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Both nonstructural proteins NS1 and NS2 of pneumonia virus of mice are inhibitors of the interferon type I and type III responses in vivo.

ABSTRACT: Infection of mice with pneumonia virus of mice (PVM) provides a convenient experimental pathogenesis model in a natural host for a human respiratory syncytial virus-related virus. Extending our previous work showing that the PVM nonstructural (NS) proteins were pathogenicity factors in mice, we identify both the NS1 and NS2 proteins as antagonists of alpha/beta interferon (IFN-?/?) and IFN-? by use of recombinant PVM (rPVM) with single and combined deletions of the NS proteins (?NS1, ?NS2, and ?NS1 ?NS2). Wild-type and NS deletion PVMs were evaluated for growth and pathogenesis by infecting knockout mice that lack functional receptors to IFN-?/?, IFN-?, or both. The absence of the receptor to IFN-?/? (IFNAR) or IFN-? (interleukin-28 receptor ? chain [IL-28R?]) individually did not reverse the attenuated virulence of the NS deletion viruses although loss of IFNAR partially restored replication efficiency. When both receptors were deleted, replication and virulence were largely rescued for rPVM ?NS1 and were significantly but not completely rescued for rPVM ?NS2. As for rPVM ?NS1 ?NS2, the effect was mostly limited to partial enhancement of replication. This indicates that both IFN-?/? and IFN-? contributed to restricting the NS deletion viruses, with the former playing the greater role. Interestingly, the replication and virulence of wild-type PVM were completely unaffected by the presence or absence of functional receptors to IFN-?/? and IFN-?, indicating that both systems are strongly suppressed during infection. However, pretreatment of mice with IFN-?/? was protective against lethal rPVM challenge, whereas pretreatment with IFN-? delayed but did not prevent disease and, in some cases, reduced mortality. The fact that virulence of rPVM lacking NS2 was not recovered completely when both interferon receptors were deleted suggests that NS2 may have further functions outside the IFN system.

SUBMITTER: Heinze B

PROVIDER: S-EPMC3126227 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Both nonstructural proteins NS1 and NS2 of pneumonia virus of mice are inhibitors of the interferon type I and type III responses in vivo.

Heinze Britta B Frey Stefanie S Mordstein Markus M Schmitt-Gräff Anette A Ehl Stephan S Buchholz Ursula J UJ Collins Peter L PL Staeheli Peter P Krempl Christine D CD

Journal of virology 20110209 9

Infection of mice with pneumonia virus of mice (PVM) provides a convenient experimental pathogenesis model in a natural host for a human respiratory syncytial virus-related virus. Extending our previous work showing that the PVM nonstructural (NS) proteins were pathogenicity factors in mice, we identify both the NS1 and NS2 proteins as antagonists of alpha/beta interferon (IFN-α/β) and IFN-λ by use of recombinant PVM (rPVM) with single and combined deletions of the NS proteins (ΔNS1, ΔNS2, and Δ ...[more]

PMID: 21307191

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Project description:Pneumonia virus of mice (PVM) strain 15 causes fatal pneumonia in mice and provides a convenient model for human respiratory syncytial virus pathogenesis and immunobiology. We prepared PVM mutants lacking the genes for nonstructural proteins NS1 and/or NS2. In Vero cells, which lack type I interferon (IFN), deletion of these proteins had no effect on the efficiency of virus growth. In IFN-competent mouse embryo fibroblasts, wild-type (wt) PVM and the DeltaNS1 virus grew efficiently and strongly inhibited the IFN response, whereas virus lacking NS2 was highly attenuated and induced high levels of IFN and IFN-inducible genes. In BALB/c mice, intranasal infection with wt PVM caused overt disease that began on day 6 and was lethal by day 9 postinoculation. In comparison, DeltaNS1 induced transient, reduced disease, and DeltaNS2 and DeltaNS12 caused no disease. Thus, NS1 and NS2 are virulence factors, with NS2 being a major antagonist of the type I IFN system. The pulmonary titers of wt PVM and DeltaNS1 were high on day 3 and increased further by day 6; in addition, expression of IFN and representative proinflammatory cytokines/chemokines and T lymphocyte-related cytokines was undetectable on day 3 but increased dramatically by day 6 coincident with the onset of disease. The titers of DeltaNS2 and DeltaNS12 were somewhat lower on day 3 and decreased further by day 6; in addition, these viruses induced a more circumscribed set of cytokines/chemokines (IFN, interleukin-6 [IL-6], and CXCL10) that were detected on day 3 and had largely subsided by day 6. Lung immunohistology revealed abundant PVM-positive pneumocytes and bronchial and bronchiolar epithelial cells in wt PVM- and DeltaNS1-infected mice on day 6 compared to few PVM-positive foci with DeltaNS2 and DeltaNS12. These results indicate that severe PVM disease is associated with high, poorly controlled virus replication driving the expression of high levels of pulmonary IFN and a broad array of cytokines/chemokines. In contrast, in the absence of NS2, there was an early, transient innate response involving moderate levels of IFN, IL-6, and CXCL10 that restricted virus replication and prevented disease.

Project description:UnlabelledInfluenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production.ImportanceInfluenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense.

Dataset Information

Both nonstructural proteins NS1 and NS2 of pneumonia virus of mice are inhibitors of the interferon type I and type III responses in vivo.

Publications

Both nonstructural proteins NS1 and NS2 of pneumonia virus of mice are inhibitors of the interferon type I and type III responses in vivo.

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