Sindbis virus and alphavirus RNAs: a bibliography

(Return to overview of bibliography)

AU - Sprecher-Goldberger S
TI - Differences between the structures of poliovirus and Sindbis virus infectious ribonucleic acids.
SO - Arch Gesamte Virusforsch 1967;20(2):225-34

AU - Pfefferkorn ER, Burge BW, Coady HM
TI - Intracellular conversion of the RNA of Sindbis virus to a double-stranded form.
SO - Virology 1967 Oct;33(2):239-49

AU - Kingsbury DW, Yershov F
TI - Absence of nucleotide sequence complementarity between Sindbis virus and Newcastle disease virus genomes.
SO - Proc Soc Exp Biol Med 1968 Mar;127(3):806-7

AU - Ben-Ishai Z, Goldblum N, Becker Y
TI - The intracellular site and sequence of Sindbis virus replication.
SO - J Gen Virol 1968 May;2(3):365-75

AU - Scheele CM, Pfefferkorn ER
TI - Inhibition of interjacent ribonucleic acid (26S) synthesis in cells infected by Sindbis virus.
SO - J Virol 1969 Aug;4(2):117-22

AU - Dobos P, Faulkner P
TI - Properties of 42S and 26S Sindbis viral ribonucleic acid species.
SO - J Virol 1969 Oct;4(4):429-38

AU - Inglot AD
TI - A rich source of replicative ribonucleic acid of Sindbis virus--a potent interferon inducer.
SO - Arch Immunol Ther Exp (Warsz) 1970;18(4):401-8

AU - Dobos P, Faulkner P
TI - Molecular weight of Sindbis virus ribonucleic acid as measured by polyacrylamide gel electrophoresis.
SO - J Virol 1970 Jul;6(1):145-7

AU - Sreevalsan T
TI - Association of viral ribonucleic acid with cellular membranes in chick embryo cells infected with Sindbis virus.
SO - J Virol 1970 Oct;6(4):438-44 M

AU - Stollar BD, Stollar V
TI - Immunofluorescent demonstration of double-stranded RNA in the cytoplasm of Sindbis virus-infected cells.
SO - Virology 1970 Sep;42(1):276-80

AU - Stollar V, Stevens TM, Shenk T
TI - RNA of uninfected and Sindbis virus-infected Aedes albopictus cells.
SO - Curr Top Microbiol Immunol 1971;55:164-9

AU - Dobos P, Arif BM, Faulkner P
TI - Denaturation of Sindbis virus RNA with dimethyl sulphoxide.
SO - J Gen Virol 1971 Jan;10(1):103-6

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 - 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 - Shenk TE, Stollar V
TI - Effect of double-stranded RNAs from Sindbis virus-infected cells on rabbit reticulocyte and chick embryo cell-free protein synthesis.
SO - Biochim Biophys Acta 1972 Dec 22;287(3):501-13

AU - Stollar V, Shenk TE, Stollar BD
TI - Double-stranded RNA in hamster, chick, and mosquito cells infected with Sindbis virus.
SO - Virology 1972 Jan;47(1):122-32 M

AU - Arif BM, Faulkner P
TI - Genome of Sindbis virus.
SO - J Virol 1972 Jan;9(1):102-9 M

AU - Poiree JC, Bonneau HP, Nicoli J
TI - [Monocatenary ribonucleic acids of an arbovirus of group A: Sindbis virus]. [French]
SO - C R Acad Sci Hebd Seances Acad Sci D 1972 Jan 31;274(5):741-4 M

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 - Simmons DT, Strauss JH
TI - Replication of Sindbis virus. II. Multiple forms of double-stranded RNA isolated from infected cells.
SO - J Mol Biol 1972 Nov 28;71(3):615-31 M

AU - Simmons DT, Strauss JH
TI - Replication of Sindbis virus. I. Relative size and genetic content of 26 s and 49 s RNA.
SO - J Mol Biol 1972 Nov 28;71(3):599-613 M

AU - Shenk TE, Stollar V
TI - Viral RNA species in BHK-21 cells infected with Sindbis virus serially passaged at high multiplicity of infection.
SO - Biochem Biophys Res Commun 1972 Oct 6;49(1):60-7

AU - Bauchart D, Poiree JC, Prioul JL, Nicoli J
TI - [Identification of specific ribonucleic acids of the sindbis virus bound to different fractions of the endoplasmic reticulum]. [French]
SO - C R Seances Soc Biol Fil 1973;167(11):1650-5

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 - Eaton BT, Faulkner P
TI - Altered pattern of viral RNA synthesis in cells infected with standard and defective Sindbis virus.
SO - Virology 1973 Jan;51(1):85-93

AU - Rosemond H, Sreevalsan T
TI - Viral RNAs associated with ribosomes in Sindbis virus-infected HeLa cells.
SO - J Virol 1973 Mar;11(3):399-415

AU - Poiree J, Bonnet J, Negre G, Nicoli J
TI - [Polyribosomes and ribonucleic acids associated with polyribosomes in cells infected by a togavirus, the Sindbis virus]. [French]
SO - Ann Microbiol (Paris) 1973 Oct;124(3):371-85

AU - Schlesinger S, Weiss B, Goran D, Schlesinger M, Cancedda R
TI - Formation of RNA and protein in cells infected with standard and defective Sindbis virus.
SO - Med Microbiol Immunol (Berl) 1974;160(4):311-29

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 - Segal S, Sreevalsan T
TI - Sindbis virus replicative intermediates: purification and characterization.
SO - Virology 1974 Jun;59(2):428-42

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 - Weiss B, Goran D, Cancedda R, Schlesinger S
TI - Defective interfering passages of Sindbis virus: nature of the intracellular defective viral RNA.
SO - J Virol 1974 Nov;14(5):1189-98

AU - Colbere F, Hannoun C
TI - [Viral specific RNA synthesis in Sindbis infected "Aedes albopictus" mosquito cells I. - Evidence for a 18S RNA species in infected cells (author's transl)]. [French]
SO - Ann Microbiol (Paris) 1974 Oct-Nov;125B(3):393-405

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 - Brinton-Darnell M, Plagemann PG
TI - Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA.
SO - J Virol 1975 Aug;16(2):420-33
AB - Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.

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 - Zavadova Z, Libikova H
TI - Comparison of the sensitivity to ultraviolet irradiation of reovirus 3 and some viruses of the Kemerovo group.
SO - Acta Virol 1975 Jan;19(1):88-90
AB - The kinetics of UV inactivation of the tick-borne Kemerovo (strain R-10) and Lipovnik (strain Lip-91) viruses which have been preliminarily classified as possible members of the Reovirus group was examined. Reovirus 3 and Sindbis virus served as reference double-stranded RNA and single-stranded RNA viruses, respectively. The parameters of UV inresembled those of Reovirus 3. This is consistent with their tentative classification as reovirus-like viruses.

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 S, Weiss B, Dohner D
TI - Defective particles in alphavirus infections.
SO - Med Biol 1975 Oct;53(5):372-9
AB - This article summarizes our studies with defective-interfering particles of Sindbis virus obtained by high multiplicity passaging of the virus in BHK cells. Cells infected with these defective passages accumulate a species of RNA (20S) at the expense of 26S RNA--the mRNA coding for the viral structural proteins. Although the structure of the RNA in defective particles remains undefined, our studies of replicative forms and replicative intermediates suggest that it is larger than the intracellular 20S RNA. The defective particles are unable to synthesize detectable amounts of viral structural proteins when they infect a cell in the absence of standard virus and they do not contribute to the stimulation of intracellular viral RNA synthesis. We have proposed a model for the mechanism of interference by these defective particles in which standard and defective RNAs compete for a limited amount of viral-specific replicase.

AU - Johnston RE, Tovell DR, Brown DT, Faulkner P
TI - Interfering passages of Sindbis virus: concomitant appearance of interference, morphological variants, and trucated viral RNA.
SO - J Virol 1975 Oct;16(4):951-8
AB - Serial passage of Sindbis at high multiplicities of infection resulted in cyclical variations in virus titer. Decreases in virus titer were correlated with the appearance of smaller-sized virions, interference and truncated viral RNA. The smaller particles were 37 nm in diameter, exclusive of the hemagglutinin spikes as compared with a diameter of 50 nm for standard virions. Passages which contained 37-nm partilces also interfered with infectious center formation by standard, plaque-purified virus. Polyacrylamide gel analysis of RNA isolated from virions present in interfering passages demonstrated the sequential appearance of three RNA species smaller than standard RNA with approximate molecular weights of 3.3 X 106, 2.7 X 106, and 2.2 X 106. The 3.3 X 106 RNA was evident in passage 5, by passage 8 both the 3.3 X 106 and 2.7 X 106 RNAs were present, and by passage 13 all three were present with the 2.2 X 106 RNA predominating.

AU - Dubin DT, Stollar V
TI - Methylation of Sindbis virus "26S" messenger RNA.
SO - Biochem Biophys Res Commun 1975 Oct 27;66(4):1373-9

AU - Guild GM, Stollar V
TI - Defective interfering particles of Sindbis virus. III. Intracellular viral RNA species in chick embryo cell cultures.
SO - Virology 1975 Sep;67(1):24-41

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 - Obijeski JF, Bishop DH, Palmer EL, Murphy FA
TI - Segmented genome and nucleocapsid of La Crosse virus.
SO - J Virol 1976 Dec;20(3):664-75
AB - La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.

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 - Petkevich AS, Dikii VV, Leont'ev NA, Galegov GA
TI - [Identification of RNAase-sensitive 20S RNA in a cell culture infected with Sindbis virus]. [RUSSIAN]
SO - Biokhimiia 1976 Nov;41(11):1983-6
AB - RNAse-sensitive 20S RNA component with molecular weight of 0.7-10(6) is found when analysing virus-specific RNAs isolated from cultured chicken embryo fibroblasts infected with Sindbis virus by means of gradient centrifugation and polyacrylamide gel electrophoresis.

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 - Dikii VV, Petkevich AS, Leont'eva NA, Galegov GA
TI - [Effect of rimantadine on the synthesis of virus-specific RNA in the culture of cells infected with Sindbis virus]. [RUSSIAN]
SO - Vopr Med Khim 1976 Nov-Dec;22(6):844-8
AB - Effect of rimanthadine (alpha-methyl-I-adamantane methylamine) on the synthesis of virus specific RNA was studied in culture of cells, infected with Syndbis virus. Rimanthadine inhibits the synthesis of virus specific RNA beginning from the 3-rd hr of infection. A magnitude of inhibition from the 3-rd to the 7-th hrs of infection was practically the same and exceeded 50%. In the presence of rimanthadine "early" RNA was formed (with sedimentation constant of 20-14 S), where radioactivity was lowered as compared with control; formation of more "late" RNA peaks (43 S, 34 S and 26 S) was completely prevented within these periods of infection.

AU - Zhdanov VM, Azadova NB
TI - [Integration and transfection of an arbovirus by mammalian cells]. [RUSSIAN]
SO - Mol Biol (Mosk) 1976 Nov-Dec;10(6):1296-302
AB - A system: L cells chronically infected with Sindbis virus was studied. Unlike acute infection wherein the mature virions are produced, the chronically infected tissue culture produces subviral structures-infectious ribonucleoproteins. Molecular hybridization experiments revealed the integration of the viral genome (DNA-transcript) into the cellular genome. Transfection experiments showed the possibility to induce the synthesis of the virus in sensitive cells treated with DNA from the chronically infected cells.

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 - Brawner TA, Lee JC, Trent DW
TI - A comparison of Saint Louis encephalitis and Sindbis virus RNA.
SO - Arch Virol 1977;54(1-2):147-51
AB - RNA isolated from purified St. Louis encephalitis and Sindbis virus particles was compared by cellulose CF-11 chromatography, and RNase T1 and pancreatic RNase A digestion. SLE RNA eluted from the cellulose CF-11 column as a molecule with very little secondary structure, while Sindbis appears to have some internal bonding. Ribonuclease digestion indicates that SLE RNA contains 2.4 per cent polyadenylic acid.

AU - Boulton RW, Westaway EG
TI - Togavirus RNA: reversible effect of urea on genomes and absence of subgenomic viral RNA in Kunjin virus-infected cells.
SO - Arch Virol 1977;55(3):201-8
AB - Electrophoretic analyses showed that no RNase-sensitive RNA smaller than the genome was specified by the flavivirus Kunjin in infected Vero cells during the period of maximum RNA and protein synthesis. In contrast, RNA extracted from Sindbis virus-infected cells under similar conditions included the expected 42S RNA (equivalent to the genome) and the smaller 26S (interjacent) RNA. Treatment of the genome of both togaviruses with 12 M urea produced a reversible (possibly conformational) change; measurement of the molecular weights of the treated RNAs by co-electrophoresis with fully denatured ribosomal RNA markers in SDS-polyacrylamide gels yielded a value of 2.1 X 10(6) if 8 M urea was incorporated in the gels and 4.2 X 10(6) if urea was omitted from the gels. These results indicate that flavivirus messenger RNA is represented solely by the intact genome of m.wt. 4.2 X 10(6).

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 - Guild GM, Flores L, Stollar V
TI - Defective interfering particles of Sindbis virus. IV. Virion RNA species and molecular weight determination of defective double-stranded RNA.
SO - Virology 1977 Mar;77(1):158-74

AU - Guild GM, Stollar V
TI - Defective interfering particles of Sindbis virus. V. Sequence relationships between SVSTD 42 S RNA and intracellular defective viral RNAs.
SO - Virology 1977 Mar;77(1):175-88

AU - Yuferov V, Grandgenett DP, Bondurant M, Riggin C, Tigges M
TI - Synthesis of Sindbis virus complementary DNA by avian myeloblastosis virus RNA-directed DNA polymerase.
SO - Biochim Biophys Acta 1978 Jul 24;519(2):348-55
AB - Sindbis virus 42 S RNA was efficiently transcribed into complementary DNA (CDNA) by avian myeloblastosis virus alphabeta DNA polymerase using oligo- (dT) or single-stranded calf thymus DNA as primers. Both of the Sindbis virus cDNA products were able to protect 60% of 125I-labeled Sindbis virus RNA, at near equal weight ratios, from RNAase A and T1 digestion. Using hybridization kinetics, the Crt 1/2 value for hybridization of the calf thymus-primed cDNA product with excess Sindbis RNA was determined to be 1.8 9 10-2 mol . s . 1-1. Thes data demonstrate that the Sindbis virus cDNA products are relatively uniform representations of Sindbis virus RNA sequences.

AU - Frey TK, Strauss JH
TI - Replication of Sindbis virus. VI. Poly(A) and poly(U) in virus-specific RNA species.
SO - Virology 1978 May 15;86(2):494-506

AU - Martin JH, Weir RC, Dalgarno L
TI - Replication of standard and defective Ross River virus in BHK cells: patterns of viral RNA and polypeptide synthesis.
SO - Arch Virol 1979;61(1-2):87-103
AB - Virus-specific macromolecule synthesis has been examined in BHK cells infected with Ross River virus. Unpassaged virus (R-0) and tenth-passage virus (R-10) have been compared. In infected cells R-0 generates i) 45S, 28S, 33S and 26S viral RNAs, ii) virus-specific precursor polypeptides of mol. wt. 127,000, 95,000 and 61,000 and iii) viral envelope proteins (mol. wts. 52,000 and 49,000) and nucleocapsid protein (mol. wt. 32,000). Thus in terms of virus-specific RNA and polypeptide synthesis, the replication of standard RRV is analogous to that of Semliki Forest virus and Sindbis virus. R-10 interferes with the replication of standard Ross River virus and generates large amounts of 19S and 24S defective RNA species; 45S and 26S RNA synthesis was not markedly affected. Defective RNAs are associated with RNAse-sensitive, 50S cytoplasmic particles which contain a variety of (mainly host) proteins but no nucleocapsid protein. No evidence for translation of defective RNAs was obtained. R-10 infection is also characterized by a relatively early shut down of host protein syntehsis and by a reduction in virus-specific polypeptide synthesis and nucleocapsid formation. The data suggest that defective Ross River virus interferes primarily at the translational level.

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 - Leone A, Shatkin AJ, Cancedda R
TI - Isolation of Sindbis virus 26 S RNA by cDNA-cellulose chromatography.
SO - FEBS Lett 1979 Apr 1;100(1):103-6

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, 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 - Kowal KJ, Stollar V
TI - Differential sensitivity of infectious and defective-interfering particles of Sindbis virus to ultraviolet irradiation.
SO - Virology 1980 May;103(1):149-57

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 - Regnery RL, Johnson KM, Kiley MP
TI - Virion nucleic acid of Ebola virus.
SO - J Virol 1980 Nov;36(2):465-9
AB - The virion nucleic acid of Ebola virus consists of a single-stranded RNA with a molecular weight of approximately 4.0 x 10(6). The virion RNA did not bind to oligodeoxythymidylic acid-cellulose under conditions known to bind RNAs rich in polyadenylic acid and was not infectious under conditions which yielded infectious RNA from Sindbis virus, suggesting that Ebola virus virion nucleic acid is a negative-stranded RNA.

AU - Rentier-Delrue F, Young NA
TI - Genomic divergence among Sindbis virus strains.
SO - Virology 1980 Oct 15;106(1):59-70

AU - Rice CM, Strauss JH
TI - Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins.
SO - Proc Natl Acad Sci U S A 1981 Apr;78(4):2062-6
AB - The nucleotide sequence of intracellular 26S mRNA of Sindbis virus has been determined by direct sequence analysis of the cDNA made to this RNA with reverse transcriptase. From this, the amino acid sequences of the encoded virus structural proteins, which include a basic capsid protein and two integral membrane glycoproteins, have been deduced. The features of these proteins as related to their functions are discussed. We suggest that three proteases are required to produce these proteins from their polyprotein precursor: a viral protease, which functions in the cytosol to release the capsid protein, signalase, which makes two cleavages to separate the glycoproteins; and a protease of the Golgi complex that cleaves after double basic residues, to process the precursor form of one of the glycoproteins. MS - GENBANK/J02363, GENBANK/J02364, GENBANK/J02365, GENBANK/J02366, GENBANK/J02367, GENBANK/V00073

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 - 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, 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 - 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 - 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 - 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 - Sagripanti JL
TI - The genome of simian hemorrhagic fever virus.
SO - Arch Virol 1984;82(1-2):61-72
AB - Techniques are described for preparing intact Simian Hemorrhagic Fever (SHF) virus RNA. SHF RNA extracted by proteinase K digestion in the presence of sodium dodecyl sulphate (SDS) has a sedimentation coefficient of 49 S compared with a reference figure of 47 S for Sindbis RNA. Purified SHF RNA in cesium sulphate gradient has a buoyant density of 1.63 g/ml similar to that of Sindbis RNA. This result leads to the conclusion that SHF RNA is single stranded. This is supported by results on RNAse sensitivity and analyses on sucrose gradients under different ionic strength conditions. Electrophoresis analyses on both hydroxymethylmercuric acid or formaldehyde containing gels, gave a value of about 5.5 X 10(6) daltons for the molecular weight of SHF RNA. No evidence of subunit structure was found. From these results, we conclude that the SHF virus genome is a single continuous chain of about 15,000 ribonucleotides.

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 - 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 - 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 - Strauss EG, Levinson R, Rice CM, Dalrymple J, Strauss JH
TI - Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.
SO - Virology 1988 May;164(1):265-74
AB - We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function. MS - GENBANK/M20303, GENBANK/M20539

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 - Schelp C, Kaaden OR
TI - Enhanced full-length transcription of Sindbis virus RNA by effective denaturation with methylmercury hydroxide.
SO - Acta Virol 1989 May;33(3):297-302
AB - 49-S Sindbis virus RNA was reverse transcribed into a complementary DNA. The RNA templates were denatured by three different methods prior to DNA synthesis. Efficient full-length transcription was only achieved after treatment with methylmercury hydroxide.

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 - 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 - Shirako Y, Niklasson B, Dalrymple JM, Strauss EG, Strauss JH
TI - Structure of the Ockelbo virus genome and its relationship to other Sindbis viruses.
SO - Virology 1991 Jun;182(2):753-64
AB - Ockelbo virus was first isolated in 1982 in Sweden. It is the causal agent of disease in humans characterized by arthritis, rash, and fever and is antigenically very closely related to Sindbis virus. We have determined the nucleotide and translated amino acid sequences of the prototype Ockelbo virus isolate (82-5) to determine the relatedness of Ockelbo virus to Sindbis virus at the genomic level and clarify the taxonomic position of Ockelbo virus within the alphavirus genus. The numbers of nucleotides and of translated amino acids in each region of the Ockelbo virus genome were exactly the same as those for the prototype AR339 strain of Sindbis virus except for three small deletions and insertions in the C-terminal half of nsP3 and for three single nucleotide insertions and deletions in the 3' untranslated region. Overall there were 672 nucleotide differences (5.7% divergence), resulting in 97 amino acid changes (2.6% divergence), between the two viruses: more than 85% of the nucleotide changes were silent. Only the C-terminal domain of nsP3 and the E2 glycoprotein showed a higher degree of amino acid substitution than the overall average. The former domain is not conserved among alphaviruses, and the latter is primarily responsible for antigenic variation. Sequence analysis of 420 nucleotides at the 3' end of a number of other Sindbis-like alphaviruses, including Karelian fever virus and South African, Indian, and Australian isolates of Sindbis virus, demonstrated that Ockelbo virus is more closely related to South African strains of Sindbis virus than it is to the prototypic Egyptian AR339 strain. Thus the South African strains, which have caused epidemic disease in humans, may have been introduced into Northern Europe by man or by migratory birds to establish Ockelbo disease there. The Indian and Australian strains form a distinct branch of the evolutionary tree and differ from prototypic AR339 Sindbis virus in 17% of the nucleotides sequenced. MS - GENBANK/M69205, GENBANK/M69207, GENBANK/M69208, GENBANK/M69209, GENBANK/M69210, GENBANK/M69211, GENBANK/M69087, GENBANK/M69088, GENBANK/M69093, GENBANK/M69096

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 - 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 - Lindsay MD, Coelen RJ, Mackenzie JS
TI - Genetic heterogeneity among isolates of Ross River virus from different geographical regions.
SO - J Virol 1993 Jun;67(6):3576-85
AB - The RNase T1 maps of 80 isolates of Ross River virus from different regions of mainland Australia and the Pacific Islands were compared. Four different clusters of isolates with greater than an estimated 5 to 6% diversity at the nucleotide level were found. There was a pattern of differences between eastern and western Australian strains; however, the pattern was disturbed by overlaps and incursants. Pacific Islands isolates belonged to the eastern Australian topotype. Our findings suggest that certain genetic types of Ross River virus predominate in different geographical regions. In contrast, populations of other important Australian arboviruses (Murray Valley encephalitis, Kunjin, and Sindbis viruses) are distributed across the Australian continent as minor variants of one strain. Our data also show that in one region, strains of Ross River virus with identical RNase T1 maps circulate during both years when epidemics occur and years when they do not. This finding suggests that Ross River virus epidemics are not dependent on the introduction or evolution of new strains of the virus. Two strains, belonging to the eastern Australian topotype, were isolated in Western Australia. It is likely that viremic humans or possibly domestic livestock travelling by aircraft were responsible for this movement.

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 - 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.

AU - Ahola T, Kaariainen L
TI - Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP.
SO - Proc Natl Acad Sci U S A 1995 Jan 17;92(2):507-11
AB - After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a covalent enzyme-GMP intermediate. We have studied the alphavirus-specific guanylyltransferase by incubating lysates from Semliki Forest and Sindbis virus-infected cells with [alpha-32P]GTP, using vaccinia virus and mock-infected cells as controls. One additional 32P-labeled protein was detected in alphavirus-infected cells but only in the presence of S-adenosylmethionine. This protein was identified as the nonstructural protein nsP1. The properties of the covalent enzyme-guanylate complex were studied with Semliki Forest virus nsP1 expressed in recombinant baculovirus-infected cells. S-Adenosylmethionine and divalent cations were required for the complex formation. The reaction was specific for guanylate nucleotides (GTP, dGTP) and was inhibited by pyrophosphate. nsP1 could be labeled with S-adenosyl[methyl-3H]methionine but only under conditions in which the nsP1-guanylate complex was formed. 7-Methyl-GMP was released from the nsP1-guanylate complex by treatment with acid or acidic hydroxylamine. Similar treatment of vaccinia virus capping enzyme released GMP. These findings suggest that in the capping of alphavirus mRNAs the guanine is methylated before linkage to the mRNA molecule.