Superantigen-activated T cells redirected by a bispecific antibody inhibit vesicular stomatitis virus replication in vitro and in vivo.

A bispecific Ab (BsAb) that binds the TCR on T cells and the G protein of the vesicular stomatitis virus (VSV) can redirect staphylococcal enterotoxin B (SEB)-activated T cells to kill VSV-infected cells and to inhibit VSV replication in vitro. Inhibition of virus replication in our system is dependent upon the specificity of the Ab for the viral protein. IFN-gamma does not play a very important role in this phenomenon, which is mainly mediated by the release of Pfp from CD8+ T cells. We have used a Stat1 knockout mouse model in which VSV infection is lethal. Infusion of staphylococcal enterotoxin-activated B T cells and bispecific Ab significantly slowed virus progression and prolonged the survival of VSV-infected Stat1 knockout mice in vivo.

Identification of a set of proteins (C' and C) encoded by the bicistronic P gene of the Indiana serotype of vesicular stomatitis virus and analysis of their effect on transcription by the viral RNA polymerase.

Previous work (C.F. Spiropoulou and S.T. Nichol, 1993, J. Virol. 67, 3103-3110) has demonstrated the existence in cells infected with the New Jersey serotype of vesicular stomatitis virus (VSV) of two small carboxy-coterminal proteins encoded by the P mRNA. These proteins have been named C' and C. We are interested in studying the function of these proteins in the virus life cycle. Toward this end, we have cloned the ORF encoding the potential C' protein of the Indiana serotype as a histidine-tagged fusion protein, purified the expressed protein from Escherichia coli, and used the fusion protein as an immunogen to raise antiserum in a rabbit. We have used this anti-C' protein serum to demonstrate that both of the predicted C' and C proteins are synthesized in cells infected with the Indiana serotype of VSV. In addition we have localized a portion of these proteins to nucleocapsids isolated from infected cells, suggesting that they may play a role in RNA synthesis. Reconstitution of the viral polymerase activity by expressing the L and P protein subunits with or without the C proteins failed to demonstrate any effect of the presence of these latter proteins on reconstituted transcription using purified nucleocapsids as templates. However, we have been able to show a dramatic stimulation of the polymerase activity in purified virions by the addition of purified C' protein to in vitro transcription reactions. Both the level and the fidelity of mRNA synthesis are stimulated by this protein. Evidence for the specificity of this effect comes from the fact that stimulation appears to be serotype specific; C' protein of the Indiana serotype stimulates transcription by purified Indiana serotype virions but has a minimal effect on transcription by purified virions of the New Jersey serotype. We are continuing our studies to determine the mechanism of this stimulation.

Analysis of human parainfluenza virus 3 receptor binding variants: evidence for the use of a specific sialic acid-containing receptor.

In order for the fusion protein (F) of the human parainfluenza virus type 3 (HPF3) to promote membrane fusion and viral entry, the haemagglutinin-neuraminidase (HN) glycoprotein must interact with its receptor. Sialoglycoconjugates are known to be the receptors for the HPF3 HN, however specific attachment factors or receptors for HPF3 have not been identified. In this report we describe the analysis of variants of HPF3 with increased fusion-promoting phenotypes that were selected by treatment with viral neuraminidase. The results suggest that for HPF3, the virus is specific in its use of sialic acid receptors; the majority of sialic-acid containing molecules are not targets for HPF3.

Inhibition of VSV genome RNA replication but not transcription by monoclonal antibodies specific for the viral P protein.

The growth of vesicular stomatitis virus requires two distinct RNA synthetic events: transcription of messenger RNA molecules and replication of the viral genome RNA. We report the use of a panel of monoclonal antibodies directed against the viral phosphoprotein P in an attempt to assess the role of this protein in RNA synthesis. Using extracts derived from virus-infected cells, we show that several anti-P monoclonal antibodies can have an inhibitory effect on genome RNA replication by binding to a soluble form of the P protein. We also show that the P protein to which one of these antibodies (6D11) is directed is not complexed with the N protein and that the amount of soluble P protein that binds to the 6D11 antibody in immunoprecipitation reactions can be increased by treating extracts with alkaline phosphatase. In addition, phosphatase treatment of infected cell extracts results in an increased level of genome RNA replication. These results suggest that a soluble subspecies of the P protein that functions in genome RNA replication exists in infected cells and that this species of the P protein is not required for transcription.

Hemagglutinin-neuraminidase of human parainfluenza 3: role of the neuraminidase in the viral life cycle.

The function of neuraminidase in the life cycle and pathogenesis of human parainfluenza virus type 3 (HPF3) was studied by analyzing a variant of HPF3 that has decreased neuraminidase enzymatic activity. The variant virus is more fusogenic than the wild-type virus during an acute infection. Cloning and sequencing of the fusion (F) and hemagglutinin-neuraminidase (HN) genes from this variant revealed a single amino acid change in the HN protein and no alterations in the F protein sequence. Analysis of the growth properties of this variant revealed a delay in release of virus particles into the supernatant. Addition of exogenous neuraminidase to the culture resulted in increased release of infections viral particles, suggesting that the viral neuraminidase is important for release of HPF3 from the infected cell surface. In addition, the behavior of the variant virus during high-multiplicity infection and in the presence of exogenous neuraminidase provided evidence that the neuraminidase of HPF3 determines the outcome of viral infection (cytopathic versus persistent) in cell culture.

Relative affinity of the human parainfluenza virus type 3 hemagglutinin-neuraminidase for sialic acid correlates with virus-induced fusion activity.

The ability of enveloped viruses to cause disease depends on their ability to enter the host cell via membrane fusion events. An understanding of these early events in infection, crucial for the design of methods of blocking infection, is needed for viruses that mediate membrane fusion at neutral pH, such as paramyxoviruses and human immunodeficiency virus. Sialic acid is the receptor for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, the HN must interact with its receptor. In the present report, two variants of HPF3 with increased fusion-promoting phenotypes were selected and used to study the function of the HN glycoprotein in membrane fusion. Increased fusogenicity correlated with single amino acid changes in the HN protein that resulted in increased binding of the variant viruses to the sialic acid receptor. These results suggest that the avidity of binding of the HN protein to its receptor regulates the level of F protein-mediated fusion and begin to define one role of the receptor-binding protein of a paramyxovirus in the membrane fusion process.

Persistent infection with human parainfluenza virus 3 in CV-1 cells: analysis of the role of defective interfering particles.

Persistent infection of cultured cells with human parainfluenza virus type 3 (HPF3), established following infection at high multiplicity, has been associated with the presence of one or more viral defective-interfering (DI) particles in addition to standard viral genomes. We recently showed that persistent infection can also be established after low multiplicity infection, a condition not generally associated with amplification of DI particles. The association of DI particle genomes with persistent infection was therefore studied after infection with low multiplicity. Persistently infected cell cultures were established after low multiplicity infection with HPF3 in the presence of exogenous bacterial neuraminidase, and viral nucleocapsid RNA was analyzed for the presence of DI genomes at each passage after infection. In addition, the timing of DI particle appearance was assessed after infection with high multiplicity, a condition known to favor the amplification of DI particles. DI particles genomes did not appear until at least seven passages of persistently infected cell cultures, after either low or high multiplicity infection. Our data suggest that DI particles are not required for establishment of persistent infection of CV-1 cells by HPF3 and that DI particles are not generated early in infection. Despite reports of the association of paramyxovirus DI particles with persistent infection in culture, the role of these particles in HPF3 persistence is unknown; our findings offer insight into the complex interplay of viral and host factors in persistent infection.

Fusion properties of cells infected with human parainfluenza virus type 3: receptor requirements for viral spread and virus-mediated membrane fusion.

Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion.

Properties of human parainfluenza virus type 3 RNA polymerase/replicase activity in vitro: consensus with other negative-stranded RNA viruses.

A cell-free system supporting transcription, replication, and nucleocapsid assembly of the genome RNA of human parainfluenza virus type 3 (HPF3) is described. Cytoplasmic extracts from infected CV-1 or BHK cells catalyzed the transcription of the entire HPF3 genome, the replication of genome RNA, and the assembly of this RNA into nucleocapsidlike structures. Newly replicated RNA was resistant to micrococcal nuclease digestion and was stable in CsCl gradients, exhibiting the density of authentic HPF3 nucleocapsids. After fractionation of the extracts, the nucleocapsid-containing pellet fraction synthesized viral mRNAs. Reconstitution with the soluble protein fraction was necessary for genome RNA replication and nucleocapsid assembly.

Fusion properties of cells persistently infected with human parainfluenza virus type 3: participation of hemagglutinin-neuraminidase in membrane fusion.

Cells persistently infected with human parainfluenza virus type 3 (HPF3) exhibit a novel phenotype. They are completely resistant to fusion with each other but readily fuse with uninfected cells. We demonstrate that the inability of these cells to fuse with each other is due to a lack of cell surface neuraminic acid. Neuraminic acid is the receptor for the HPF3 hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. Uninfected CV-1 cells were treated with neuraminidase and then tested for their ability to fuse with the persistently infected (pi) cells. Neuraminidase treatment totally abolished cell fusion. To extend this result, we used a cell line deficient in sialic acid and demonstrated that these cells, like the neuraminidase-treated CV-1 cells, were unable to fuse with pi cells. We then tested whether mimicking the agglutinating function of the HN molecule with lectins would result in cell fusion. We added a panel of five lectins to the neuraminic acid-deficient cells and showed that binding of these cells to the pi cells did not result in fusion; the lectins could not substitute for interaction of neuraminic acid with the HN molecule in promoting membrane fusion. These results provide compelling evidence that the HN molecule of HPF3 and its interaction with neuraminic acid participate in membrane fusion and that cell fusion is mediated by an interaction more complex than mere juxtaposition of the cell membranes.

The 1:1 N-NS protein complex of vesicular stomatitis virus is essential for efficient genome replication.

We studied the effect pH had on the N-NS protein complex to determine its role in vesicular stomatitis virus (VSV) genome replication, as we had previously shown that VSV genome replication in vitro requires the interaction of the viral N and NS proteins into a 1:1 complex. A previous report showed that the growth of VSV in L cells was sensitive to the pH of the environment (M. Fiszman, J. B. Leaute, C. Chany, and M. Girard, J. Virol. 13:801-808, 1974). We hypothesized that low pH might disrupt the N-NS protein complex, and so we investigated the molecular events leading to inhibition of viral RNA replication in vitro from extracts that were prepared from VSV-infected cells incubated at pH 6.6. We found that viral genome RNA synthesis in vitro was reduced when infected cells were maintained at pH 6.6. Through immunoprecipitation analysis of the viral soluble protein pool, we found that a complex that usually exists between the N and NS proteins at pH 7.4 was altered in extracts from infected cells maintained at pH 6.6, and this was responsible for the observed effects on viral replication. The effect of low pH on the N-NS protein complex could not be abolished by increasing the concentration of the altered complex, indicating that the effects is more than simply a decrease in the level of the protein complex in the cell. Our data provide additional evidence that the 1:1 N-NS protein complex, and not the N protein alone, serves as the substrate for viral RNA replication in vivo.

In vitro assembly of a functional nucleocapsid from the negative-stranded genome RNA of a defective interfering particle of vesicular stomatitis virus.

The template for transcription and replication of negative-stranded RNA viruses is a ribonucleoprotein structure, the nucleocapsid. We have developed a system that supports assembly of the negative-stranded RNA genome of a defective interfering (DI) particle of vesicular stomatitis virus (VSV) into a nucleocapsid in vitro. This system uses extracts from wild-type VSV-infected cells as a source of proteins to encapsidate the RNA. In vitro assembled nucleocapsids were compared to in vivo-derived nucleocapsids by the following characteristics: nuclease resistance of the encapsidated RNA, CsCl density banding of labeled RNA in a position coincident with nucleocapsids, correct sedimentation rate in sucrose gradients, the presence of the nucleocapsid protein on the nucleocapsids, and the infectivity of the in vitro assembled nucleocapsids. We conclude that the system we present is capable of assembling the isolated genome of a rhabdovirus DI particle into nucleocapsids indistinguishable from those produced during the course of intracellular DI replication.

Kinetic, quantitative, and functional analysis of multiple forms of the vesicular stomatitis virus nucleocapsid protein in infected cells.

Multiple forms of the vesicular stomatitis virus nucleocapsid protein N have been detected in infected cells. One form is complexed with the viral NS protein in a 1:1 molar ratio, and the other forms are distinguished by their more rapid sedimentation rates on glycerol gradients. I performed a series of experiments designed to analyze the relationships between these forms of the N protein. Pulse-chase experiments demonstrate that the N protein is made first as the form which binds to the NS protein, forming a 1-to-1 molar complex, and that with increasing times of chase it is either assembled into nucleocapsids or converted to the two higher sedimenting forms. Using a newly developed quantitative immunoblotting procedure, I have quantitated the three differentially sedimenting species of the N protein and have shown that at later times postinfection (6 to 7 h), the faster-sedimenting forms of the N protein account for as much as 50% of the soluble N protein in the cell. The activity of these forms has been assessed, with only the 1-to-1 molar N-NS complex demonstrating the ability to support the replication and encapsidation of viral genomic RNA. A model for the conversion of the N protein from the active N-NS complex into the other forms of the protein is presented, and the possible function of the N-protein self-complexes is discussed.

Viral proteins required for the in vitro replication of vesicular stomatitis virus defective interfering particle genome RNA.

The viral proteins required for VSV RNA replication have been partially purified. With the use of monoclonal antibodies specific for the VSV N protein we have identified a putative N/NS complex present in the soluble protein fraction of infected cells. The complex is stable upon partial purification, contains the N and NS proteins in a 1:1 molar ratio, and has an elongated shape based on its hydrodynamic properties. Depletion of the N/NS complex from the infected cell soluble protein fraction results in the loss of the ability of this fraction to support RNA replication suggesting that the complex is required for this reaction. The ability to support viral genome RNA replication indeed cochromatographs with the N/NS protein complex through several steps of purification. Only the N protein of the N/NS complex appears to be bound to RNA during encapsidation with the release of NS protein.

Quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels onto positively charged nylon membranes.

A method for the reproducible and quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a single sheet of either Zetabind or Gene Screen Plus membranes is presented. This procedure uses commercially available equipment and includes three crucial parameters: the omission of methanol from the transfer buffer, the use of thin (0.75-mm) resolving gels, and a newly developed protocol for pretreatment of the polyacrylamide gel after electrophoresis and before electroblotting. This combination of parameters yields a blot that both qualitatively and quantitatively reflects the proteins in the original polyacrylamide gel.

In vitro replication of Sendai virus wild-type and defective interfering particle genome RNAs.

A system for studying the in vitro replication of the genome RNAs of Sendai virus and its defective interfering particle DI-H has been developed. Cytoplasmic extracts of baby hamster kidney cells infected with wild-type Sendai virus or coinfected with wild-type Sendai virus plus DI-H were prepared after lysolecithin treatment at 12 h postinfection. The extracts supported the transcription of six viral mRNAs as well as the replication of the Sendai virus 50S (wild-type) and 14S DI-H genome RNAs and their encapsidation into nucleocapsids in the absence of de novo protein synthesis. RNA replication in vitro represented more than 50% of total RNA synthesis, a relative level higher than that found in the infected cell. The proteins required for Sendai virus RNA replication were present in a soluble protein pool at the time of extract preparation. Depletion of the protein pool by prior treatment of infected cells with cycloheximide inhibited subsequent in vitro genome replication without affecting transcription. The cytoplasmic extract may be separated by high-speed centrifugation into two components: the Sendai virus wild-type and DI-H nucleocapsid templates containing the RNA and associated NP, L, and P proteins and the soluble protein fraction containing primarily the P, NP, and M viral proteins with trace amounts of the L, HN, Fo, and nonstructural C proteins. The isolated intracellular DI-H nucleocapsid template alone cannot replicate its RNA, but when recombined with the Sendai virus soluble protein fraction it catalyzes the replication and encapsidation of viral RNAs. The initiation of RNA replication in vitro can be demonstrated because detergent-disrupted purified DI-H virions replicate both positive- and negative-strand RNAs in the presence, but not in the absence, of the soluble protein fraction from an extract of infected cells.

A simple method for the preparation of extracts from animal cells which catalyze efficient in vitro protein synthesis.

A rapid, simple procedure is described for the preparation of cell-free extracts of both uninfected and virus-infected pig kidney and baby hamster kidney cells which are very active in the in vitro translation of not only endogenous mRNA, but also of exogenous mRNA added to extracts that had been depleted of endogenous mRNA by treatment with micrococcal nuclease. This procedure appears to be adaptable with only minor variations to many eukaryotic cell lines and should greatly facilitate in vitro molecular studies of protein synthesis and gene regulation at the level of translation.

Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.

A system for studying the in vitro replication of the RNA genomes of both wild-type vesicular stomatitis virus (VSV) and its defective interfering particle MS-T has been developed. After lysolecithin treatment of cells infected with VSV or VSV plus MS-T, a cell-free cytoplasmic extract is prepared which will support VSV mRNA synthesis and the synthesis of the 42S wild-type or 19S MS-T genome RNAs. The genome-length RNAs synthesized in vitro are assembled into RNase-resistant nucleocapsids. The level of 42S RNA synthesis in vitro (6-13% of total RNA synthesis) reflects the level of replication in vivo. Although the extracts of VSV-infected cells can also support the synthesis of VSV proteins, RNA replication is not dependent on de novo protein synthesis but utilizes the preformed soluble proteins present in the infected cell at the time the extract is prepared. The initiation of genomic RNA during in vitro replication can be demonstrated because detergent-disrupted, purified MS-T particles will replicate their RNA when added to either a total cytoplasmic extract from VSV-infected cells or the soluble protein fraction derived from such an extract.

Infection with paramyxoviruses stimulates synthesis of cellular polypeptides that are also stimulated in cells transformed by Rous sarcoma virus or deprived of glucose.

Infection of several types of cultured cells with the paramyxoviruses simian virus 5 and Sendai virus stimulates synthesis of four polypeptides (I-IV) with molecular weights of approximately 99,000, 97,000, 86,000, and 78,000, respectively. That these are host polypeptides encoded in cellular mRNAs has been shown by the inhibition of their synthesis by actinomycin D and by the similarity of the peptide maps of them and of polypeptides with the same electrophoretic mobility from uninfected cells. Peptide mapping as well as identical migration in polyacrylamide gels has also indicated that polypeptides I, II, and IV are the same as plasma membrane polypeptides whose synthesis is enhanced in cells transformed by Rous sarcoma virus and in normal cells by glucose deprivation or treatment with 2-deoxyglucose. Polypeptides I and II appear to be the same polypeptides, with the observed differences in migration reflecting the glycosylation of polypeptide I, a relationship previously shown to exist between polypeptides in glucose-deprived and glucose-fed cells. Infection with paramyxoviruses does not significantly increase the transport of glucose by cells, and the maintenance of a high concentration of glucose in the medium does not prevent the enhanced synthesis of these polypeptides. This is in contrast to the situation in transformed cells in which stimulation of synthesis of these polypeptides is secondary to depletion of glucose in the medium due to increased glucose uptake by the cells. Thus, although paramyxovirus infection and transformation by Rous sarcoma virus result in stimulation of the synthesis of the same membrane polypeptides, the mechanism of stimulation differs.

Polypeptide synthesis in simian virus 5-infected cells.

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F(0)) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F(1) and F(2). No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed.