Structural characterization and biological function of bivalent binding of CD2AP to intrinsically disordered domain of chikungunya virus nsP3 protein.

Alphavirus nsP3 proteins contain long, intrinsically disordered, hypervariable domains, HVD, which serve as hubs for interaction with many cellular proteins. Here, we have deciphered the mechanism and function of HVD interaction with host factors in alphavirus replication. Using NMR spectroscopy, we show that CHIKV HVD contains two SH3 domain-binding sites. Using an innovative chemical shift perturbation signature approach, we demonstrate that CD2AP interaction with HVD is mediated by its SH3-A and SH3-C domains, and this leaves the SH3-B domain available for interaction with other cellular factor(s). This cooperative interaction with two SH3 domains increases binding affinity to CD2AP and possibly induces long-range allosteric effects in HVD. Our data demonstrate that BIN1, CD2AP and SH3KBP1 play redundant roles in initiation of CHIKV replication. Point mutations in both CHIKV HVD binding sites abolish its interaction with all three proteins, CD2AP, BIN1 and SH3KBP1. This results in strong inhibition of viral replication initiation.

Sindbis Virus Infection Causes Cell Death by nsP2-Induced Transcriptional Shutoff or by nsP3-Dependent Translational Shutoff.

Sindbis virus (SINV) is a representative member of the Alphavirus genus in the Togaviridae family. The hallmark of SINV replication in vertebrate cells is a rapid development of the cytopathic effect (CPE), which usually occurs within 24 h postinfection. Mechanistic understanding of CPE might lead to development of new prophylactic vaccines and therapeutic means against alphavirus infections. However, development of noncytopathic SINV variants and those of other Old World alphaviruses was always highly inefficient and usually resulted in selection of mutants demonstrating poor replication of the viral genome and transcription of subgenomic RNA. This likely caused a nonspecific negative effect on the rates of CPE development. The results of this study demonstrate that CPE induced by SINV and likely by other Old World alphaviruses is a multicomponent process, in which transcriptional and translational shutoffs are the key contributors. Inhibition of cellular transcription and translation is determined by SINV nsP2 and nsP3 proteins, respectively. Defined mutations in the nsP2-specific peptide between amino acids (aa) 674 and 688 prevent virus-induced degradation of the catalytic subunit of cellular-DNA-dependent RNA polymerase II and transcription inhibition and make SINV a strong type I interferon (IFN) inducer without affecting its replication rates. Mutations in the nsP3 macrodomain, which were demonstrated to inhibit its mono-ADP-ribosylhydrolase activity, downregulate the second component of CPE development, inhibition of cellular translation, and also have no effect on virus replication rates. Only the combination of nsP2- and nsP3-specific mutations in the SINV genome has a dramatic negative effect on the ability of virus to induce CPE.IMPORTANCE Alphaviruses are a group of important human and animal pathogens with worldwide distribution. Their characteristic feature is a highly cytopathic phenotype in cells of vertebrate origin. The molecular mechanism of CPE remains poorly understood. In this study, by using Sindbis virus (SINV) as a model of the Old World alphaviruses, we demonstrated that SINV-specific CPE is redundantly determined by viral nsP2 and nsP3 proteins. NsP2 induces the global transcriptional shutoff, and this nuclear function can be abolished by the mutations of the small, surface-exposed peptide in the nsP2 protease domain. NsP3, in turn, determines the development of translational shutoff, and this activity depends on nsP3 macrodomain-associated mono-ADP-ribosylhydrolase activity. A combination of defined mutations in nsP2 and nsP3, which abolish SINV-induced transcription and translation inhibition, in the same viral genome does not affect SINV replication rates but makes it noncytopathic and a potent inducer of type I interferon.

Both RIG-I and MDA5 detect alphavirus replication in concentration-dependent mode.

Alphaviruses are a family of positive-strand RNA viruses that circulate on all continents between mosquito vectors and vertebrate hosts. Despite a significant public health threat, their biology is not sufficiently investigated, and the mechanisms of alphavirus replication and virus-host interaction are insufficiently understood. In this study, we have applied a variety of experimental systems to further understand the mechanism by which infected cells detect replicating alphaviruses. Our new data strongly suggest that activation of the antiviral response by alphavirus-infected cells is determined by the integrity of viral genes encoding proteins with nuclear functions, and by the presence of two cellular pattern recognition receptors (PRRs), RIG-I and MDA5. No type I IFN response is induced in their absence. The presence of either of these PRRs is sufficient for detecting virus replication. However, type I IFN activation in response to pathogenic alphaviruses depends on the basal levels of RIG-I or MDA5.

Early events in alphavirus replication determine the outcome of infection.

Alphaviruses are a group of important human and animal pathogens. They efficiently replicate to high titers in vivo and in many commonly used cell lines of vertebrate origin. They have also evolved effective means of interfering with development of the innate immune response. Nevertheless, most of the alphaviruses are known to induce a type I interferon (IFN) response in vivo. The results of this study demonstrate that the first hours postinfection play a critical role in infection spread and development of the antiviral response. During this window, a balance is struck between virus replication and spread in vertebrate cells and IFN response development. The most important findings are as follows: (i) within the first 2 to 4 h postinfection, alphavirus-infected cells become unable to respond to IFN-ß, and this occurs before the virus-induced decrease in STAT1 phosphorylation in response to IFN treatment. (ii) Most importantly, very low, subprotective doses of IFN-ß, which do not induce the antiviral response in uninfected cells, have a very strong stimulatory effect on the cells' ability to express type I IFN and activate interferon-stimulated genes during subsequent infection with Sindbis virus (SINV). (iii) Small changes in SINV nsP2 protein affect its ability to inhibit cellular transcription and IFN release. Thus, the balance between type I IFN induction and the ability of the virus to develop further rounds of infection is determined in the first few hours of virus replication, when only low numbers of cells and infectious virus are involved.

Functional Sindbis virus replicative complexes are formed at the plasma membrane.

Formation of virus-specific replicative complexes (RCs) in infected cells is one of the most intriguing and important processes that determine virus replication and ultimately their pathogenesis on the molecular and cellular levels. Alphavirus replication was known to lead to formation of so-called type 1 cytopathic vacuoles (CPV1s), whose distinguishing feature is the presence of numerous membrane invaginations (spherules) and accumulation of viral nonstructural proteins (nsPs) at the cytoplasmic necks of these spherules. These CPV1s, modified endosomes and lysosomes, were proposed as the sites of viral RNA synthesis. However, our recent studies have demonstrated that Sindbis virus (SINV)-specific, double-stranded RNA (dsRNA)- and nonstructural protein (nsP)-containing RCs are initially formed at the plasma membrane. In this new study, we present extensive evidence that (i) in cells of vertebrate origin, at early times postinfection, viral nsPs colocalize with spherules at the plasma membrane; (ii) viral dsRNA intermediates are packed into membrane spherules and are located in their cavities on the external surface of the plasma membrane; (iii) formation of the membrane spherules is induced by the partially processed nonstructural polyprotein P123 and nsP4, but synthesis of dsRNA is an essential prerequisite of their formation; (iv) plasma membrane-associated dsRNA and protein structures are the active sites of single-stranded RNA (ssRNA) synthesis; (v) at late times postinfection, only a small fraction of SINV nsP-containing complexes are relocalized into the cytoplasm on the endosome membrane. (vi) pharmacological drugs inhibiting different endocytotic pathways have either only minor or no negative effects on SINV RNA replication; and (vii) in mosquito cells, at any times postinfection, dsRNA/nsP complexes and spherules are associated with both endosomal/lysosomal and plasma membranes, suggesting that mechanisms of RC formation may differ in cells of insect and vertebrate origins.

Random insertion mutagenesis of sindbis virus nonstructural protein 2 and selection of variants incapable of downregulating cellular transcription.

Sindbis virus nonstructural protein 2 (SINV nsP2) is an important determinant of virus pathogenesis and downregulation of virus-induced cell response. This protein efficiently inhibits transcription of cellular messenger and ribosomal RNAs and, thus, is capable of inhibiting the activation of genes whose products are involved in development of the antiviral response. Alphavirus nsP2 has a number of predicted functional domains, some of which were confirmed by crystal structure. Our current study demonstrated that none of the putative or known structural domains alone or their combinations was capable of functioning in transcription inhibition. By using random, transposon-mediated mutagenesis, we generated a library of SINV nsP2 variants having short peptide insertions and selected those that lost the ability to inhibit cellular transcription and cause a cytopathic effect. Insertions abrogating the nuclear functions of the protein were found in the three different functional nsP2 domains. Some of the mutated protein variants retained the enzymatic functions required for replication of the viral genome. Such viruses were capable of efficient, productive replication in cells defective in interferon (IFN) signaling but were attenuated and incapable of spreading in cells with an intact type I IFN response. These results revealed new information about the structure of SINV nsP2 and interaction of its domains.

Chimeric alphavirus vaccine candidates protect mice from intranasal challenge with western equine encephalitis virus.

We developed two types of chimeric Sindbis virus (SINV)/western equine encephalitis virus (WEEV) alphaviruses to investigate their potential use as live virus vaccines against WEE. The first-generation vaccine candidate, SIN/CO92, was derived from structural protein genes of WEEV strain CO92-1356, and two second-generation candidates were derived from WEEV strain McMillan. For both first- and second-generation vaccine candidates, the nonstructural protein genes were derived from SINV strain AR339. Second-generation vaccine candidates SIN/SIN/McM and SIN/EEE/McM included the envelope glycoprotein genes from WEEV strain McMillan; however, the amino-terminal half of the capsid, which encodes the RNA-binding domain, was derived from either SINV or eastern equine encephalitis virus (EEEV) strain FL93-939. All chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in 6-week-old mice. Vaccinated mice developed little or no detectable disease and showed little or no evidence of challenge virus replication; however, all developed high titers of neutralizing antibodies. Upon intranasal challenge with high doses of virulent WEEV strains, mice vaccinated with >or=10(5)PFU of SIN/CO92 or >or=10(4)PFU of SIN/SIN/McM or SIN/EEE/McM were completely protected from disease. These findings support the potential use of these live-attenuated vaccine candidates as safe and effective vaccines against WEE.

Chimeric alphavirus vaccine candidates for chikungunya.

Chikungunya virus (CHIKV) is an emerging alphavirus that has caused major epidemics in India and islands off the east coast of Africa since 2005. Importations into Europe and the Americas, including one that led to epidemic transmission in Italy during 2007, underscore the risk of endemic establishment elsewhere. Because there is no licensed human vaccine, and an attenuated Investigational New Drug product developed by the U.S. Army causes mild arthritis in some vaccinees, we developed chimeric alphavirus vaccine candidates using either Venezuelan equine encephalitis attenuated vaccine strain TC-83, a naturally attenuated strain of eastern equine encephalitis virus (EEEV), or Sindbis virus as a backbone and the structural protein genes of CHIKV. All vaccine candidates replicated efficiently in cell cultures, and were highly attenuated in mice. All of the chimeras also produced robust neutralizing antibody responses, although the TC-83 and EEEV backbones appeared to offer greater immunogenicity. Vaccinated mice were fully protected against disease and viremia after CHIKV challenge.

A new role for ns polyprotein cleavage in Sindbis virus replication.

One of the distinguishing features of the alphaviruses is a sequential processing of the nonstructural polyproteins P1234 and P123. In the early stages of the infection, the complex of P123+nsP4 forms the primary replication complexes (RCs) that function in negative-strand RNA synthesis. The following processing steps make nsP1+P23+nsP4, and later nsP1+nsP2+nsP3+nsP4. The latter mature complex is active in positive-strand RNA synthesis but can no longer produce negative strands. However, the regulation of negative- and positive-strand RNA synthesis apparently is not the only function of ns polyprotein processing. In this study, we developed Sindbis virus mutants that were incapable of either P23 or P123 cleavage. Both mutants replicated in BHK-21 cells to levels comparable to those of the cleavage-competent virus. They continuously produced negative-strand RNA, but its synthesis was blocked by the translation inhibitor cycloheximide. Thus, after negative-strand synthesis, the ns proteins appeared to irreversibly change conformation and formed mature RCs, in spite of the lack of ns polyprotein cleavage. However, in the cells having no defects in alpha/beta interferon (IFN-alpha/beta) production and signaling, the cleavage-deficient viruses induced a high level of type I IFN and were incapable of causing the spread of infection. Moreover, the P123-cleavage-deficient virus was readily eliminated, even from the already infected cells. We speculate that this inability of the viruses with unprocessed polyprotein to productively replicate in the IFN-competent cells and in the cells of mosquito origin was an additional, important factor in ns polyprotein cleavage development. In the case of the Old World alphaviruses, it leads to the release of nsP2 protein, which plays a critical role in inhibiting the cellular antiviral response.

Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence.

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.

Production of pseudoinfectious yellow fever virus with a two-component genome.

Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.

Comparative analysis of the alphavirus-based vectors expressing Rift Valley fever virus glycoproteins.

During the last decade, alphaviruses became widely used for expression of heterologous genetic information and development of recombinant vaccines against a variety of human and animal pathogens. In this study, we compared a number of vectors based on the genome of Sindbis (SINV) and Venezuelan equine encephalitis (VEEV) viruses for their ability to express the Rift Valley fever virus (RVFV) envelope glycoprotein Gn and induce a protective immune response against RVFV infection. Our results suggest that (i) application of VEEV-based expression systems appears to be advantageous, when compared to similar systems designed on the basis of the SINV genome. (ii) Alphavirus-specific E3 and E2 proteins and furin-specific cleavage sites can be used for engineering secreted forms of the proteins. (iii) Alphaviruses can be modified for expression of the large fragments of heterologous proteins on the surface of chimeric, infectious viral particles. Thus, alphavirus-based expression systems may have the potential for a broader application beyond their current use as replicons or double-subgenomic vectors.

Development of Sindbis viruses encoding nsP2/GFP chimeric proteins and their application for studying nsP2 functioning.

Sindbis virus (SINV) is one of almost 30 currently known alphaviruses. In infected cells, it produces only a few proteins that function in virus replication and interfere with the development of the antiviral response. One of the viral nonstructural proteins, nsP2, not only exhibits protease and RNA helicase activities that are directly involved in viral RNA replication but also plays critical roles in the development of transcriptional and translational shutoffs in the SINV-infected cells. These multiple activities of nsP2 complicate investigations of this protein's functions and further understanding of its structure. Using a transposon-based approach, we generated a cDNA library of SINV genomes with a green fluorescent protein (GFP) gene randomly inserted into nsP2 and identified a number of sites that can be used for GFP cloning without a strong effect on virus replication. Recombinant SIN viruses encoding nsP2/GFP chimeric protein were capable of growth in tissue culture and interfering with cellular functions. SINV, expressing GFP in the nsP2, was used to isolate nsP2-specific protein complexes formed in the cytoplasm of the infected cells. These complexes contained viral nsPs, all of the cellular proteins that we previously coisolated with SINV nsP3, and some additional protein factors that were not found before in detectable concentrations. The random insertion library-based approach, followed by the selection of the viable variants expressing heterologous proteins, can be applied for mapping the domain structure of the viral nonstructural and structural proteins, cloning of peptide tags for isolation of the protein-specific complexes, and studying their formation by using live-cell imaging. This approach may also be applicable to presentation of additional antigens and retargeting of viruses to new receptors.

The Old World and New World alphaviruses use different virus-specific proteins for induction of transcriptional shutoff.

Alphaviruses are widely distributed throughout the world. During the last few thousand years, the New World viruses, including Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), evolved separately from those of the Old World, i.e., Sindbis virus (SINV) and Semliki Forest virus (SFV). Nevertheless, the results of our study indicate that both groups have developed the same characteristic: their replication efficiently interferes with cellular transcription and the cell response to virus replication. Transcriptional shutoff caused by at least two of the Old World alphaviruses, SINV and SFV, which belong to different serological complexes, depends on nsP2, but not on the capsid protein, functioning. Our data suggest that the New World alphaviruses VEEV and EEEV developed an alternative mechanism of transcription inhibition that is mainly determined by their capsid protein, but not by the nsP2. The ability of the VEEV capsid to inhibit cellular transcription appears to be controlled by the amino-terminal fragment of the protein, but not by its protease activity or by the positively charged RNA-binding domain. These data provide new insights into alphavirus evolution and present a plausible explanation for the particular recombination events that led to the formation of western equine encephalitis virus (WEEV) from SINV- and EEEV-like ancestors. The recombination allowed WEEV to acquire capsid protein functioning in transcription inhibition from EEEV-like virus. Identification of the new functions in the New World alphavirus-derived capsids opens an opportunity for developing new, safer alphavirus-based gene expression systems and designing new types of attenuated vaccine strains of VEEV and EEEV.

Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses.

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.

Inhibition of transcription and translation in Sindbis virus-infected cells.

Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. The natural transmission cycle of alphaviruses requires their presence at high concentrations in the blood of amplification hosts for efficient infection of mosquito vectors. The high-titer viremia development implies multiple rounds of infection that proceed in the background of the developing antiviral cell response aimed at blocking virus spread on an organismal level. Therefore, as for many viruses, if not most of them, alphaviruses have evolved mechanisms directed toward downregulating different components of the antiviral cell reaction and increasing viremia to a level sufficient for the next round of transmission. Using Sindbis virus (SIN) as a model, we demonstrated that (i) the replication of wild-type SIN strongly affects major cellular processes, e.g., transcription and translation of mRNAs; (ii) transcriptional and translational shutoffs are distinctly independent events, and their development can be differentially manipulated by creating different mutations in SIN nonstructural protein nsP2; and (iii) inhibition of transcription, but not translation, is a critical mechanism that SIN employs to suppress the expression of cellular viral stress-inducible genes in cells of vertebrate origin. Downregulation of transcription of all of the cellular mRNAs appears to be a very efficient means of reducing the development of an antiviral response. The ability to cause transcriptional shutoff may partially determine SIN host range and replication in particular tissues.

Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells.

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.

Venezuelan equine encephalitis virus in the guinea pig model: evidence for epizootic virulence determinants outside the E2 envelope glycoprotein gene.

Epizootic strains of Venezuelan equine encephalitis virus (VEEV) cause epidemics by exploiting equines as highly efficient amplification hosts for mosquito transmission. Although phylogenetic studies indicate that epizootic VEEV strains emerge via mutation from enzootic progenitors that are incapable of efficient equine amplification, the molecular mechanism(s) involved remain enigmatic. The convergent evolution of E2 envelope glycoprotein mutations suggests that they are critical to VEEV emergence, but little is known about the role of non-envelope genes. We used the guinea pig, the small animal model that best predicts the ability to generate equine viremia, to assess the role of envelope versus other mutations in the epizootic phenotype. Using reciprocal chimeric viruses generated by swapping the envelope genes of closely related epizootic IC and enzootic ID strains, infections of guinea pigs demonstrated that envelope and non-envelope genes and sequences both contributed to virulence. However, early replication in lymphoid tissues appeared to be primarily envelope dependent.

Dual mechanisms of pestiviral superinfection exclusion at entry and RNA replication.

For many viruses, primary infection has been shown to prevent superinfection by a homologous second virus. In this study, we investigated superinfection exclusion of bovine viral diarrhea virus (BVDV), a positive-sense RNA pestivirus. Cells acutely infected with BVDV were protected from superinfection by homologous BVDV but not with heterologous vesicular stomatitis virus. Superinfection exclusion was established within 30 to 60 min but was lost upon passaging of persistently infected cells. Superinfecting BVDV failed to deliver a translatable genome into acutely infected cells, indicating a block in viral entry. Deletion of structural protein E2 from primary infecting BVDV abolished this exclusion. Bypassing the entry block by RNA transfection revealed a second block at the level of replication but not translation. This exclusion did not require structural protein expression and was inversely correlated with the level of primary BVDV RNA replication. These findings suggest dual mechanisms of pestivirus superinfection exclusion, one at the level of viral entry that requires viral glycoprotein E2 and a second at the level of viral RNA replication.

Gene therapy for malignant glioma using Sindbis vectors expressing a fusogenic membrane glycoprotein.

BACKGROUND: Malignant glioma has a dismal prognosis. It was previously shown that glioma cells are efficiently killed when they express a gene coding for a hyperfusogenic mutant of the gibbon ape leukemia virus envelope glycoprotein (GALV.fus). However, production of viral vectors expressing GALV.fus has proven problematic because the transgene is toxic to vector-producing cells of human origin. We reasoned that Sindbis-virus-based vectors might be ideal for GALV.fus gene transfer because high-titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa laminin receptor. In addition, Sindbis virus nonstructural proteins are potent inducers of apoptosis, and Sindbis vector RNAs expressing fusogenic viral proteins have been shown to spread from cell-to-cell in membrane-formed infectious particles. METHODS: Sindbis virus replicon-containing particles were generated by co-transfecting vector and helper RNAs into baby hamster kidney (BHK-21) cells. Packaged beta-galactosidase and GALV.fus expressing Sindbis vectors were used to infect glioma cell lines, which were then compared for syncytial cytopathic effect, cell killing, and release of infectious virus-like particles containing the vector genome. Finally, the efficacy of GALV.fus and beta-galactosidase Sindbis vectors was compared in an orthotopic intracerebral U87 glioma xenograft model in nude mice. RESULTS: High-titer stocks (>10(9) infectious units (iu)/ml) of the GALV.fus and beta-galactosidase vectors were obtained. Glioma cells infected with the GALV.fus vector formed large syncytia which died rapidly by apoptosis and released infectious membrane-formed particles that could transfer vector genomes to uninfected cells. The GALV.fus vector had significantly greater antitumor therapeutic potency than the beta-galactosidase vector in the U87 glioma xenograft model. CONCLUSIONS: Sindbis vectors expressing GALV.fus can be packaged into infectious viral particles to high titer, they exhibit potent bystander cytopathic potential and are active against U87 glioma xenografts. Sindbis-virus-based replicons appear to be efficient vector systems for delivery and expression of fusogenic membrane glycoproteins.