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Glycine 184 in nonstructural protein NS1 determines the virulence of influenza A virus strain PR8 without affecting the host interferon response.

ABSTRACT: The nonstructural protein NS1 of influenza A virus counteracts the interferon (IFN) system and thereby promotes viral replication. NS1 has acquired different mechanisms to limit induction of IFN. It prevents double-stranded RNA (dsRNA) and RIG-I-mediated activation of interferon regulatory factor 3 (IRF3), and it blocks posttranscriptional processing of cellular mRNAs by binding to the cleavage and polyadenylation specificity factor (CPSF). Using a mouse-adapted A/PR/8/34 virus and reverse genetics to introduce specific mutations in NS1 which eliminate one or both functions, we determined the relative contributions of these two activities of NS1 to viral virulence in mice. We found that a functional RNA-binding motif was required for IFN suppression and virulence. Restoration of CPSF binding in the NS1 protein of wild-type A/PR/8/34 virus, which cannot bind CPSF due to mutations in the central binding motif at positions 103 and 106, resulted in enhanced virulence. Surprisingly, if CPSF binding was abolished by substituting glycine for arginine at position 184 in the classical NS1-CPSF binding motif, the mutant virus replicated much more slowly in mice, although the mutated NS1 protein continued to repress the IFN response very efficiently. Our results show that a functional RNA-binding motif is decisive for NS1 of A/PR/8/34 virus to suppress IFN induction. They further demonstrate that in addition to its contribution to CPSF binding, glycine 184 strongly influences viral virulence by an unknown mechanism which does not involve the IFN system.

SUBMITTER: Steidle S

PROVIDER: S-EPMC3004359 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Glycine 184 in nonstructural protein NS1 determines the virulence of influenza A virus strain PR8 without affecting the host interferon response.

Steidle Sabine S Martínez-Sobrido Luis L Mordstein Markus M Lienenklaus Stefan S García-Sastre Adolfo A Stäheli Peter P Kochs Georg G

Journal of virology 20101006 24

The nonstructural protein NS1 of influenza A virus counteracts the interferon (IFN) system and thereby promotes viral replication. NS1 has acquired different mechanisms to limit induction of IFN. It prevents double-stranded RNA (dsRNA) and RIG-I-mediated activation of interferon regulatory factor 3 (IRF3), and it blocks posttranscriptional processing of cellular mRNAs by binding to the cleavage and polyadenylation specificity factor (CPSF). Using a mouse-adapted A/PR/8/34 virus and reverse genet ...[more]

PMID: 20926573

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Project description:Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus. IMPORTANCE:Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence, the pathogenicity of the circulating viruses is highly relevant. Here, we analyzed amino acid variability in the NS1 proteins from human seasonal viruses and the effect of the mutations in innate immune responses and virus pathogenesis. A previously unidentified mutation in the dsRNA-binding domain decreased NS1-mediated general inhibition of host protein synthesis and the interaction of the protein with CPSF30. This mutation led to increased innate immune responses after viral infection, augmented IFN sensitivity, and virus attenuation in mice. Interestingly, using PBMCs, the subject infected with the virus encoding the attenuating mutation induced decreased antiviral responses, suggesting why this subject could be infected with this virus.

Dataset Information

Glycine 184 in nonstructural protein NS1 determines the virulence of influenza A virus strain PR8 without affecting the host interferon response.

Publications

Glycine 184 in nonstructural protein NS1 determines the virulence of influenza A virus strain PR8 without affecting the host interferon response.

Similar Datasets

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