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Dennis Brown - One of the best experts on this subject based on the ideXlab platform.
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Role of the Vacuolar-ATPase in Sindbis Virus Infection
Journal of virology, 2010Co-Authors: Sabrina R. Hunt, Raquel Hernandez, Dennis BrownAbstract:Bafilomycin A(1) is a specific inhibitor of the vacuolar-ATPase (V-ATPase), which is responsible for pH homeostasis of the cell and for the acidification of endosomes. Bafilomycin A(1) has been commonly used as a method of inhibition of infection by Viruses known or suspected to follow the path of receptor-mediated endocytosis and low-pH-mediated membrane fusion. The exact method of entry for Sindbis Virus, the prototype alphaVirus, remains undetermined. To further investigate the role of the V-ATPase in Sindbis Virus infection, the effects of bafilomycin A(1) on the infection of BHK and insect cells by Sindbis Virus were studied. Bafilomycin A(1) was found to block the expression of a Virus-encoded reporter gene in both infection and transfection of BHK cells. The inhibitory effects of bafilomycin A(1) were found to be reversible. The results suggest that in BHK cells in the presence of bafilomycin A(1), Virus RNA enters the cell and is translated, but replication and proper folding of the product proteins requires the function of the V-ATPase. Bafilomycin A(1) had no significant effect on the outcome of infection in insect cells.
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Infection of cells by Sindbis Virus at low temperature.
Virology, 2007Co-Authors: Gongbo Wang, Raquel Hernandez, Keith Weninger, Dennis BrownAbstract:Sindbis Virus, which belongs to the family Togaviridae genus AlphaVirus infects a variety of vertebrate and invertebrate cells. The initial steps of Sindbis Virus infection involve attachment, penetration and uncoating. Two different pathways of infection have been proposed for AlphaViruses. One proposed mechanism involves receptor mediated virion endocytosis followed by membrane fusion triggered by endosome acidification. This Virus-host membrane fusion model, well established by influenza Virus, has been applied to other unrelated membrane-containing Viruses including AlphaViruses. The other mechanism proposes direct penetration of the cell plasma membrane by the Virus glycoproteins in the absence of membrane fusion. This alternate model is supported by both ultrastructural [Paredes, A.M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., Brown, D.T., 2004. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion. Virology 324(2), 373-386] and biochemical [Koschinski, A., Wengler, G., Wengler, G., and Repp, H., 2005. Rare earth ions block the ion pores generated by the class II fusion proteins of alphaViruses and allow analysis of the biological functions of these pores. J. Gen. Virol. 86(Pt. 12), 3311-3320] studies. We have examined the ability of Sindbis Virus to infect Baby Hamster Kidney (BHK) cells at temperatures which block endocytosis. We have found that under these conditions Sindbis Virus infects cells in a temperature- and time-dependent fashion.
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unit 15b 1 Sindbis Virus propagation quantification and storage
Current protocols in microbiology, 2005Co-Authors: Raquel Hernandez, Christine Sinodis, Dennis BrownAbstract:The prototype of the AlphaViruses, Sindbis Virus, has a broad host range. In nature, Sindbis Virus shuttles from an insect vector to a vertebrate host and back to the insect vector in a complex transmission cycle. Sindbis Virus must, therefore, be able to replicate in two biochemically and genetically divergent hosts, invertebrates and vertebrates. In the laboratory, Sindbis grows to high titers in a large variety of cultured cells of both vertebrate and invertebrate origin. Sindbis Virus is easily titered for infectivity on several mammalian cell lines as well as certain mosquito cells. Full-length cDNA clones of several strains of Sindbis Virus are available from which infectious RNA can be synthesized, making possible the genetic manipulation of the Virus. Transfection of mammalian and insect cells by electroporation has facilitated expression of RNA mutants in the cell lines of choice. Curr. Protoc. Microbiol. 16:15B.1.1-15B.1.41. © 2010 by John Wiley & Sons, Inc. Keywords: Sindbis Virus; SVHR; plaque assay; focus assay; MTT titration; infection; transfection; insect cell culture; mammalian cell culture; AlphaVirus
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Comparison of Sindbis Virus-Induced Pathology in Mosquito and Vertebrate Cell Cultures
Virology, 1998Co-Authors: Adam R. Karpf, Dennis BrownAbstract:Abstract We have compared Sindbis Virus-induced cytopathology in vertebrate and mosquito (Aedes albopictus) cell cultures. It has been shown that vertebrate cells undergo apoptosis when infected by Sindbis Virus and this was confirmed here using hamster cells (BHK). The occurrence of cell death in Sindbis Virus-infectedA. albopictuscells is a cell clone-specific phenomenon and, unlike in BHK cell cultures, mosquito cell death does not correlate with a large induction of apoptosis, as determined by assays testing for DNA fragmentation or reduced cellular DNA content. Cell cycle distribution changes were observed in Sindbis Virus-infected BHK and C7-10 cell cultures, and the changes are distinct, both in the time of induction and the types of perturbations. In Sindbis Virus-infected BHK cells, the major cell cycle profile change is the early accumulation of cells with sub-G1 DNA content and a corresponding reduction in the proportion of cells in G1 and G2/M. For Sindbis Virus-infected C7-10 cells, the major perturbations are an increased proportion of cells showing G2/M or polyploid DNA content and a reduction in the proportion of G1 and S phase cells. These data suggest that the pathology induced in mosquito cell cultures by Sindbis Virus infection may be distinct from the pathology which appears in vertebrate cell cultures.
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Superinfection exclusion of alphaViruses in three mosquito cell lines persistently infected with Sindbis Virus.
Journal of virology, 1997Co-Authors: Adam R. Karpf, Ellen G Strauss, James H. Strauss, Edith M. Lenches, Dennis BrownAbstract:Three Aedes albopictus (mosquito) cell lines persistently infected with Sindbis Virus excluded the replication of both homologous (various strains of Sindbis) and heterologous (Aura, Semliki Forest, and Ross River) alphaViruses. In contrast, an unrelated flaviVirus, yellow fever Virus, replicated equally well in uninfected and persistently infected cells of each line. Sindbis Virus and Semliki Forest Virus are among the most distantly related alphaViruses, and our results thus indicate that mosquito cells persistently infected with Sindbis Virus are broadly able to exclude other alphaViruses but that exclusion is restricted to members of the alphaVirus genus. Superinfection exclusion occurred to the same extent in three biologically distinct cell clones, indicating that the expression of superinfection exclusion is conserved among A. albopictus cell types. Superinfection of persistently infected C7-10 cells, which show a severe cytopathic effect during primary Sindbis Virus infection, by homologous Virus does not produce cytopathology, consistent with the idea that cytopathology requires significant levels of viral replication. A possible model for the molecular basis of superinfection exclusion, which suggests a central role for the alphaVirus trans-acting protease that processes the nonstructural proteins, is discussed in light of these results.
Sondra Schlesinger - One of the best experts on this subject based on the ideXlab platform.
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Properties of Sindbis Virus vectors produced with a chimeric split helper system.
International journal of molecular medicine, 2005Co-Authors: Anna Ketola, Sondra Schlesinger, Jarmo WahlforsAbstract:We have evaluated a chimeric, two-component Sindbis Virus packaging system. As expected, use of this combination of two modified helper RNA species prevented formation of infection competent Sindbis Viruses as analyzed by serial passaging. We observed, however, that vectors produced using this method were able to spread in BHK cell cultures and formed clusters of transgene positive cells that did not display cytopathic effects for up to 3 days post-transduction. Formation of spreading Sindbis Virus vectors required only one of the helper components--the chimera with a deleted Ross River Virus capsid and the Sindbis Virus envelope glycoproteins. Spreading was also demonstrated in two rat glioma cell lines, 9L and BT4C, showing that this phenomenon was not limited to BHK cells. Our results warrant further characterization of split helper Sindbis Virus vectors and imply their utility in gene therapy approaches where spreading of transgene expression and consequently high gene transfer rate could be beneficial.
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845. Properties of Sindbis Virus Vectors Produced with a Chimeric Split Helper System
Molecular Therapy, 2005Co-Authors: Anna Ketola, Sondra Schlesinger, Jarmo WahlforsAbstract:Top of pageAbstract We have evaluated a chimeric, two-component Sindbis Virus packaging system (Frolov et al, 1997, J. Virol. 71, 2819-2829). As expected, use of this combination of two modified helper RNA species prevented formation of infection competent Sindbis Viruses as analyzed by serial passaging. We observed, however, that vectors produced using this method were able to spread in BHK cell cultures and formed clusters of transgene positive cells that did not display cytopathic effects for up to 3 days post-transduction. Formation of spreading Sindbis Virus vectors required only one of the helper components |[ndash]| the chimera with a deleted Ross River Virus capsid and the Sindbis Virus envelope glycoproteins. Spreading was also demonstrated in two rat glioma cell lines, 9L and BT4C, showing that this phenomenon was not limited to BHK cells. Our results warrant further characterization of split-helper Sindbis Virus vectors and imply their utility in gene therapy approaches where spreading of transgene expression and consequently high gene transfer rate could be beneficial.
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Regulated Expression of a Sindbis Virus Replicon by HerpesVirus Promoters
Journal of virology, 1999Co-Authors: Lidia Ivanova, Sondra Schlesinger, Paul D. OlivoAbstract:We describe the use of herpesVirus promoters to regulate the expression of a Sindbis Virus replicon (SINrep/LacZ). We isolated cell lines that contain the cDNA of SINrep/LacZ under the control of a promoter from a herpesVirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. Wild-type Sindbis Virus and replicons derived from this Virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and β-galactosidase only after infection with a herpesVirus. Vero cell lines in which the expression of SINrep/LacZ was regulated by the herpes simplex Virus type 1 (HSV-1) infected-cell protein 8 promoter were generated. One Vero cell line (V3-45N) contained, in addition to the SINrep/LacZ cDNA, a Sindbis Virus-defective helper cDNA which provides the structural proteins for packaging the replicon. Infection of V3-45N cells with HSV-1 resulted in the production of packaged SINrep/LacZ replicons. HSV-1 induction of the Sindbis Virus replicon and packaging and spread of the replicon led to enhanced expression of the reporter gene, suggesting that this type of cell could be used to develop sensitive assays to detect herpesViruses. We also isolated a mink lung cell line that was transformed with SINrep/LacZ cDNA under the control of the promoter from the human cytomegaloVirus (HCMV) early gene UL45. HCMV carries out an abortive infection in mink lung cells, but it was able to induce the SINrep/LacZ replicon. These results, and those obtained with an HSV-1 mutant, demonstrate that this type of signal amplification system could be valuable for detecting herpesViruses for which a permissive cell culture system is not available.
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a plasmid based self amplifying Sindbis Virus vector
Human Gene Therapy, 1995Co-Authors: Hans Herweijer, Jeffrey S Latendresse, Phillip Williams, Guofeng Zhang, Istvan Danko, Sondra Schlesinger, Jon A WolffAbstract:Sindbis Virus was used as a self-amplifying eukaryotic expression vector. A recombinant cDNA genome of this (+)-strand RNA Virus was placed under the transcriptional control of a Rous sarcoma Virus LTR (RSV) promoter. Transfection of this plasmid construct into mammalian cell lines (3T3, HepG2, and 293 cells) resulted in expression of the luciferase reporter gene. High-expression levels were also measured after transfection into primary rat myoblasts. In differentiated myotubes, expression levels generated by the Sindbis Virus vector were up to 200 times higher than those obtained with a conventional RSV expression vector. In vivo expression was detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable by day 16. This self-amplifying expression vector can be used for generating high-level expression of transgenes in vitro and in vivo. Its transient nature in vivo could allow for safe, short-term delivery of gene products in gene therapy protocols. It should facilitate the study of Sindbis and other RNA Viruses.
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Comparison of the effects of Sindbis Virus and Sindbis Virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.
Journal of virology, 1994Co-Authors: Ilya Frolov, Sondra SchlesingerAbstract:Abstract 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.
Beth Levine - One of the best experts on this subject based on the ideXlab platform.
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protection against fatal Sindbis Virus encephalitis by beclin a novel bcl 2 interacting protein
Journal of Virology, 1998Co-Authors: Xiao Huan Liang, Hui Hui Jiang, Linda Kleeman, Gerald W Gordon, James E Goldman, Gail Berry, Brian Herman, Beth LevineAbstract:bcl-2, the prototypic cellular antiapoptotic gene, decreases Sindbis Virus replication and Sindbis Virus-induced apoptosis in mouse brains, resulting in protection against lethal encephalitis. To investigate potential mechanisms by which Bcl-2 protects against central nervous system Sindbis Virus infection, we performed a yeast two-hybrid screen to identify Bcl-2-interacting gene products in an adult mouse brain library. We identified a novel 60-kDa coiled-coil protein, Beclin, which we confirmed interacts with Bcl-2 in mammalian cells, using fluorescence resonance energy transfer microscopy. To examine the role of Beclin in Sindbis Virus pathogenesis, we constructed recombinant Sindbis Virus chimeras that express full-length human Beclin (SIN/beclin), Beclin lacking the putative Bcl-2-binding domain (SIN/beclinΔBcl-2BD), or Beclin containing a premature stop codon near the 5′ terminus (SIN/beclinstop). The survival of mice infected with SIN/beclin was significantly higher (71%) than the survival of mice infected with SIN/beclinΔBcl-2BD (9%) or SIN/beclinstop (7%) (P < 0.001). The brains of mice infected with SIN/beclin had fewer Sindbis Virus RNA-positive cells, fewer apoptotic cells, and lower viral titers than the brains of mice infected with SIN/beclinΔBcl-2BD or SIN/beclinstop. These findings demonstrate that Beclin is a novel Bcl-2-interacting cellular protein that may play a role in antiviral host defense.
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Sindbis Virus Induces Apoptosis through a Caspase-Dependent, CrmA-Sensitive Pathway
Journal of virology, 1998Co-Authors: Victor E. Nava, Beth Levine, Antony Rosen, Michael A. Veliuona, Rollie J. Clem, J. Marie HardwickAbstract:Sindbis Virus infection of cultured cells and of neurons in mouse brains leads to programmed cell death exhibiting the classical characteristics of apoptosis. Although the mechanism by which Sindbis Virus activates the cell suicide program is not known, we demonstrate here that Sindbis Virus activates caspases, a family of death-inducing proteases, resulting in cleavage of several cellular substrates. To study the role of caspases in Virus-induced apoptosis, we determined the effects of specific caspase inhibitors on Sindbis Virus-induced cell death. CrmA (a serpin from cowpox Virus) and zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) inhibited Sindbis Virus-induced cell death, suggesting that cellular caspases facilitate apoptosis induced by Sindbis Virus. Furthermore, CrmA significantly increased the rate of survival of infected mice. These inhibitors appear to protect cells by inhibiting the cellular death pathway rather than impairing Virus replication or by inhibiting the nsP2 and capsid viral proteases. The specificity of CrmA indicates that the Sindbis Virus-induced death pathway is similar to that induced by Fas or tumor necrosis factor alpha rather than being like the death pathway induced by DNA damage. Taken together, these data suggest a central role for caspases in Sindbis Virus-induced apoptosis.
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Expression of a biologically active antiviral antibody using a Sindbis Virus vector system
Molecular immunology, 1997Co-Authors: Xiao Huan Liang, Hui Hui Jiang, Beth LevineAbstract:Abstract Monoclonal antibodies to the Sindbis Virus E2 envelope glycoprotein protect mice against lethal encephalitis and mediate viral clearance from neurons. To facilitate structure-function analyses of anti-E2 mAbs, we developed an expression system that can be used for the construction of genetically engineered anti-E2 mAbs. We constructed recombinant Sindbis/immunoglobulin gene chimeric Viruses that express heavy and light chains of an anti-E2 monoclonal antibody, R6. We used a PCR-based strategy to clone the entire rearranged heavy and light chain genes from R6 hybridoma cell cDNA into a double subgenomic Sindbis Virus vector. The recombinant Viruses, SIN R6L and SIN R6H , were generated by transfecting BHK-21 cells with in vitro transcribed RNA from Sindbis Virus/R6 light chain and Sindbis Virus/R6 heavy chain cDNA clones, respectively. Twelve hours after co-infection of BHK cells with SIN R6L and SIN R6H , the tissue culture supernatant contained up to 1.4 mg/ml of recombinant R6 IgG. The heavy and light chains of recombinant R6 were associated as judged by co-purification on protein A G sepharose and co-electrophoresis of non-reduced proteins. The ELISA reactivity to Sindbis Virus antigen was similar for recombinant R6 and R6 purified from ascites fluid. Furthermore, the in vivo biologic activity of recombinant R6 was similar to that of R6 purified from ascites; recombinant R6 treatment completely protected Balb cJ mice from paralysis and death due to infection with neuroadapted Sindbis Virus and also resulted in the clearance of infectious Virus from the brains of immunodeficient scid mice persistently infected with wild-type Sindbis Virus. Thus, the co-infection of BHK cells with SIN R6H and SIN R6H leads to the expression, assembly, and secretion of a biologically active recombinant antiviral antibody. Our results suggest that the Sindbis Virus vector system is a simple and powerful tool for the production of functional, genetically engineered antibodies.
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Dominant inhibitory Ras delays Sindbis Virus-induced apoptosis in neuronal cells.
Journal of virology, 1996Co-Authors: Andrew K. Joe, Giovanna Ferrari, Hui Hui Jiang, Xiao Huan Liang, Beth LevineAbstract:Mature neurons are more resistant than dividing cells or differentiating neurons to Sindbis Virus-induced apoptotic death. Therefore, we hypothesized that mitogenic signal transduction pathways may influence susceptibility to Sindbis Virus-induced apoptosis. Since Ras, a 21-kDa GTP-binding protein, plays an important role in cellular proliferation and neuronal differentiation, we investigated the effect of an inducible dominant inhibitory Ras on Sindbis Virus-induced death of a rat pheochromocytoma cell line, PC12 cells. Dexamethasone induction of dominant inhibitory Ras (Ha Ras(Asn17)) expression in transfected PC12 cell lines (MMTV-M17-21 and GSrasDN6 cells) resulted in a marked delay in Sindbis Virus-induced apoptosis, compared with infected, uninduced cells. The delay in death after Sindbis Virus infection in induced versus uninduced PC12 cells was not associated with differences in viral titers or viral infectivity. No delay in Sindbis Virus-induced apoptosis was observed in Ha Ras(Asn17)-transfected PC12 cells if dexamethasone induction was initiated less than 12 h before Sindbis Virus infection or in wild-type PC12 cells infected with a chimeric Sindbis Virus construct that expresses Ha Ras(Asn17). The delay in Sindbis Virus-induced apoptosis in induced Ha Ras(Asn17)-transfected PC12 cells was associated with a decrease in cellular DNA synthesis as measured by 59-bromo-29-deoxyuridine incorporation. Thus, in PC12 cells, inducible dominant inhibitory Ras inhibits cellular proliferation and delays Sindbis Virus-induced apoptosis. These findings suggest that a Ras-dependent signaling pathway is a determinant of neuronal susceptibility to Sindbis Virus-induced apoptosis.
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Effect of E2 envelope glycoprotein cytoplasmic domain mutations on Sindbis Virus pathogenesis.
Journal of virology, 1996Co-Authors: Beth Levine, Hui Hui Jiang, Linda Kleeman, Grace YangAbstract:The cytoplasmic domain of the E2 envelope glycoprotein is important in Sindbis Virus assembly, but little is known about its role in the pathogenesis of Sindbis Virus encephalitis. To investigate its role in viral pathogenesis, we constructed six recombinant Viruses containing site mutations in the E2 cytoplasmic domain, using the neurovirulent background strain, TE12. Our findings demonstrate that the E2 cytoplasmic domain is a determinant of Sindbis Virus growth and neurovirulence in suckling mice as well as persistent infection in weanling scid mice. They also suggest that the tyrosine, serine, or threonine residues are not essential for replication in mouse brain or anti-E2 monoclonal antibody-mediated restriction of Sindbis Virus replication.
Jarmo Wahlfors - One of the best experts on this subject based on the ideXlab platform.
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Properties of Sindbis Virus vectors produced with a chimeric split helper system.
International journal of molecular medicine, 2005Co-Authors: Anna Ketola, Sondra Schlesinger, Jarmo WahlforsAbstract:We have evaluated a chimeric, two-component Sindbis Virus packaging system. As expected, use of this combination of two modified helper RNA species prevented formation of infection competent Sindbis Viruses as analyzed by serial passaging. We observed, however, that vectors produced using this method were able to spread in BHK cell cultures and formed clusters of transgene positive cells that did not display cytopathic effects for up to 3 days post-transduction. Formation of spreading Sindbis Virus vectors required only one of the helper components--the chimera with a deleted Ross River Virus capsid and the Sindbis Virus envelope glycoproteins. Spreading was also demonstrated in two rat glioma cell lines, 9L and BT4C, showing that this phenomenon was not limited to BHK cells. Our results warrant further characterization of split helper Sindbis Virus vectors and imply their utility in gene therapy approaches where spreading of transgene expression and consequently high gene transfer rate could be beneficial.
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845. Properties of Sindbis Virus Vectors Produced with a Chimeric Split Helper System
Molecular Therapy, 2005Co-Authors: Anna Ketola, Sondra Schlesinger, Jarmo WahlforsAbstract:Top of pageAbstract We have evaluated a chimeric, two-component Sindbis Virus packaging system (Frolov et al, 1997, J. Virol. 71, 2819-2829). As expected, use of this combination of two modified helper RNA species prevented formation of infection competent Sindbis Viruses as analyzed by serial passaging. We observed, however, that vectors produced using this method were able to spread in BHK cell cultures and formed clusters of transgene positive cells that did not display cytopathic effects for up to 3 days post-transduction. Formation of spreading Sindbis Virus vectors required only one of the helper components |[ndash]| the chimera with a deleted Ross River Virus capsid and the Sindbis Virus envelope glycoproteins. Spreading was also demonstrated in two rat glioma cell lines, 9L and BT4C, showing that this phenomenon was not limited to BHK cells. Our results warrant further characterization of split-helper Sindbis Virus vectors and imply their utility in gene therapy approaches where spreading of transgene expression and consequently high gene transfer rate could be beneficial.
Raquel Hernandez - One of the best experts on this subject based on the ideXlab platform.
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Role of the Vacuolar-ATPase in Sindbis Virus Infection
Journal of virology, 2010Co-Authors: Sabrina R. Hunt, Raquel Hernandez, Dennis BrownAbstract:Bafilomycin A(1) is a specific inhibitor of the vacuolar-ATPase (V-ATPase), which is responsible for pH homeostasis of the cell and for the acidification of endosomes. Bafilomycin A(1) has been commonly used as a method of inhibition of infection by Viruses known or suspected to follow the path of receptor-mediated endocytosis and low-pH-mediated membrane fusion. The exact method of entry for Sindbis Virus, the prototype alphaVirus, remains undetermined. To further investigate the role of the V-ATPase in Sindbis Virus infection, the effects of bafilomycin A(1) on the infection of BHK and insect cells by Sindbis Virus were studied. Bafilomycin A(1) was found to block the expression of a Virus-encoded reporter gene in both infection and transfection of BHK cells. The inhibitory effects of bafilomycin A(1) were found to be reversible. The results suggest that in BHK cells in the presence of bafilomycin A(1), Virus RNA enters the cell and is translated, but replication and proper folding of the product proteins requires the function of the V-ATPase. Bafilomycin A(1) had no significant effect on the outcome of infection in insect cells.
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Infection of cells by Sindbis Virus at low temperature.
Virology, 2007Co-Authors: Gongbo Wang, Raquel Hernandez, Keith Weninger, Dennis BrownAbstract:Sindbis Virus, which belongs to the family Togaviridae genus AlphaVirus infects a variety of vertebrate and invertebrate cells. The initial steps of Sindbis Virus infection involve attachment, penetration and uncoating. Two different pathways of infection have been proposed for AlphaViruses. One proposed mechanism involves receptor mediated virion endocytosis followed by membrane fusion triggered by endosome acidification. This Virus-host membrane fusion model, well established by influenza Virus, has been applied to other unrelated membrane-containing Viruses including AlphaViruses. The other mechanism proposes direct penetration of the cell plasma membrane by the Virus glycoproteins in the absence of membrane fusion. This alternate model is supported by both ultrastructural [Paredes, A.M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., Brown, D.T., 2004. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion. Virology 324(2), 373-386] and biochemical [Koschinski, A., Wengler, G., Wengler, G., and Repp, H., 2005. Rare earth ions block the ion pores generated by the class II fusion proteins of alphaViruses and allow analysis of the biological functions of these pores. J. Gen. Virol. 86(Pt. 12), 3311-3320] studies. We have examined the ability of Sindbis Virus to infect Baby Hamster Kidney (BHK) cells at temperatures which block endocytosis. We have found that under these conditions Sindbis Virus infects cells in a temperature- and time-dependent fashion.
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unit 15b 1 Sindbis Virus propagation quantification and storage
Current protocols in microbiology, 2005Co-Authors: Raquel Hernandez, Christine Sinodis, Dennis BrownAbstract:The prototype of the AlphaViruses, Sindbis Virus, has a broad host range. In nature, Sindbis Virus shuttles from an insect vector to a vertebrate host and back to the insect vector in a complex transmission cycle. Sindbis Virus must, therefore, be able to replicate in two biochemically and genetically divergent hosts, invertebrates and vertebrates. In the laboratory, Sindbis grows to high titers in a large variety of cultured cells of both vertebrate and invertebrate origin. Sindbis Virus is easily titered for infectivity on several mammalian cell lines as well as certain mosquito cells. Full-length cDNA clones of several strains of Sindbis Virus are available from which infectious RNA can be synthesized, making possible the genetic manipulation of the Virus. Transfection of mammalian and insect cells by electroporation has facilitated expression of RNA mutants in the cell lines of choice. Curr. Protoc. Microbiol. 16:15B.1.1-15B.1.41. © 2010 by John Wiley & Sons, Inc. Keywords: Sindbis Virus; SVHR; plaque assay; focus assay; MTT titration; infection; transfection; insect cell culture; mammalian cell culture; AlphaVirus
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Sindbis Virus: propagation, quantification, and storage.
Current protocols in microbiology, 2005Co-Authors: Raquel Hernandez, Christine Sinodis, Dennis T BrownAbstract:The prototype of the AlphaViruses, Sindbis Virus, has a broad host range. In nature, Sindbis Virus shuttles from an insect vector to a vertebrate host and back to the insect vector in a complex transmission cycle. Sindbis Virus must, therefore, be able to replicate in two biochemically and genetically divergent hosts, invertebrates and vertebrates. In the laboratory, Sindbis grows to high titers in a large variety of cultured cells of both vertebrate and invertebrate origin. Sindbis Virus is easily titered for infectivity on several mammalian cell lines as well as certain mosquito cells. Full-length cDNA clones of several strains of Sindbis Virus are available from which infectious RNA can be synthesized, making possible the genetic manipulation of the Virus. Transfection of mammalian and insect cells by electroporation has facilitated expression of RNA mutants in the cell lines of choice.
