Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30

Abstract

BackgroundThe genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species.MethodsNS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed.ResultsEach of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-β promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.ConclusionsThe F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.

Identification of Adaptive Mutations in the Influenza A Virus Non-Structural 1 Gene That Increase Cytoplasmic Localization and Differentially Regulate Host Gene Expression

“…RNA binding as well as RIG-I and the E3 ligase Riplet binding [41] inhibit signaling and IFN activation in mouse cells. In a recent report by Dankar et al (2013), NS1 residues 103L and 106I of the A/HK/156/1997 (H5N1) HPAI strain were found to increase NS1 localization to the cytoplasm of infected mouse cells, and also to mediate increased binding to RIG-I, which correlated with decreased IFN-β production in infected mouse cells [24]. Thus the increased distribution of NS1 to the cytoplasm observed for MA NS1 mutants may also contribute to inhibition of type I IFN production.…”

“…NS1 aa residues 103L and 106I have also been linked to cytoplasmic localization of the H5N1 highly pathogenic avian influenza (HPAI) isolate A/HK/156/1997 NS1 protein in mouse cells, when expressed on the PR8 viral backbone [24]. With respect to these findings, it is possible the NS1 protein contains an additional domain that influences nuclear export or cytoplasmic retention spanning from aa 98 to 106.…”

Fluorescence-Activated Cell Sorting-Based Analysis Reveals an Asymmetric Induction of Interferon-Stimulated Genes in Response to Seasonal Influenza A Virus

“…7). We did not investigate CPSF30 binding of the NS1 proteins from PR/8, HK/97, and BrevM/18, as previous studies showed that BrevM/18 NS1 binds to CPSF30, whereas the NS1 proteins of PR/8 and HK/97 cannot (27,49). These results established a direct and, most likely, causal link between the capacities of IAV NS1 proteins from seasonal strains to bind to CPSF30 and limit ISG15 expression in infected cells.…”

“…NS1 proteins expressed by seasonal H1N1 and H3N2 strains efficiently interacted with CPSF30. In contrast, the mallard/Ger (H3N2) and Anhui/2013 (H7N9) NS1 proteins were poor interactors, and others had previously determined similar failures for the NS1 proteins of highly and lowly pathogenic avian H5 subtype viruses (49,54).…”

Mammalian Adaptive Mutations of the PA Protein of Highly Pathogenic Avian H5N1 Influenza Virus

“…Therefore, viruses with mutations that facilitate efficient transmission and replication in humans could cause a pandemic. Many amino acid substitutions have been shown to contribute to the adaptation of avian H5N1 viruses to mammalian hosts (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). In particular, the PB2, PB1, and PA subunits of the polymerase complex play a role in virus pathogenicity and efficient viral growth in mammals.…”