D. F. Young

Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics

Two canine isolates of simian virus 5 (SV5), termed CPI+ and CPI-, were examined for their ability to react with a bank of monoclonal antibodies (MAbs) that had been previously raised against a human isolate of SV5. CPI- virus was originally isolated from the brain of a gnotobiotic dog infected with CPI+ virus and establishes persistent infections more readily than CPI+ in vitro. Of more than 50 MAbs tested, only one (P-k) reacted with CPI+ but not CPI-, enabling distinction between the two canine isolates. It had been shown previously that MAb P-k reacts with an epitope common to both the P and V proteins. In order to characterize further the epitope binding site of this MAb the P/V genes of CPI+ and CPI- were sequenced. There were four nucleotide differences between CPI+ and CPI-, three of which resulted in predicted amino acid substitutions. Synthetic peptides corresponding to regions encompassing these changes were made and radioimmune competition assays were used to identify the epitope binding site of MAb P-k. Sequence comparison of the P/V gene of CPI+ with the published sequence of a monkey isolate of SV5 (W3) revealed 14 nucleotide differences with five amino acid substitutions. The only amino acid substitution observed between CPI+, CPI- and W3 which altered the predicted secondary structures of the P and V proteins was a leucine to proline change that induced a predicted beta-turn and resulted in the loss of binding of MAb P-k.

Isolation and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates.

Hybridomas secreting monoclonal antibodies to simian virus 5 (SV5) were obtained following immunization of mice with purified preparations of a human isolate (LN) of SV5. Immune precipitation studies showed that these monoclonal antibodies had specificities for the haemagglutinin-neuraminidase (HN), fusion (F), nucleo-, matrix and phospho- (P) proteins of SV5. By use of a radioimmune competition assay the monoclonal antibodies to the HN protein were assigned to four groups, members of which recognized different antigenic sites on the protein. All the anti-HN antibodies and the anti-F antibody neutralized virus infectivity. The 54 monoclonal antibodies obtained were used to determine whether there were antigenic differences between five human, two canine and one simian isolate of SV5. Although most of the monoclonal antibodies reacted with all isolates, a few did reveal antigenic differences in the HN, F and P proteins. Furthermore, analysis by SDS-PAGE showed that while the electrophoretic mobilities of most of the virus polypeptides of these isolates were similar some differences could be detected. In particular the P protein showed the most marked mobility differences between the human, canine and simian isolates. Slight differences in the mobility of the F1 glycoprotein could also be visualized.

The p127 Subunit (DDB1) of the UV-DNA Damage Repair Binding Protein Is Essential for the Targeted Degradation of STAT1 by the V Protein of the Paramyxovirus Simian Virus 5

ABSTRACT The V protein of simian virus 5 (SV5) blocks interferon signaling by targeting STAT1 for proteasome-mediated degradation. Here we present three main pieces of evidence which demonstrate that the p127 subunit (DDB1) of the UV damage-specific DNA binding protein (DDB) plays a central role in this degradation process. First, the V protein of an SV5 mutant which fails to target STAT1 for degradation does not bind DDB1. Second, mutations in the N and C termini of V which abolish the binding of V to DDB1 also prevent V from blocking interferon (IFN) signaling. Third, treatment of HeLa/SV5-V cells, which constitutively express the V protein of SV5 and thus lack STAT1, with short interfering RNAs specific for DDB1 resulted in a reduction in DDB1 levels with a concomitant increase in STAT1 levels and a restoration of IFN signaling. Furthermore, STAT1 is degraded in GM02415 (2RO) cells, which have a mutation in DDB2 (the p48 subunit of DDB) which abolishes its ability to interact with DDB1, thereby demonstrating that the role of DDB1 in STAT1 degradation is independent of its association with DDB2. Evidence is also presented which demonstrates that STAT2 is required for the degradation of STAT1 by SV5. These results suggest that DDB1, STAT1, STAT2, and V may form part of a large multiprotein complex which leads to the targeted degradation of STAT1 by the proteasome.

Single Amino Acid Substitution in the V Protein of Simian Virus 5 Differentiates Its Ability To Block Interferon Signaling in Human and Murine Cells

ABSTRACT Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N100D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N100D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N100D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N100D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N100D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.

Virus replication in engineered human cells that do not respond to interferons.

ABSTRACT The V protein of the paramyxovirus simian virus 5 blocks interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. Here we report on the isolation of human cell lines that express the V protein and can no longer respond to IFN. A variety of viruses, particularly slow-growing wild-type viruses and vaccine candidate viruses (which are attenuated due to mutations that affect virus replication, virus spread, or ability to circumvent the IFN response), form bigger plaques and grow to titers that are increased as much as 10- to 4,000-fold in these IFN-nonresponsive cells. We discuss the practical applications of using such cells in vaccine development and manufacture, virus diagnostics and isolation of newly emerging viruses, and studies on host cell tropism and pathogenesis.

Heterocellular induction of interferon by negative-sense RNA viruses

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

Deep Sequencing Analysis of Defective Genomes of Parainfluenza Virus 5 and Their Role in Interferon Induction

ABSTRACT Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and “copyback” DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.

Failure to activate the IFN-β promoter by a paramyxovirus lacking an interferon antagonist☆

It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-β promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-β promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this system.

Immunization with solid matrix-antibody-antigen complexes containing surface or internal virus structural proteins protects mice from infection with the paramyxovirus, simian virus 5.

A mouse model system has been developed to examine the ability of purified virus proteins to protect mice from infection with the paramyxovirus simian virus 5. The system is based on the infection of mouse lungs by intranasal administration of infectious virus. The relative amounts of virus proteins and nucleic acid present within infected lungs were estimated either by Western blot analysis of disrupted lung tissues or by in situ hybridization studies using cryostat sections of infected lungs. During a normal time course of infection in non-immunized mice increasing amounts of virus protein and nucleic acid were detected in the lungs until 3 days post-infection (p.i.). Thereafter the amount of virus present within the lungs remained relatively constant until 7 days p.i. when there was a rapid decrease. Cytotoxic T cells, but not neutralizing antibody, could be detected at the time when the amount of virus within the lungs was decreasing. Prior immunization of mice with solid matrix-antibody-antigen (SMAA) complexes containing either surface or internal virus structural proteins reduced the amount of virus replication within infected lungs, the greatest degree of protection being observed when nucleoprotein or matrix protein was used to immunize the mice. There was no correlation between the degree of protection observed and the level of neutralizing antibody present in immunized animals; no neutralizing antibody was detected in mice immunized with internal virus proteins even at the time of sacrifice 5 days p.i. We have previously shown that immunization of mice with SMAA complexes containing either surface or internal virus structural proteins can induce cytotoxic T cells and thus conclude that the most likely explanation for the protection observed in immunized mice is through the induction of cytotoxic T cells.

Activation of the beta interferon promoter by paramyxoviruses in the absence of virus protein synthesis

Conflicting reports exist regarding the requirement for virus replication in interferon (IFN) induction by paramyxoviruses. Our previous work has demonstrated that pathogen-associated molecular patterns capable of activating the IFN-induction cascade are not normally generated during virus replication, but are associated instead with the presence of defective interfering (DI) viruses. We demonstrate here that DIs of paramyxoviruses, including parainfluenza virus 5, mumps virus and Sendai virus, can activate the IFN-induction cascade and the IFN-β promoter in the absence of virus protein synthesis. As virus protein synthesis is an absolute requirement for paramyxovirus genome replication, our results indicate that these DI viruses do not require replication to activate the IFN-induction cascade.