(Return to overview of bibliography)
(Return to overview of bibliography)
AU - Levis R
au - Huang H
au - Schlesinger S
TI - Engineered defective interfering RNAs of Sindbis virus express bacterial chloramphenicol acetyltransferase in avian cells.
SO - Proc Natl Acad Sci U S A 1987 Jul;84(14):4811-5
AB - We are investigating the feasibility of using the positive-strand RNA virus Sindbis virus and its defective interfering (DI) particles as vectors for introducing foreign genes into cells. In previous work we showed by deletion mapping of a cloned cDNA derived from one of the DI RNAs that only nucleotides at the 3' and 5' termini of the RNA are essential for the DI RNA to be amplified after it is transfected into cells in the presence of helper virus. As a first step in developing a vector we replaced 75% of the internal nucleotides of this DI cDNA with foreign sequences including the bacterial chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. DI RNAs transcribed from this cDNA were replicated and packaged by helper Sindbis virus and became a major viral RNA species in infected cells by the third passage after transfection. They were also translated to produce enzymatically active CAT. CAT activity was measured at passage 3 but could also be detected in transfected cells. DI RNAs containing the CAT gene were translated in vivo and in vitro to produce two polypeptides immunoprecipitable by anti-CAT antibodies. One polypeptide was identical in size to the authentic CAT polypeptide; the other was the size expected for a protein initiated at an upstream, viral-specific AUG in frame with the CAT AUG. These studies establish that DI genomes of Sindbis virus can tolerate the insertion and direct the expression of at least one foreign gene.
AU - Xiong C
au - Levis R
au - Shen P
au - Schlesinger S
au - Rice CM
au - Huang HV
TI - Sindbis virus: an efficient, broad host range vector for gene expression in animal cells.
SO - Science 1989 Mar 3;243(4895):1188-91
AB - Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.
AU - Bredenbeek PJ
au - Frolov I
au - Rice CM
au - Schlesinger S
TI - Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs.
SO - J Virol 1993 Nov;67(11):6439-46
AB - Since the recovery of infectious RNA transcripts from full-length cDNA clones, alphavirus genome RNAs have been engineered to allow expression of heterologous RNAs and proteins. The highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant RNAs are self-replicating (replicons) and can be introduced into cells as naked RNA, but they require trans complementation to be packaged and released from cells as infectious virion particles. In this report, we describe a series of defective Sindbis virus helper RNAs which can be used for packaging Sindbis virus RNA replicons. The defective helper RNAs contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter which drives expression of the structural protein genes. In cells cotransfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from the replicon RNA allow replication and transcription of the defective helper RNA to produce the virion structural proteins. A series of defective helper RNAs were compared for the ability to package the replicon RNA as well as for the ability to be replicated and packaged. One defective helper RNA not only packaged the replicon but also was itself encapsidated and would be useful under conditions in which extensive amplification is advantageous. Other defective helper RNAs were able to package the replicon efficiently but were packaged very poorly themselves. These helpers should be useful for applications in which expression of the viral structural proteins or virus spread is not desired.
AU - Geigenmuller-Gnirke U
au - Weiss B
au - Wright R
au - Schlesinger S
TI - Complementation between Sindbis viral RNAs produces infectious particles with a bipartite genome.
SO - Proc Natl Acad Sci U S A 1991 Apr 15;88(8):3253-7
AB - Sindbis virus, the type member of the alpha-viruses, is an enveloped virus containing a nonsegmented positive-strand RNA genome. We show that the nonstructural and the structural genes can function to produce infectious virus particles when they are expressed on two different RNA segments. The nonstructural genes are translated from an RNA in which the structural genes have been replaced by the chloramphenicol acetyltransferase gene [Xiong, C., Levis, R., Shen, P., Schlesinger, S., Rice, C. M. & Huang, H. V. (1989) Science 243, 1188-1191]. The structural genes are encoded in a defective-interfering RNA but are translated from a subgenomic RNA. Both segments contain the cis-acting sequences required for replication and packaging and are copackaged. This type of genome provides a model for an ancestral intermediate between alphaviruses and the multipartite positive-strand RNA viruses of plants. These different viruses show sequence similarities in their replicative proteins and are thought to have evolved from a common ancestor.
AU - Raju R
au - 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 - Perri S
au - Driver DA
au - Gardner JP
au - Sherrill S
au - Belli BA
au - Dubensky TW Jr
au - Polo JM
TI - Replicon vectors derived from Sindbis virus and Semliki forest virus that establish persistent replication in host cells.
SO - J Virol 2000 Oct;74(20):9802-7
AB - Alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant replicons. This work expands the utility of noncytopathic replicons and the understanding of how alphavirus replicons establish persistent replication in cultured cells.
AU - Polo JM
au - Belli BA
au - Driver DA
au - Frolov I
au - Sherrill S
au - Hariharan MJ
au - Townsend K
au - Perri S
au - Mento SJ
au - Jolly DJ
au - Chang SM
au - Schlesinger S
au - Dubensky TW Jr
TI - Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors.
SO - Proc Natl Acad Sci U S A 1999 Apr 13;96(8):4598-603
AB - Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10(7) infectious units/ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors.
AU - Dubensky TW Jr
au - Driver DA
au - Polo JM
au - Belli BA
au - Latham EM
au - Ibanez CE
au - Chada S
au - Brumm D
au - Banks TA
au - Mento SJ
au - Jolly DJ
au - Chang SM
TI - Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer.
SO - J Virol 1996 Jan;70(1):508-19
AB - Several DNA-based Sindbis virus vectors were constructed to investigate the feasibility and potential applications for initiating the virus life cycle in cells transfected directly with plasmid DNA. These vectors, when transfected into mammalian cells, have been used to produce virus, to express heterologous genes, and to produce infectious vector particles. This approach involved the conversion of a self-replicating vector RNA (replicon) into a layered DNA-based expression system. The first layer includes a eukaryotic RNA polymerase II expression cassette that initiates nuclear transcription of an RNA which corresponds to the Sindbis virus vector replicon. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. The Sindbis virus DNA vectors expressed reporter genes in transfected cells at levels that were comparable to those of in vitro-transcribed RNA replicons and were approximately 10-fold higher than the levels produced by conventional RNA polymerase II-dependent plasmids in which the promoter and reporter gene were linked directly. Reporter gene expression was also observed in rodent muscle following injection with Sindbis virus DNA vectors. In a second application, packaged vector particles were produced in cells cotransfected with complementing replicon and defective helper DNAs. The Sindbis virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.
AU - Weiss BG
au - 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 - Schlesinger S
au - 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 - Levis R
au - Schlesinger S
au - 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 - Frolov I
au - 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 - Frolov I
au - Schlesinger S
TI - Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.
SO - J Virol 1994 Mar;68(3):1721-7
AB - Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.
AU - Jackson AC
au - Bowen JC
au - Downe AE
TI - Experimental infection of Aedes aegypti (Diptera: Culicidae) by the oral route with Sindbis virus.
SO - J Med Entomol 1993 Mar;30(2):332-7
AB - The infectivity, dissemination, and transmissibility of wild-type Sindbis (SIN) virus were studied in Aedes aegypti (L). There was an initial decline in the viral titer of whole mosquitoes for 3 d after ingestion of virus, followed by a gradual increase to a maximal level by day 6. Immunoperoxidase staining of Ae. aegypti for viral antigen showed infection of midgut epithelial cells on day 1, of the fat body by day 3, and of the brain by day 4. By day 5, there was infection of the foregut, hindgut, Malpighian tubules, ovariole sheaths, Johnston's organ, thoracic ganglia, ventral nerve cord, and salivary glands. Viral antigen was not detected in the flight muscles and was found only in ovariole sheaths of the ovaries; germinal tissue was not infected. The transmission rate from SIN-infected Ae. aegypti to neonatal mice was 40%. A comparison of Ae. aegypti infected with SIN and with a neuroadapted strain of Sindbis virus (NSIN), which is more neurovirulent than SIN to mice after intracerebral inoculation, did not reveal significant differences in infectivity, dissemination, or transmissibility. The important differences between SIN and NSIN in a mouse model were not reflected in the infection of Ae. aegypti by the oral route.
AU - Strauss JH
au - Wang KS
au - Schmaljohn AL
au - Kuhn RJ
au - Strauss EG
TI - Host-cell receptors for Sindbis virus.
SO - Arch Virol Suppl 1994;9:473-84
AB - Sindbis virus has a very wide host range, infecting many species of mosquitoes and other hematophagous insects and infecting many species of higher vertebrates. We have used two approaches to study host cell receptors used by Sindbis virus to enter cells. Anti-idiotype antibodies to neutralizing antibodies directed against glycoprotein E2 of the virus identified a 63-kDa protein as a putative receptor in chicken cells. In a second approach, monoclonal antibodies identified a 67 kDa protein, believed to be a high affinity laminin receptor, as a putative receptor in mammalian cells and in mosquito cells. We conclude that the virus attains its very wide host range by two mechanisms. In one mechanism, the virus is able to use more than one protein as a receptor. In a second mechanism, the virus utilizes proteins as receptors that are highly conserved across the animal kingdom.
AU - Corsini J
au - Traul DL
au - Wilcox CL
au - Gaines P
au - Carlson JO
TI - Efficiency of transduction by recombinant Sindbis replicon virus varies among cell lines, including mosquito cells and rat sensory neurons.
SO - BioTechniques 1996 Sep;21:492-497
AB - Recombinant alphaviruses have been used as vehicles for delivery and expression of heterologous genes in mammalian, avian and insect cell lines. We have used a Sindbis replicon virus (SINreplac) able to express the E. coli lacZ gene to compare the efficiency of transduction in one insect, six mammalian cell lines and cultured rat dorsal neurons which apparently express beta-galactosidase over a 30-day time period. Results show that different celll lines were transduced with varying degrees of efficiency and that this efficiency could be improved in some cell lines by packaging the replicon with a helper derived from a more neurovirulent strain of Sindbis.
AU - Olivo PD
au - Frolov I
au - Schlesinger S
TI - A cell line that expresses a reporter gene in response to infection by Sindbis virus: a prototype for detection of positive strand RNA viruses.
SO - Virology 1994 Jan;198(1):381-4
AB - We describe a stably transformed cell line (BHKSINLuc2) that contains a defective Sindbis virus genome under the control of a Rous sarcoma virus promoter and the luciferase gene downstream of the viral subgenomic RNA promoter. This cell line expresses high levels of luciferase activity following infection with Sindbis virus and provides a sensitive assay for titering variants of Sindbis virus that lack the structural protein genes, in particular, Sindbis virus replicons that express heterologous proteins. Cell lines such as this may be of value for detection of positive-strand RNA viruses.
AU - Xiong C
Grieve RB
Kim K
Boothroyd JC
TI - Expression of Toxoplasma gondii P30 as fusions with glutathione S-transferase in animal cells by Sindbis recombinant virus.
SO - Mol Biochem Parasitol 1993 Sep;61(1):143-8
AU - Pugachev KV
au - Mason PW
au - Shope RE
au - Frey TK
TI - Double-subgenomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induce significant protection in mice against lethal JEV infection.
SO - Virology 1995 Oct 1;212(2):587-94
AB - A series of double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding the immunogenic proteins of Japanese encephalitis virus (JEV) [prM-E, prM-E-NS1, NS1-NS2A, 80%E (encodes the amino-terminal 80% part of E), and NS1] were constructed and analyzed for their ability to confer protective immunity in mice against lethal challenge with neurovirulent JEV. The cassettes were introduced into both 5' [second subgenomic promoter of the vector precedes the SIN structural open reading frame (SP-ORF)] and 3' (the promoter follows the SP-ORF) dsSIN vectors. The longest cassette (prM-E-NS1) was 3.2 kb in length, which is remarkable for such a small vector virus as SIN (SIN genome is roughly 11.8 kb in length). The level of expression of JEV proteins appeared similar for both 5' and 3' recombinants. In general, the stability of the recombinants obtained was found to be low (expression was lost following one to five passages at low multiplicity of infection, depending on the recombinant). However, 5' recombinants containing longer cassettes (prM-E-NS1, prM-E, NS1-NS2A) were more stable than the corresponding 3' recombinants. Intraperitoneal inoculation of mice with 10(7) PFU of dsSIN-JEV recombinants induced antibodies against JEV proteins and low titers of JEV-neutralizing antibodies were produced by mice inoculated with recombinants expressing 80%E, prM-E, and prM-E-NS1. A single immunization of mice with the dsSIN-prM-E or dsSIN-prM-E-NS1 recombinants provided 40-65% protection against peripheral lethal challenge with 10(4) LD50 of neurovirulent JEV. The results demonstrate that genetically engineered togaviruses can be successfully used as vaccine vectors.
AU - Pugachev KV
au - Mason PW
au - Frey TK
TI - Sindbis vectors suppress secretion of subviral particles of Japanese encephalitis virus from mammalian cells infected with SIN-JEV recombinants.
SO - Virology 1995 May 10;209(1):155-66
AB - Double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding prM-E or a C-terminally truncated form of E of Japanese encephalitis virus (JEV) were constructed. The products were efficiently expressed in both mammalian and mosquito cell lines infected with the dsSIN recombinants. However, suppression of prM-E secretion from mammalian cells infected with dsSIN-prM-E recombinants was observed. This suppression was more pronounced late in infection (< 5% of total product was secreted during an 8-hr chase) than early in infection (15% secretion during a 6-hr chase). In comparison, a vaccinia virus-prM-E recombinant (vP829) described previously (E. Konishi et al. (1991) Virology 185, 401-410) was shown to secrete 35-50% of total product during a 6- to 8-hr chase both early and late in infection. In contrast, secretion of prM-E from dsSIN-prM-E-infected mosquito (C6/36) cells was found to be efficient (> 50% during an 8-hr chase). The prM-E secreted from both mammalian and mosquito cells was in the form of subviral particles as determined by velocity gradient centrifugation, sensitivity to nonionic detergent, and analysis of processing of N-linked glycans. The truncated E protein expressed by the dsSIN recombinants was secreted efficiently from both mammalian and mosquito cells. Coinfection experiments with the dsSIN-JEV recombinants + wild-type vaccinia virus and vP829 + SIN demonstrated that the reduced level of secretion of subviral particles exhibited by the dsSIN-JEV recombinants was due to an inhibitory effect of the dsSIN vectors. Furthermore, this inhibitory effect was accounted for by the SIN nonstructural proteins since SIN replicons that express prM-E cassette in place of the SIN structural protein open reading frame exhibited a low level of subviral particle secretion. No self-propagating infectious particles were produced in cells transfected with SIN replicons that encode the JEV prM-E cassette. The suppression of subviral particle secretion was apparently correlated with the inhibition of cell protein synthesis which is mediated in SIN-infected vertebrate cells by expression of the SIN nonstructural proteins.
AU - Johanning FW
au - Conry RM
au - LoBuglio AF
au - Wright M
au - Sumerel LA
au - Pike MJ
au - Curiel DT
TI - A Sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo.
SO - Nucleic Acids Res 1995 May 11;23(9):1495-501
AB - The direct intramuscular delivery of naked plasmid DNA has been demonstrated to allow expression of encoded heterologous genes in the target myocytes. The method has been employed to elicit immunization based upon delivery of antigen encoding plasmid DNA. For application in the context of achieving anti-tumor immunization against antigenic transforming oncoproteins, delivery of plasmid DNAs encoding these molecules would create significant potential safety hazards. As an alternative to DNA polynucleotide vectors, we explored the utility of mRNA vehicles for inducing foreign gene expression in muscle cells in vivo. Synthetic reporter-gene encoding mRNA transcripts were derived for this analysis. The Sindbis virus vector was also used to derive luciferase mRNA transcripts which possessed self-replication capacity. In these studies, it could be shown that the replicative vector was capable of directing significantly elevated levels of reporter gene expression in myocytes compared to a non-replicative mRNA species. In addition, the replicative species was capable of achieving significantly prolonged levels of in vivo gene expression compared to non-replicative mRNA. Both of these characteristics will make replicative mRNA vectors of utility for polynucleotide-based immunization protocols.
AU - Gardner JP
au - Frolov I
au - Perri S
au - Ji Y
au - MacKichan ML
au - zur Megede J
au - Chen M
au - Belli BA
au - Driver DA
au - Sherrill S
au - Greer CE
au - Otten GR
au - Barnett SW
au - Liu MA
au - Dubensky TW
au - Polo JM
TI - Infection of Human Dendritic Cells by a Sindbis Virus Replicon Vector Is Determined by a Single Amino Acid Substitution in the E2 Glycoprotein.
SO - J Virol 2000 Dec 15;74(24):11849-11857
AB - The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55(Gag) elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.
AU - Coppola MA
au - Lam TM
au - Strawbridge RR
au - Green WR
TI - Recognition of endogenous ecotropic murine leukaemia viruses by anti-AKR/gross virus cytotoxic T lymphocytes (CTL): epitope variation in a CTL-resistant virus.
SO - J Gen Virol 1995 Mar;76 ( Pt 3):635-41
AB - AKR/Gross virus-specific cytotoxic T lymphocytes (CTL) from C57BL/6 (B6) mice are H-2Kb-restricted and recognize epitopes encoded by the prototype endogenous ecotropic murine leukaemia virus (Emv) AKR623. Four CTL epitopes have been identified by the use of synthetic peptides corresponding to AKR623-encoded amino acid sequences. Here we present both functional and nucleotide sequence data indicating that three closely related Emv share all of these CTL epitopes. We also found that one other murine leukaemia virus (MuLV) was not susceptible to lysis by these CTL. This is the ecotropic component of the LP-BM5 virus complex that causes murine AIDS. Nucleotide sequencing revealed that three of the four epitopes, including the immunodominant peptide, are altered in this virus. The other epitope was unchanged. These data implied that the inability of anti-AKR/Gross virus CTL to lyse cells infected with the LP-BM5 ecotropic (BM5eco) MuLV was due to the functional loss of three of the four CTL epitopes. Using recombinant vaccinia and Sindbis virus vectors, we have shown that the BM5eco-encoded form of the immunodominant epitope, which differs only by an arginine for lysine substitution at the N-terminal residue, fails to induce a CTL response in B6 mice. Immunization with BM5eco-infected cells also failed to induce MuLV-specific CTL. In light of the long in vivo passage history of the LP-BM5 complex in B6 mice, our results are consistent with a contribution of CTL-mediated immune selection to the evolution of the BM5eco MuLV.
AU - Hahn CS
au - Hahn YS
au - Braciale TJ
au - Rice CM
TI - Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation.
SO - Proc Natl Acad Sci U S A 1992 Apr 1;89(7):2679-83
AB - Sindbis virus (SIN) is a small positive-strand enveloped RNA virus that infects a broad range of vertebrate and insect cells. A SIN vector (called dsSIN), designed for transient expression of heterologous RNAs and proteins, was engineered by inserting a second subgenomic mRNA promoter sequence into a nonessential region of the SIN genome. By using this vector, dsSIN recombinants have been constructed that express either bacterial chloramphenicol acetyltransferase, a truncated form of the influenza hemagglutinin (HA), or mini-genes encoding two distinct immunodominant cytotoxic T lymphocyte (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of approximately 10(6)-10(7) chloramphenicol acetyltransferase polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either a truncated form of HA or the immunodominant influenza CTL epitopes. This SIN expression system allows the generation of high-titered recombinant virus stocks in a matter of days and should facilitate mapping and mutational analysis of class I major histocompatibility complex-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation.
AU - London SD
au - Schmaljohn AL
au - Dalrymple JM
au - Rice CM
TI - Infectious enveloped RNA virus antigenic chimeras.
SO - Proc Natl Acad Sci U S A 1992 Jan 1;89(1):207-11
AB - Random insertion mutagenesis has been used to construct infectious Sindbis virus structural protein chimeras containing a neutralization epitope from a heterologous virus, Rift Valley fever virus. Insertion sites, permissive for recovery of chimeric viruses with growth properties similar to the parental virus, were found in the virion E2 glycoprotein and the secreted E3 glycoprotein. For the E2 chimeras, the epitope was expressed on the virion surface and stimulated a partially protective immune response to Rift Valley fever virus infection in vivo. Besides providing a possible approach for developing live attenuated vaccine viruses, insertion of peptide ligands into virion surface proteins may ultimately allow targeting of virus infection to specific cell types.
AU - Lovett AE
au - Hahn CS
au - Rice CM
au - Frey TK
au - Wolinsky JS
TI - Rubella virus-specific cytotoxic T-lymphocyte responses: identification of the capsid as a target of major histocompatibility complex class I-restricted lysis and definition of two epitopes.
SO - J Virol 1993 Oct;67(10):5849-58
AB - The role of major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T lymphocytes in immunity to rubella virus (RV) infection is unknown. Lymphocytes of RV-immune individuals were prestimulated on an RV-infected MHC class I-matched (or partially matched) fibroblast monolayer which generated CD8+ lymphoblasts capable of lysing RV-infected fibroblast targets in a class I-restricted manner. Using an infectious Sindbis virus (SV) vector which expressed the RV capsid protein (SV/RubC), lymphocytes from 17 of 22 RV-immune individuals prestimulated on RV-infected fibroblast monolayers lysed SV/RubC-infected fibroblast targets. A sequence within the amino terminus of the capsid protein that was previously shown to contain immunodominant class II-restricted T-cell epitopes was evaluated for class I-restricted epitopes. Fibroblast targets pulsed with synthetic peptides representing subsequences within C1 to C29 (subscripts indicate amino acid positions) were lysed effectively when the targets and effectors matched at multiple class I alleles. By limiting the number of matching class I alleles, an A2-restricted epitope was identified within C9 to C22 and an epitope that could be presented by multiple class I molecules was identified within C11 to C29. A sequence such as C1 to C29 which contains both MHC class I- and MHC class II-restricted epitopes recognized by a heterologous human population may serve as a component of an effective synthetic vaccine.
AU - Piper RC
au - Slot JW
au - Li G
au - Stahl PD
au - James DE
TI - Recombinant Sindbis virus as an expression system for cell biology. [Review]
SO - Methods Cell Biol 1994;43 Pt A:55-78
AU - Li G
au - Barbieri MA
au - Stahl PD
TI - Myristoylation cannot functionally replace the isoprenylation of Rab5.
SO - Arch Biochem Biophys 1995 Jan 10;316(1):529-34
AB - C-terminal isoprenylation is necessary for the small GTPase Rab5 to associate with early endosomes and to exert its regulatory function in endocytosis. In this study, we tested whether Rab5 could retain its membrane association and biological function if the isoprenylation were replaced by another type of lipid modification (myristoylation). Rab5 mutants were constructed that contained both isoprenylation and myristoylation (Gag-Rab5), myristoylation only (Gag-Rab5 delta C4), and neither of the modifications (Rab5 delta C4), respectively. These mutants and wild-type Rab5 were expressed, via a Sindbis virus vector, in cultured BHK-21 cells and their membrane association and biological activity (stimulation of endocytosis) were examined. Wild-type Rab5 was isoprenylated, membrane associated, and biologically active. With additional myristoylation, Gag-Rab5 showed increased affinity for membranes but decreased biological activity. Rab5 delta C4 contained no lipid modifications, failed to associate with membranes, and had no biological activity. With myristoylation (Gag-Rab5 delta C4), there was a significant increase in membrane association (approximately 30%). However, this increased membrane association did not result in any recovery of Rab5 activity. In light of these data, we conclude that N-terminal myristoylation cannot functionally replace the C-terminal isoprenylation of Rab5. Furthermore, myristoylation of Rab5 (Gag-Rab5) interferes with its normal function.
AU - Li G
au - Barbieri MA
au - Colombo MI
au - Stahl PD
TI - Structural features of the GTP-binding defective Rab5 mutants required for their inhibitory activity on endocytosis.
SO - J Biol Chem 1994 May 20;269(20):14631-5
AB - Rab5 is a Ras-like small GTPase that regulates early events of endocytosis. Previous work indicates that two GTP-binding defective Rab5 mutants (Rab5:S34N and Rab5:N133I) are dominant inhibitors of endocytosis. In this report, we have initiated experiments to address the structural features necessary for the inhibitory activity of these two Rab5 mutants. Second-site mutations were introduced into Rab5:S34N and Rab5:N133I, respectively, and the resulting double mutants were expressed in cultured BHK-21 cells via a Sindbis virus expression vector. Endocytic activity of the cells was monitored by following the uptake of a fluid-phase endocytic marker (horseradish peroxidase). The effects of the Rab5 mutants on endosome fusion in vitro were also examined. Truncation of the C-terminal isoprenylation motif CCSN abolished the inhibitory activity of both Rab5:S34N and Rab5:N133I. The same held true when the secondary mutation was a substitution mutation (F57S) in the effector domain. Another substitution mutation in this region (I53A) had no effect on the inhibitory activity of either Rab5:S34N or Rab5:N133I. The final mutation (R81A) was created immediately downstream of the second GTP binding motif (WDTAGQER), i.e. in the loop 4 region based on the structural model of Ras. This mutation greatly decreased the isoprenylation of Rab5:N133I and its inhibitory activity on endocytosis. It is believed that Rab5 function requires protein-protein interactions with Rab5-specific regulators and effectors. Some of these interactions are disrupted by Rab5:S34N and Rab5:N133I. By analogy to Ras, both Rab5:S34N and Rab5:N133I are likely to sequester a Rab5-specific guanine nucleotide exchange factor. This interaction requires the effector domain Phe57 residue and C-terminal isoprenylation of Rab5.
AU - Li G
au - Stahl PD
TI - Post-translational processing and membrane association of the two early endosome-associated rab GTP-binding proteins (rab4 and rab5).
SO - Arch Biochem Biophys 1993 Aug 1;304(2):471-8
AB - The two early endosome-associated rab GTP-binding proteins, rab4 and rab5, are suggested to regulate endocytosis. In this report, we examined post-translational processing and membrane association of the two rab proteins. Human rab4 and rab5 were expressed in chicken embryo fibroblasts using a Sindbis virus expression vector. Cells were labeled with either [35S]methionine or [3H]mevalonolactone. Cell lysates were immunoprecipitated with antisera specific for rab4 and rab5, respectively, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that both rab4 and rab5 contained at least three forms: the precursor form, the isoprenylated intermediate, and the isoprenylated mature form of faster mobility. The rab5 intermediate comigrated with the precursor form, whereas the rab4 intermediate migrated slightly faster than the precursor form. The intermediate form of rab4, but not rab5, accumulated in the presence of a chymotrypsin-like protease inhibitor (N-acetyl Tyr ethyl ester), suggesting that proteolysis was required for generation of the mature form. Furthermore, the intermediate and mature forms of rab4, but not rab5, were carboxyl-methylated as demonstrated by incorporation of alkali-labile counts from [methyl-3H]methionine. Membrane association of the distinct rab4 and rab5 forms was examined by subcellular fractionation and Triton X-114 partitioning. The precursor forms were found in the cytosol and partitioned into the aqueous phase. The mature forms were membrane-associated and partitioned into the detergent phase. Unexpectedly, the isoprenylated intermediate forms of both rab4 and rab5 partitioned exclusively into the aqueous phase. Taken together, the data indicate that the entire post-translational processing, which includes isoprenylation, carboxyl methylation (rab4), and possibly proteolysis, confers the competency for membrane association of rab4 and rab5.
AU - Li G
au - Stahl PD
TI - Structure-function relationship of the small GTPase rab5.
SO - J Biol Chem 1993 Nov 15;268(32):24475-80
AB - Overexpression of rab5 via a Sindbis virus vector resulted in a 2-3-fold stimulation of horseradish peroxidase uptake in BHK-21 cells. Based on this functional assay of rab5 activity, we conducted extensive mutational analysis of the structure-function relationship of rab5. A total of 21 deletion and substitution mutations were created and their effects on rab5 activity were examined. Deletion of the entire C-terminal tetrapeptide motif CCSN abolished rab5 activity. A mutant with the last three residues deleted, however, showed residual rab5 activity. Truncation of only two residues from the C terminus had no effect on the biological activity of rab5. A mutant containing a 4-residue deletion from the N terminus retained full activity in comparison with wild-type rab5. N-terminal deletion of 19 residues only partially blocked rab5 activity. Substitution mutations in the guanine nucleotide binding motifs showed dramatic effects on rab5 function. In addition to the previously reported N133I mutation, the S34N mutation also resulted in a guanine nucleotide binding defective form that was a dominant inhibitor of endogenous rab5 activity. The Q79L mutation (the ras equivalent Q61L decreases intrinsic and GTPase-activating protein-stimulated GTPase activities), however, had no effect on rab5 activity. The S35N mutation, which is immediately downstream of the first GTP/GDP binding motif, decreased guanine nucleotide binding by approximately 4-fold and partially inactivated rab5. Mutations in several other conserved residues (K22A, F57Y, and R81A) also resulted in partial loss of rab5 activity. Eight mutations in and around the putative effector domain had little effect on rab5 activity. In light of these data, the structure-function relationship of rab5 is discussed and compared with that of ras, the prototype of small GTPases.
AU - Piper RC
au - Tai C
au - Slot JW
au - Hahn CS
au - Rice CM
au - Huang H
au - James DE
TI - The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus.
SO - J Cell Biol 1992 May;117(4):729-43
AB - GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.
AU - Piper RC
au - Tai C
au - Kulesza P
au - Pang S
au - Warnock D
au - Baenziger J
au - Slot JW
au - Geuze HJ
au - Puri C
au - James DE
TI - GLUT-4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration.
SO - J Cell Biol 1993 Jun;121(6):1221-32
AB - Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated that the cytoplasmic NH2 terminus of GLUT-4 contains important targeting information that mediates intracellular sequestration of this isoform (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies, the amino acid constituents of the GLUT-4 NH2-terminal targeting domain have been identified. GLUT-4 constructs containing NH2-terminal deletions or alanine substitutions within the NH2 terminus were expressed in CHO cells using a Sindbis virus expression system. Deletion of eight amino acids from the GLUT-4 NH2 terminus or substituting alanine for phenylalanine at position 5 in GLUT-4 resulted in a marked accumulation of the transporter at the plasma membrane. Mutations at other amino acids surrounding Phe5 also caused increased cell surface expression of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 was also localized to clathrin lattices and this colocalization was abolished when either the first 13 amino acids were deleted or when Phe5 was changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function independently of the glucose transporter structure this domain was inserted into the cytoplasmic tail of the H1 subunit of the asialoglycoprotein receptor. H1 with the GLUT-4 NH2 terminus was predominantly localized to an intracellular compartment similar to GLUT-4 and was sequestered more from the cell surface than was the wild-type H1 protein. It is concluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic machinery located at the cell surface.
AU - Piper RC
au - James DE
au - Slot JW
au - Puri C
au - Lawrence JC Jr
TI - GLUT4 phosphorylation and inhibition of glucose transport by dibutyryl cAMP.
SO - J Biol Chem 1993 Aug 5;268(22):16557-63
AB - To investigate the mechanism responsible for the inhibition of glucose transport by dibutyryl cAMP (Bt2cAMP), two different transporter isoforms (GLUT1 and GLUT4) and several GLUT1/4 chimeric transporters were expressed in Chinese hamster ovary (CHO) cells by using a Sindbis virus expression system. Bt2cAMP inhibited GLUT4-mediated 2-deoxy[3H]glucose (2DOG) uptake by 50% but was without effect on GLUT1-mediated uptake. When the subcellular distribution of GLUT4 was assessed by quantitative immunocytochemistry, neither the overall concentration of GLUT4 nor the regional distribution of GLUT-4 within the plasma membrane was found to be altered by Bt2cAMP. Thus, inhibition of 2DOG uptake by Bt2cAMP appears to be due to a decrease in transporter activity rather than a decrease in the number of transporters exposed at the plasma membrane. By using chimeric transporters, a region of GLUT4 necessary for the inhibitory effect of Bt2cAMP was localized to the last 29 amino acids in the COOH terminus. This intracellular region contains the site (Ser488) phosphorylated in vitro by cAMP-dependent protein kinase (cAdPK). Changing Ser488 to an Ala abolished phosphorylation of GLUT4; however, the inhibitory effect of Bt2cAMP on glucose transport was not diminished by this mutation. Therefore, phosphorylation of GLUT4 was not required for the inhibition. The effects of other nucleotides on GLUT4 transport activity were assessed to investigate the role of cAdPK. Uptake of 2DOG by GLUT4 was inhibited by 8-bromo-AMP, but not by 8-bromo-cAMP, suggesting that the inhibitory effect did not involve activation of cAdPK. Results consistent with this interpretation were obtained with CHO cells (line 10248), which express a cAdPK that is resistant to activation by cAMP. No difference in the concentrations of Bt2cAMP required to inhibit GLUT4-mediated transport was observed in normal CHO cells and 10248 cells. The results presented suggest that the inhibitory effects of Bt2cAMP could be mediated by direct binding of a nucleotide to GLUT4 at a site involving the intracellular COOH terminus of the transporter.
AU - Jiang W
au - Venugopal K
au - Gould EA
TI - Intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant Sindbis virus.
SO - J Virol 1995 Feb;69(2):1044-9
AB - A single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. To see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant Sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. The variable genes and interchain linker encoding the single-chain antibody were cloned into a double subgenomic Sindbis virus expression vector to generate recombinant Sindbis virus. Infection with this recombinant Sindbis virus provided high-level cytoplasmic expression of the antibody fragment in mammalian cells. We demonstrate (i) that the antibody fragment was antigen binding and (ii) that louping ill virus infectivity was significantly reduced in the presence of intracellular antibody expressed by the superinfecting recombinant Sindbis virus.
AU - Dubuisson J
au - Hsu HH
au - Cheung RC
au - Greenberg HB
au - Russell DG
au - Rice CM
TI - Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses.
SO - J Virol 1994 Oct;68(10):6147-60
AB - Hepatitis C virus (HCV) encodes two putative virion glycoproteins (E1 and E2) which are released from the polyprotein by signal peptidase cleavage. In this report, we have characterized the complexes formed between E1 and E2 (called E1E2) for two different HCV strains (H and BK) and studied their intracellular localization. Vaccinia virus and Sindbis virus vectors were used to express the HCV structural proteins in three different cell lines (HepG2, BHK-21, and PK-15). The kinetics of association between E1 and E2, as studied by pulse-chase analysis and coprecipitation of E2 with an anti-E1 monoclonal antibody, indicated that formation of stable E1E2 complexes is slow. The times required for half-maximal association between E1 and E2 were 60 to 85 min for the H strain and more than 165 min for the BK strain. In the presence of nonionic detergents, two forms of E1E2 complexes were detected. The predominant form was a heterodimer of E1 and E2 stabilized by noncovalent interactions. A minor fraction consisted of heterogeneous disulfide-linked aggregates, which most likely represent misfolded complexes. Posttranslational processing and localization of the HCV glycoproteins were examined by acquisition of endoglycosidase H resistance, subcellular fractionation, immunofluorescence, cell surface immunostaining, and immunoelectron microscopy. HCV glycoproteins containing complex N-linked glycans were not observed, and the proteins were not detected at the cell surface. Rather, the proteins localized predominantly to the endoplasmic reticular network, suggesting that some mechanism exists for their retention in this compartment.
AU - Stabell EC
au - Olivo PD
TI - A truncated herpes simplex virus origin binding protein which contains the carboxyl terminal origin binding domain binds to the origin of replication but does not alter its conformation.
SO - Nucleic Acids Res 1993 Nov 11;21(22):5203-11
AB - We have studied the DNA binding properties of a polypeptide consisting of the carboxyl terminal 37% of UL9, the herpes simplex virus type 1 (HSV-1) origin of replication binding protein. Using a Sindbis virus expression system, we expressed and partially purified this truncated form of UL9 (UL9CT) which contains the site-specific DNA binding domain. UL9CT specifically recognized UL9 binding sites on a 200 base pair DNA fragment containing the HSV origin ori(s) and appeared to bind as a dimer to each site. DNAse I footprint analysis showed that UL9CT protected the two high affinity binding sites of ori(s), but unlike full-length UL9, UL9CT did not induce a conformational change in the origin. Addition of anti-UL9CT antibody to the UL9CT-origin complex, however, caused a conformational change in the origin to be evident. Our results suggest that a domain, or domains, in the amino terminus are necessary for a UL9-induced origin conformational change to occur and that UL9-UL9 interactions between binding sites are involved.
AU - Huang MJ
au - Summers J
TI - Infection initiated by the RNA pregenome of a DNA virus.
SO - J Virol 1991 Oct;65(10):5435-9
AB - We describe experiments demonstrating that after transfection into permissive cells, the RNA pregenome of an avian hepadnavirus, the duck hepatitis B virus, is infectious. Using a Sindbis virus expression vector, we showed that cytoplasmic synthesis of the pregenome resulted in hepadnaviral DNA synthesis. Moreover, complete infectious virus was produced from cells transfected with hepadnaviral pregenomic RNA. We conclude that the pregenome of hepadnaviruses can express all the proteins required for DNA synthesis as well as serve as a template for reverse transcription and that DNA resulting from pregenome expression can be utilized to establish a productive infection in pregenome-transfected cells.
(see also expression of antisense of the small segment of LaCrosse virus.)
AU - Higgs S
au - Olson KE
au - Klimowski L
au - Powers AM
au - Carlson JO
au - Possee RD
au - Beaty BJ
TI - Mosquito sensitivity to a scorpion neurotoxin expressed using an infectious Sindbis virus vector.
SO - Insect Mol Biol 1995 May;4(2):97-103
AB - The scorpion, Androctonus australis Hector, produces an insect-specific toxin (AaHIT) encoded by the Scotox gene. To assess the toxicity of AaHIT for mosquitoes, we have taken a novel approach to express the Scotox gene in vivo. We have engineered a double subgenomic Sindbis (dsSIN) virus that contains the Scotox gene in the viral genome and intrathoracically inoculated the virus (TE/3'2J/Scotox) into mosquitoes (Aedes aegypti, Ae. triseriatus and Culex pipiens), houseflies (Musca domestica) and ticks (Dermacentor andersoni). Mosquitoes, which normally show no pathologic effects from Sindbis (SIN) virus infections, died 1-5 days after infection with TE/3'2J/Scotox virus. Neither flies nor ticks were killed. The mosquitocidal action of AaHIT in mosquitoes makes AaHIT a potential candidate for inclusion in molecular-based methods of mosquito control. The expression of an arthropod gene in vivo demonstrates the utility of dsSIN expression vectors for future use to examine and potentially disrupt endogenous gene functions in mosquitoes.
AU - Olson KE
au - Higgs S
au - Hahn CS
au - Rice CM
au - Carlson JO
au - Beaty BJ
TI - The expression of chloramphenicol acetyltransferase in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes using a double subgenomic recombinant Sindbis virus.
SO - Insect Biochem Mol Biol 1994 Jan;24(1):39-48
AB - Genomic RNA was transcribed in vitro from the double subgenomic recombinant Sindbis (SIN) virus expression vector, pTE/3'2J/CAT, and transfected into BHK-21 cells to generate recombinant virus stocks. TE/3'2J/CAT virus was used to infect C6/36 (Aedes albopictus) cells and adult female Aedes triseriatus. When C6/36 cells were infected with TE/3'2J/CAT virus at a multiplicity of infection (MOI) of greater than 20, 100% of the cells expressed CAT. The number of CAT polypeptides expressed per cell at 24 h post infection (pi) was 8.3 x 10(5). Approximately 4.0 log10TCID50 of the TE/3'2J/CAT virus was intrathoracically inoculated into adult female mosquitoes. Titers greater than 6.0 log10TCID50/ml were detected within 4 days pi and declined to less than 4.0 log10TCID50/ml 20 days following inoculation. CAT activity was detected within 2 days (8 x 10(-5) units of CAT/mosquito or 1.4 x 10(10) CAT polypeptides), peaked at day 6 (4 x 10(-3) units of CAT/mosquito or 7.2 x 10(11) CAT polypeptides), and remained at peak levels to day 20. Immunofluorescence and CAT activity assays were used to localize CAT expression in infected mosquitoes and demonstrated that CAT was present in neural, midgut, ovarian, and salivary gland tissues. Alphavirus-based expression vectors should be useful for expressing heterologous genes in mosquito cells as well as adult mosquitoes.
AU - Olson KE
au - Carlson JO
au - Beaty BJ
TI - Expression of the chloramphenicol acetyltransferase gene in Aedes albopictus (C6/36) cells using a non-infectious Sindbis virus expression vector.
SO - Insect Mol Biol 1992;1(1):49-52
AB - Genomic RNA was transcribed in vitro from the non-infectious Sindbis (SIN) virus expression vector (pTRCAT) and introduced into C6/36 (Aedes albopictus) cells by liposome-mediated transfections. The chloramphenicol acetyltransferase (CAT) polypeptide expressed within cells was detected by an indirect immunofluorescent assay directly into 24-well polystyrene tissue culture plates. Approximately 1 in 1000 C6/36 cells showed fluorescence when the transfection was optimized. CAT enzyme activity was also assayed; in C6/36 cells CAT expression was detected as early as 8 h after transfection, peaked at 24 h, and could still be detected at 7 days. At 24 h posttransfection each transfected C6/36 cell was calculated to express 1.3 x 10(7) CAT polypeptides.
AU - Powers AM
au - Olson KE
au - Higgs S
au - Carlson JO
au - Beaty BJ
TI - Intracellular immunization of mosquito cells to LaCrosse virus using a recombinant Sindbis virus vector.
SO - Virus Res 1994 Apr;32(1):57-67
AB - A cDNA of the small RNA genome segment of La Crosse (LAC) virus was inserted, in an antisense orientation, into a double subgenomic Sindbis (dsSIN) virus expression vector generating pTE/3'2J/ANTI-S (15,000bp). In vitro transcription of the pTE/3'2J/ANTI-S template generated genomic RNA that was electrotransfected into BHK-21 cells to produce virus. Northern blot analysis of RNA isolated from infected Aedes albopictus (C6/36) cells showed that the TE/3'2J/ANTI-S virus produced a subgenomic mRNA of the appropriate size, indicating transcription of the LAC cDNA segment. C6/36 cells were infected with either TE/3'2J/ANTI-S, TE/3'2J (a dsSIN virus with no LAC insert), or wild type Sindbis (SIN, strain AR339) viruses and subsequently challenged with LAC virus. LAC virus titers were determined using a capture antibody ELISA. Mosquito cells infected with TE/3'2J/ANTI-S virus yielded at least 4 log10 TCID50/ml less LAC virus than cells infected with either TE/3'2J or AR339 SIN viruses. The use of the infectious SIN virus expression vectors provides a novel approach for high level cytoplasmic expression of genes or sequences of interest in arthropod cells, and for evaluating strategies for intracellular immunization against arboviruses.
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