Griffith D. Parks

Naturally Occurring Substitutions in the P/V Gene Convert the Noncytopathic Paramyxovirus Simian Virus 5 into a Virus That Induces Alpha/Beta Interferon Synthesis and Cell Death

ABSTRACT The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-α/β) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI−) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-α/β signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI− had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI− were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI− mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI− but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-β promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI− but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI− mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI− growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI− by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI− induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-α/β synthesis, and virus-induced apoptosis.

Paramyxovirus-Induced Shutoff of Host and Viral Protein Synthesis: Role of the P and V Proteins in Limiting PKR Activation

ABSTRACT The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI− mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI− mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI− virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2α. WT SV5 was a poor activator of the eIF-2α kinase protein kinase R (PKR). By contrast, the P/V-CPI− mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI− mutant was restored to ∼50% that of control HeLa cells. By contrast, the rates of P/V-CPI− viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI− mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI− mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

Role for the Phosphoprotein P Subunit of the Paramyxovirus Polymerase in Limiting Induction of Host Cell Antiviral Responses

ABSTRACT Six amino acid substitutions in the shared N-terminal region of the P subunit of the viral polymerase and the accessory V protein convert the noncytopathic paramyxovirus simian virus 5 (SV5), which is a poor inducer of host cell responses, into a P/V mutant (P/V-CPI-) that induces high levels of apoptosis, interferon-beta (IFN-beta), and proinflammatory cytokines. In this study, we addressed the question of whether these new mutant phenotypes are due to the presence of an altered P protein or of an altered V protein or of both proteins. By the use of the P/V-CPI- mutant as a backbone, new mutant viruses were engineered to express the wild-type (WT) V protein (+V-wt) or WT P protein (+P-wt) from an additional gene inserted between the HN and L genes. In human epithelial cell lines, the +V-wt virus showed reduced activation of apoptosis and lower secretion of IFN-beta and proinflammatory cytokines compared to the parental P/V-CPI- virus. The presence of a V protein lacking the C-terminal cysteine-rich domain (corresponding to the SV5 I protein) did not reduce these host cell responses to P/V-CPI- infection. Unexpectedly, the +P-wt virus, which expressed a WT P subunit of the viral polymerase, also induced much lower levels of host cell responses than the parental P/V-CPI- mutant. For both +V-wt and +P-wt viruses, reduced levels of IFN-beta synthesis correlated with reduced IRF-3 dimerization and nuclear localization of IRF-3 and NF-κB, suggesting that the WT P and V proteins acted at an early stage in antiviral pathways. Host cell responses induced by the various P/V mutants directly correlated with levels of viral mRNA accumulation but not with steady-state levels of genomic RNA. Our results support the hypothesis that WT P and V proteins limit induction of antiviral responses by controlling the production of key viral inducers. A model is presented for the mechanism by which both the P subunit of the viral polymerase and the V accessory protein contribute to the ability of a paramyxovirus to limit activation of antiviral responses.

Differential mechanisms of complement-mediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus

The complement system is an important component of the innate immune response to virus infection. The role of human complement pathways in the in vitro neutralization of three closely related paramyxoviruses, Simian Virus 5 (SV5), Mumps virus (MuV) and Human Parainfluenza virus type 2 (HPIV2) was investigated. Sera from ten donors showed high levels of neutralization against HPIV2 that was largely complement-independent, whereas nine of ten donor sera were found to neutralize SV5 and MuV only in the presence of active complement pathways. SV5 and MuV neutralization proceeded through the alternative pathway of the complement cascade. Electron microscopy studies and biochemical analyses showed that treatment of purified SV5 with human serum resulted in C3 deposition on virions and the formation of massive aggregates, but there was relatively little evidence of virion lysis. Treatment of MuV with human serum also resulted in C3 deposition on virions, however in contrast to SV5, MuV particles were lysed by serum complement and there was relatively little aggregation. Assays using serum depleted of complement factors showed that SV5 and MuV neutralization in vitro was absolutely dependent on complement factor C3, but was not dependent on downstream complement factors C5 or C8. Our results indicate that even though antibodies exist that recognize both SV5 and MuV, they are mostly non-neutralizing and viral inactivation in vitro occurs through the alternative pathway of complement. The implications of our work for development of paramyxovirus vectors and vaccines are discussed.

Increased Readthrough Transcription across the Simian Virus 5 M-F Gene Junction Leads to Growth Defects and a Global Inhibition of Viral mRNA Synthesis

ABSTRACT Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50- to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ∼1.5- to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5′ end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.

IFNγ-producing, virus-specific CD8+ effector cells acquire the ability to produce IL-10 as a result of entry into the infected lung environment

It has become clear that T cells with the potential to negatively regulate the immune response are normal constituents of the immune system. These cells often mediate their effects through the production of immunosuppressive factors. At present our understanding of how these cells are generated is limited. Here we report the presence of a population of IL-10-producing, virus-specific CD8+ T cells in the lungs of mice following acute respiratory infection. These cells were only found at minimal levels in the spleen and draining lymph node; instead they were restricted primarily to the infected lung tissue. A major finding from this study is demonstration that the ability to produce IL-10 can be acquired by IFNgamma-producing effector cells following entry into the infected lung. These studies suggest IL-10 production is the result of further differentiation of an antigen-specific CD8+ T cell that is governed by signals present in infected lung tissue.

A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5.

ABSTRACT Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo.

Altered Function in CD8+ T Cells following Paramyxovirus Infection of the Respiratory Tract

ABSTRACT For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in ∼85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.

High Avidity CD8+ T Cells Are the Initial Population Elicited Following Viral Infection of the Respiratory Tract

Following intranasal administration, the model paramyxovirus simian virus 5 (SV5) establishes an infection in the respiratory tract of mice, which is subsequently cleared by CD8+ T cells. In this study, we sought to understand the maturation of the antiviral immune response over time by assessing the functional avidity of the responding T cells and the expansion of immunodominant populations. Surprisingly, we determined that the initial response to Ag at day 3 (d3) in the mediastinal lymph node was exclusively high avidity. However, by d5 postinfection, low avidity cells were ∼50% of the responding T cell population. Following secondary exposure to SV5, high avidity CD8+ T cells again are the exclusive cell type present at early times postinfection (d2). Similarly, high avidity cells were preferentially elicited at d3 following infection with the unrelated vaccinia virus. We also made the observation that the immunodominance profile has not been established at d3 postinfection with SV5. However, by d5 a clear immunodominance pattern arises and is permanently maintained. These data indicate that high avidity cells are the predominant population responding at early times postinfection following respiratory infection with SV5 or vaccinia virus. However, as the response progresses, low avidity cells are activated/expanded to a greater extent compared with high avidity cells.

Replication-independent activation of human plasmacytoid dendritic cells by the paramyxovirus SV5 Requires TLR7 and autophagy pathways.

The paramyxovirus Simian Virus 5 (SV5) is a poor inducer of interferon (IFN) secretion in all cell types tested so far, including primary epithelial cells and primary human myeloid dendritic cells. SV5 is hypothesized to limit induction of antiviral responses through control of viral gene expression and production of the V protein antagonist. Plasmacytoid dendritic cells (pDCs) are known to uniquely express toll-like receptor (TLR)-7 and are a main producer of IFN-alpha among peripheral blood mononuclear cells in response to many viruses. Here, we tested whether SV5 would remain a poor inducer of IFN in primary human pDCs. The efficiency of SV5 infection of pDCs could be increased by an increasing multiplicity of infection. pDCs infected by both live and UV-inactivated SV5 induced large amounts of IFN-alpha secretion and resulted in upregulation of maturation markers CD80 and CD86. However, IL-6 secretion was not induced by SV5 infection. When TLR7 signaling was inhibited, SV5 induced less IFN secretion and CD80 expression, and there was a corresponding increase in number of infected cells. Similar effects were seen with inhibitors of cellular autophagy pathways, suggesting that the SV5 activation of pDC requires access to the cytoplasm and autophagic sampling of cytoplasmic contents. These results have implications for control of SV5 infections in vivo and for development of SV5 as a vaccine vector.