus genus. Infectious RNA can be transcribed from this clone using SP6 or T7 RNA polymerase. The rescued virus has properties indistinguishable from those of the T48 strain of Ross River virus. We have used this clone, together with a full-length cDNA clone of Sindbis virus, to construct chimeric plasmids in which the 5' and the 3' nontranslated regions of the Sindbis and Ross River genomes were exchanged. The nontranslated regions of the two viral genomes differ in both size and sequence although they maintain specific conserved sequence elements. Virus was recovered from all four chimeras. Chimeras containing heterologous 3' nontranslated regions had replicative efficiencies equal to those of the parents. In contrast, the chimeras containing heterologous 5' nontranslated regions were defective in RNA synthesis and virus production, and the severity of the defect was dependent upon the host. Replication of a virus containing a heterologous 5' nontranslated region may be inefficient due to the formation of defective protein-RNA complexes, whereas, the presumptive complexes formed between host or virus proteins and the 3' nontranslated region to promote RNA synthesis appear to function normally in the chimeras.

AU - Raju R, Huang HV
TI - Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two subgenomic mRNA promoters.
SO - J Virol 1991 May;65(5):2501-10
AB - Four types of Sindbis virus vectors, each carrying two promoters for subgenomic mRNA synthesis, were designed to measure relative promoter strengths and to survey potential contextual effects on promoter strengths. One of the promoters in each vector was used as the reference promoter, while the other was the one being tested. We used these vectors to measure the relative strengths of four promoters: the minimal promoter, an extended sequence believed to have full promoter activity, and two mutant promoters, one with an inactivating 3-nucleotide insertion called CR4.1 and the other with a 4-nucleotide deletion called delta 4. The strengths of the promoters were measured by quantitating the RNA transcribed from each promoter in vivo and also by assaying for chloramphenicol acetyltransferase activity encoded by one of the two transcripts. We found that the relative strengths of the promoters were similar in different contexts. The complete promoter was 6-fold more active, the delta 4 promoter was (surprisingly) about twice as active, and the CR4.1 promoter was 100-fold less active than the minimal promoter. At least two contextual effects were identified that can alter the activity of one or both promoters in the vectors. One effect is that given identical promoters, the 3'-proximal promoter on the minus-strand template can be more active than the 5'-proximal promoter. This may be due to preferential association of one or more components of the transcription complex for the 3' end of the minus-strand template. A second effect is promoter competition, particularly when the promoters are closely spaced.

AU - Nakhasi HL, Cao XQ, Rouault TA, Liu TY
TI - Specific binding of host cell proteins to the 3'-terminal stem-loop structure of rubella virus negative-strand RNA.
SO - J Virol 1991 Nov;65(11):5961-7
AB - At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed.

AU - Kuhn RJ, Griffin DE, Zhang H, Niesters HG, Strauss JH
TI - Attenuation of Sindbis virus neurovirulence by using defined mutations in nontranslated regions of the genome RNA.
SO - J Virol 1992 Dec;66(12):7121-7
AB - We examined a panel of Sindbis virus mutants containing defined mutations in the 5' nontranslated region of the genome RNA, in the 3' nontranslated region, or in both for their growth in cultured cells and virulence in newborn mice. In cultured cells, these viruses all had defects in RNA synthesis and displayed a wide range of growth rates. The growth properties of the mutants were often very different in mouse cells from those in chicken cells or in mosquito cells. We hypothesize that host factors, presumably proteins, interact with these nontranslated regions to promote viral replication and that the mammalian protein and the chicken or mosquito protein are sufficiently divergent that alterations in the viral RNA sequence can affect the interactions with these different host proteins in different ways. Some of the mutants were temperature sensitive for plaque formation, whereas one mutant was slightly cold sensitive in its growth in chicken cells. Upon inoculation into mice, viruses that grew well in cultured mouse cells retained their virulence, but mice that succumbed usually had extended survival times. One virulent mutant that grew slightly less well in cultured mouse cells than did the parental virus produced eight times as much virus in mouse brain following intracerebral inoculation, suggesting that changes in these regulatory regions may have tissue-specific as well as host-specific effects. Viruses that were severely crippled in their growth in mouse cells in culture were usually, but not always, attenuated in their virulence. In particular, temperature sensitivity was correlated with attenuation. The effect of two mutations was found to be cumulative, and double mutants that contained mutations in both the 5' and 3' nontranslated regions were more attenuated than was either single mutant. Three of four double mutants tested were severely crippled for virus production in cultured cells and were avirulent for mice, even when inoculated intracerebrally.

AU - Pardigon N, Strauss JH
TI - Cellular proteins bind to the 3' end of Sindbis virus minus-strand RNA.
SO - J Virol 1992 Feb;66(2):1007-15
AB - Forty-four nucleotides at the 5' terminus of the genomic RNA of Sindbis virus can form a stable stem-loop structure and have been shown previously to be important for viral replication. The structure formed by the complement of this sequence at the 3' end of the minus-strand RNA has been proposed to be a promoter for RNA replication and as such might be bound in a specific fashion by proteins of either cellular or viral origin. Short oligonucleotide probes (either 62 or 132 nucleotides) representing the 3'-terminal sequence of the minus strand were prepared. When added to extracts from infected or uninfected cells, these probes were bound by cellular proteins, as evidenced by a shift in the electrophoretic mobility of the (labeled) oligonucleotide. Competition experiments confirmed the specificity of the interaction. Proteins of apparent molecular sizes 42 and 44 kDa, and to a lesser extent 52 kDa, could be cross-linked to the minus-sense probes by UV irradiation. A mutant minus-strand probe identical to the longer probe except for a single-nucleotide deletion corresponding to nucleotide 5 in the genomic RNA, which is lethal for the virus, was also found to bind the same proteins as the wild-type probe. The half-life of the mutant probe-cellular protein complex was threefold longer than that of the wild-type complex, however, indicating that the mutant probe was bound more tightly than the wild-type probe. We hypothesize that the binding of cellular factors may be transiently required for initiation of transcription of plus-strand RNA from the minus-strand template and that overly tight binding of such factors is deleterious for RNA replication.

AU - Hertz JM, Huang HV
TI - Utilization of heterologous alphavirus junction sequences as promoters by Sindbis virus.
SO - J Virol 1992 Feb;66(2):857-64
AB - We used Sindbis virus, an alphavirus, as a model to study the evolution of the recognition of viral cis-acting sequences. During the life cycle of alphaviruses, a full-length minus-strand RNA is made and serves as a template for both genomic RNA replication and subgenomic mRNA transcription. Transcription initiates at an internal promoter site, the junction sequence, to produce a subgenomic mRNA. The junction sequences of alphaviruses are highly conserved, but they do contain a number of base differences. These could have been essentially neutral mutations during evolution, such that any of the contemporary sequences can be recognized efficiently by any of the alphaviruses. Alternately, the changes could have resulted in significant functional divergence, such that the contemporary viruses can no longer recognize heterologous junction sequences as promoters. To distinguish between these possibilities, we constructed Sindbis virus derivatives with two subgenomic mRNA promoters. One is the wild-type Sindbis virus promoter used for expression of the structural proteins. The other is either the minimal Sindbis virus promoter or the corresponding junction sequences from other alphaviruses, which are placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. RNA analyses were used to determine the relative promoter strengths of the various junction sequences. The results showed that all but two were recognized as promoters by Sindbis virus. CAT enzyme assays were used to measure the accumulation of CAT protein made from mRNAs transcribed by using the heterologous junction sequences as promoters. Most of the viruses expressed amounts of CAT enzyme within 10-fold of each other. The two viruses with junction sequences that were not recognized as promoters did not give significant CAT expression. We conclude that, with respect to Sindbis virus, the junction sequences are functionally conserved; i.e., most of the contemporary nucleotide differences in the junction sequences are neutral or near-neutral mutations. The functional conservation suggests that neither the cis-acting sequence nor the cognate binding site of the transcription factor can change independently. This type of coupled evolution between cis-acting sequences and their cognate viral protein binding sites may be a general phenomenon. For example, it explains the ubiquitous presence of conserved cis-acting sequences in each of the families of RNA viruses. There are implications of this hypothesis for the design of antiviral drugs.

AU - Reyes GR, Huang CC, Tam AW, Purdy MA
TI - Molecular organization and replication of hepatitis E virus (HEV). [Review]
SO - Arch Virol Suppl 1993;7:15-25
AB - The recently characterized fecal-orally transmitted agent of hepatitis E (formerly known as enterically transmitted non-A, non-B hepatitis) has been determined to be a new type of positive strand RNA virus. The complete sequencing of four different geographic isolates of the hepatitis E virus (HEV) has confirmed a similar genetic organization not previously recognized in nonenveloped positive strand RNA viruses. The approximately 7.5 kb RNA genome (including polyA tail) has nonstructural genes located at the 5' end and structural genes at the 3' end. Expression of these viral genes occurs in at least 3 different forward open reading frames. The largest open reading frame begins 27 nucleotides (nt) downstream of the apparent noncoding 5' end and extends 5,079 nt. Multiple nonstructural gene motifs/domains have been recognized in this 5' ORF1 including a methyltransferase, a papain-like protease, a helicase and the RNA-dependent, RNA polymerase. The second major ORF2 begins 37nt downstream of ORF1 and extends 1980 nt before terminating 65 nt upstream of the polyadenylation site. A third ORF of only 369 nt was identified by immunoscreening experiments as encoding an immunogenic epitope of the virus. Expression of the downstream ORF2 may occur through internal subgenomic RNA initiation at a sequence element found to have homology to internal RNA initiation sequences in Sindbis virus. This element in the HEV genome maps near the apparent 5' end of one of two identified subgenomic messages. The genomic organization and expression of HEV will be discussed and a hypothesis presented regarding the viral replication strategy. [References: 35]

AU - Pardigon N, Lenches E, Strauss JH
TI - Multiple binding sites for cellular proteins in the 3' end of Sindbis alphavirus minus-sense RNA.
SO - J Virol 1993 Aug;67(8):5003-11
AB - The 3' end of Sindbis virus minus-sense RNA was tested for its ability to bind proteins in mosquito cell extracts, using labeled riboprobes that represented different parts of this region. We found four domains in the first 250 nucleotides that could bind the same 50- and 52-kDa proteins, three with high affinity and one with low affinity, whereas tested domains outside this region did not bind these proteins. The first binding domain was found in the first 60 nucleotides, which represents the complement of the 5'-nontranslated region, the second in the next 60 nucleotides, the third in the following 60 nucleotides, and the fourth between nucleotides 194 and 249 (all numbering is 3' to 5'). The relative binding constants, Kr, of the first, second, and fourth sites were similar, whereas that of domain 2 was fivefold less. Deletion mapping of the first domain showed that the first 10 nucleotides were critical for binding. Deletion of nucleotides 2 to 4, deletion or replacement of nucleotide 5, or deletion of the first 15 nucleotides was deleterious for binding, deletion of nucleotides 10 to 15, 26 to 40, or 41 to 55 had little effect on the binding, and deletion of nucleotides 15 to to 25 increased the binding affinity. We also found that the corresponding riboprobes derived from two other alphaviruses, Ross River virus and Semliki Forest virus, and from rubella virus were also able to interact with the 50- and 52-kDa proteins. The Kr value for the Semliki Forest virus probe was similar to that for the Sindbis virus probe, while that for the Ross River virus probe was four times greater. The rubella virus probe was bound only weakly, consistent with the fact that mosquito cells are not permissive for rubella virus replication. We suggest that the binding of the 50- and 52-kDa proteins to the 3' end of alphavirus minus-sense RNA represents an important step in the initiation of RNA replication.

AU - Li G, Rice CM
TI - The signal for translational readthrough of a UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon.
SO - J Virol 1993 Aug;67(8):5062-7
AB - The nucleotide sequences surrounding termination codons influence the efficiency of translational readthrough. In this report, we examined the sequence requirement for efficient readthrough of the UGA codon in the Sindbis virus genomic RNA which regulates production of the putative viral RNA polymerase, nsP4. The UGA codon and its neighboring nucleotide sequences were subcloned into a heterologous coding context, and readthrough efficiency was measured by cell-free translation of RNA transcripts in rabbit reticulocyte lysates. The CUA codon immediately downstream of the UGA codon was found to be sufficient for efficient translational readthrough. Further mutagenesis of residues in the CUA triplet demonstrated that mutations at the second or third residues following the UGA codon (U and A, respectively) had little effect on readthrough efficiency. In contrast, replacement of the cytidine residue immediately downstream of the UGA codon with any of the other three nucleotides (U, A, or G) dramatically reduced the readthrough efficiency from approximately 10% to less than 1%. These results show that a simple sequence context can allow efficient readthrough of UGA codons in a mammalian translation system. Interestingly, compilation studies of nucleotide sequences surrounding eukaryotic termination codons indicate a strong bias against cytidine residues immediately 3' to UGA termination codons. Taken together with our results, this bias may reflect a selective pressure for efficient translation termination for most eukaryotic gene products.

AU - Geigenmuller-Gnirke U, Nitschko H, Schlesinger S
TI - Deletion analysis of the capsid protein of Sindbis virus: identification of the RNA binding region.
SO - J Virol 1993 Mar;67(3):1620-6
AB - The capsid protein of Sindbis virus has multiple functions in the life cycle of the virus. One essential function is to interact with the genomic RNA of the virus to form the nucleocapsid. The experiments described in this article define a region of the protein that is required for binding to Sindbis virus RNA. The assay we used measured the binding of in vitro-translated proteins to RNA on the basis of their migration with the RNA during electrophoresis in an agarose gel. Binding to RNA showed specificity; more protein bound to an RNA containing the previously defined packaging signal in Sindbis virus RNAs than to a similar RNA lacking this sequence. We were able to produce a variety of deleted forms of the capsid protein by constructing cDNAs with in-frame deletions throughout the coding region of the capsid protein gene. These cDNAs were then transcribed into mRNAs and translated in vitro. C-terminal deletions in the capsid protein were obtained by preparing transcripts from cDNAs linearized at sites within the coding region. Our studies identified a 32-amino-acid region that is essential for the specificity in RNA binding, and they defined a 68-amino-acid minimal sequence which displays almost the complete specific RNA binding activity of the intact Sindbis virus capsid protein containing 264 amino acids.

AU - Weaver SC, Hagenbaugh A, Bellew LA, Netesov SV, Volchkov VE, Chang GJ, Clarke DK, Gousset L, Scott TW, Trent DW, et al
TI - A comparison of the nucleotide sequences of eastern and western equine encephalomyelitis viruses with those of other alphaviruses and related RNA viruses [published erratum appears in Virology 1994 Aug 1;202(2):1083].
SO - Virology 1993 Nov;197(1):375-90
AB - The complete nucleotide sequence of a 1982 Florida strain of eastern equine encephalomyelitis (EEE) virus, and partial sequence of the nonstructural protein genes of western equine encephalomyelitis (WEE) virus, were determined. The EEE virus genome was 11,678 nucleotides in length, excluding the cap nucleotide and poly(A) tail, and the nucleotide composition was 28% A, 24% G, 25% C, and 23% U. The organization of both EEE and WEE virus genomes was like that of other alphaviruses and included a termination codon between the nsP3 and nsP4 genes. Codon usage for 10 of 20 amino acids was nonrandom in the EEE genome, and dinucleotide CpG-containing codons were underutilized in both genomes. The slight CpG deficiency was similar to that seen in other alphaviruses and plant viruses in the alphavirus-like group, but less than that of poliovirus and yellow fever virus. This slight deficiency may reflect adaptation for replication in both CpG-deficient vertebrates, as well as insects which do not have CpG-deficient genomes. Phylogenetic analyses using nonstructural protein amino acid sequences indicated that alphaviruses evolved from a common ancestor which existed a few thousand years ago. An intercontinental introduction of an ancestral virus from the Old to New World, or vice versa, probably resulted in two main extant groups: one includes New World (EEE and Venezuelan equine encephalitis) viruses, while the other includes Old World (Sindbis, Middelburg, O'nyong-nyong, Ross River, and Semliki Forest) viruses. The position of WEE virus in the phylogenetic trees indicated that, in addition to its capsid gene (C. S. Hahn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5997-6001), WEE virus acquired its nonstructural genes from an EEE-like ancestor during recombination. MS - GENBANK/U01034, GENBANK/U01065

AU - Schlesinger S, Weiss BG
TI - Recombination between Sindbis virus RNAs.
SO - Arch Virol Suppl 1994;9:213-20
AB - The Sindbis virus RNA genome is divided into two modules--one coding for the nonstructural protein genes and the other coding for the structural protein genes. In our studies of recombination, the two parental RNAs were defective in different modules. Analysis of the recombinant RNAs demonstrated that the parental RNAs each contributed its intact module and that the crossovers occurred within the defective modules. The recombinational events giving rise to infectious virion RNAs could create deletions, rearrangements or insertions as long as they occurred outside of the functional module. These crossovers produced RNA genomes that contained two functional subgenomic RNA promoters.

AU - Pogue GP, Huntley CC, Hall TC
TI - Common replication strategies emerging from the study of diverse groups of positive-strand RNA viruses. [Review]
SO - Arch Virol Suppl 1994;9:181-94
AB - Studies using brome mosaic virus (BMV), Sindbis virus and poliovirus have provided evidence that disparate groups of plant and animal positive strand RNA viruses have remarkably similar replication strategies. The conservation of several functional domains within virus-encoded nonstructural proteins implies that, although the precise character of these and interacting host components varies for each virus, they employ similar mechanisms for RNA replication. For (+) strand replication, similarities in cis-acting sequence motifs and RNA secondary structures within 5' termini of genomic (+) strands have been identified and have been shown to participate in binding of host factors. The model presented for replication of BMV RNA suggests that binding of these factors to internal control region (ICR) sequence motifs in the double-stranded replication intermediate releases a single-stranded 3' terminus on the (-) strand that may be essential for initiation of genomic (+) strand synthesis. ICR sequences internal to the BMV genome were also found to be required for efficient replication. Asymmetric production of excess genomic (+) over (-) strand RNA, characteristic of all (+) strand viruses, may be accomplished through transition of the replicase from competence for (-) to (+) strand synthesis by the recruitment of additional host factors. [References: 25]

AU - Frolov I, Schlesinger S
TI - Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon.
SO - J Virol 1994 Dec;68(12):8111-7
AB - One incentive for developing the alphavirus Sindbis virus as a vector for the expression of heterologous proteins is the very high level of viral structural proteins that accumulates in infected cells. Although replacement of the structural protein genes by a heterologous gene should lead to an equivalent accumulation of the heterologous protein, the Sindbis virus capsid protein is produced at a level 10- to 20-fold higher than that of any foreign protein. Chimeric mRNAs which contain the first 275 nucleotides of the Sindbis virus 26S mRNA fused to the lacZ gene are also translated at the higher level. The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin-like structure; deletions in this region destroy the activity. These sequences enhance translation in infected cells but have the opposite effect in uninfected cells. Furthermore, translation of this RNA in infected cells is suppressed by a second viral RNA lacking the hairpin-like structure, but translation of the latter RNA is not affected. We propose that the hairpin-like structure presents a barrier to the movement of the ribosomes during translation of mRNA. In infected cells, under conditions in which this mRNA is essentially the only RNA being translated, a slowdown in the transit of the ribosomes gives factors present at low concentrations a chance to bind to the translation complex and permits a high level of functional complexes to be formed. In uninfected cells and in infected cells translating two different viral subgenomic mRNAs, a pause in the movement of the ribosomes along the RNA is no longer an advantage, because the required factors are now usurped by other translation complexes.

AU - Rumenapf T, Strauss EG, Strauss JH
TI - Subgenomic mRNA of Aura alphavirus is packaged into virions.
SO - J Virol 1994 Jan;68(1):56-62
AB - Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined.

AU - Wang CY, Dominguez G, Frey TK
TI - Construction of rubella virus genome-length cDNA clones and synthesis of infectious RNA transcripts.
SO - J Virol 1994 Jun;68(6):3550-7
AB - Plasmids containing a complete cDNA copy of the rubella virus (RUB) genomic RNA were constructed. Transfection into cell culture of genome-length RNA transcribed in vitro from one of these cDNA clones, Robo102, resulted in the production of virus which preserved the genetic and phenotypic characteristics of the parental virus from which the cDNA clone was derived. Prior to construction of the RUB genome-length cDNA clones, the 5'-terminal sequence of the RUB genomic RNA was determined to be 5'CAAUGG...3' following the cap structure. Analysis of the specific infectivity of RUB genomic RNA isolated from virions revealed that in Vero cells, the specific infectivity of RUB genomic RNA is roughly equivalent to that of Sindbis virus genomic RNA. In RUB virion RNA preparations, the subgenomic RNA was detected. It was demonstrated that subgenomic RNA was packaged into RUB virions; however, the presence of the subgenomic RNA was not essential for infectivity of the genomic RNA.

AU - Rumenapf T, Strauss EG, Strauss JH
TI - Aura virus is a New World representative of Sindbis-like viruses.
SO - Virology 1995 Apr 20;208(2):621-33
AB - Aura virus is an alphavirus present in Brazil and Argentina that is serologically related to Sindbis virus (present throughout the Old World) and to Western equine encephalitis (WEE) virus (present in the Americas). We have previously shown that WEE is a recombinant virus whose glycoproteins and part of whose 3' nontranslated region (NTR) are derived from a Sindbis-like virus, but the remainder of whose genome is derived from Eastern equine encephalitis (EEE) virus. We show here that Aura virus is a Sindbis-like virus that shares considerable organizational and sequence identity with Sindbis virus. Certain nucleotide sequence elements present in Aura RNA that are believed to function as promoters are almost identical to their Sindbis counterparts, repeated elements in the 3' nontranslated region are shared with Sindbis virus, and important antigenic epitopes are conserved between the two viruses. Despite their close relationship, the two viruses have diverged significantly, sharing 73% amino acid sequence identity in the nonstructural proteins and 62% identity in the structural proteins. This is about the same as the identities between EEE and Venezuelan equine encephalitis virus, whose promoter elements, 3' NTRs, and antigenic epitopes have diverged more radically, such that these two viruses are considered to belong to different subgroups. Importantly, the glycoproteins of WEE are more closely related to those of Sindbis than to those of Aura virus. From this we propose that an ancestral Sindbis-like virus present in the Americas (probably South America) diverged 1000-2000 years ago into a lineage that gave rise to Aura virus and a lineage that gave rise to Sindbis virus and to the Sindbis-like parent of WEE. At some time after this divergence, a Sindbis-like virus belonging to the latter lineage was transferred to the Old World where it gave rise to Sindbis viruses distributed throughout the Old World, and in a separate event a Sindbis-like virus belonging to the same lineage underwent recombination with EEE to give rise to WEE.