Identification of adaptive mutations in the influenza A virus non-structural 1 gene that increase cytoplasmic localization and differentially regulate host gene expression.

Nicole Forbes,Mohammed Selman,Jian Jun Jia,Martin Pelchat,Earl G Brown,Alain Stintzi

doi:10.1371/journal.pone.0084673

Abstract

The NS1 protein of influenza A virus (IAV) is a multifunctional virulence factor. We have previously characterized gain-of-function mutations in the NS1 protein arising from the experimental adaptation of the human isolate A/Hong Kong/1/1968(H3N2) (HK) to the mouse. The majority of these mouse adapted NS1 mutations were demonstrated to increase virulence, viral fitness, and interferon antagonism, but differ in binding to the post-transcriptional processing factor cleavage and polyadenylation specificity factor 30 (CPSF30). Because nuclear trafficking is a major genetic determinant of influenza virus host adaptation, we assessed subcellular localization and host gene expression of NS1 adaptive mutations. Recombinant HK viruses with adaptive mutations in the NS1 gene were assessed for NS1 protein subcellular localization in mouse and human cells using confocal microscopy and cellular fractionation. In human cells the HK wild-type (HK-wt) virus NS1 protein partitioned equivalently between the cytoplasm and nucleus but was defective in cytoplasmic localization in mouse cells. Several adaptive mutations increased the proportion of NS1 in the cytoplasm of mouse cells with the greatest effects for mutations M106I and D125G. The host gene expression profile of the adaptive mutants was determined by microarray analysis of infected mouse cells to show either high or low extents of host-gene regulation (HGR or LGR) phenotypes. While host genes were predominantly down regulated for the HGR group of mutants (D2N, V23A, F103L, M106I+L98S, L98S, M106V, and M106V+M124I), the LGR phenotype mutants (D125G, M106I, V180A, V226I, and R227K) were characterized by a predominant up regulation of host genes. CPSF30 binding affinity of NS1 mutants did not predict effects on host gene expression. To our knowledge this is the first report of roles of adaptive NS1 mutations that impact intracellular localization and regulation of host gene expression.

Highlights

The influenza A virus (IAV) NS1 protein possesses multiple functions that support virus replication
We have previously shown that NS1 mutations selected upon mouse adaptation of the A/Hong Kong/1/1968 H3N2 virus (MA NS1 mutations) are adaptive, and associated with increased virulence in the mouse, decreased IFNb production in the mouse lung, and increased viral replication, both in vivo and in vitro, of which the latter was associated with enhanced viral gene expression at the level of viral mRNA and protein synthesis [21]
Mouse cells infected with NS1 mutants D125G and M106I+L98S, and to a lesser extent M106I and V226I, significantly increased the observed frequency of NS1 staining within the cytoplasm to up to 64% (p,0.001; D125G) (Figure 1 and S2)

Summary

Introduction

The influenza A virus (IAV) NS1 protein possesses multiple functions that support virus replication. NS1 can engage in many functions due to its ability to translocate to both the nucleus and cytoplasm of infected cells and interact with numerous cellular and viral factors including RNA. Cytoplasmic activities include blocking viral RNA detection by RIG-I signalling of type I interferon (IFN) induction as well as inhibition of IFN-stimulated antiviral proteins, suppression of the host cell apoptotic response, and enhancement of viral protein synthesis [1]. NS1 localization is governed by two known nuclear localization signals (NLS) (NLS1: aa 34–38, NLS2: aa 203–237), which interact with the cellular protein importin a [4] and induce rapid nuclear localization following translation [5]. The protein is detected in both the nucleus and the cytoplasm [6], which is attributed to interaction of its NES element (137–147) [6] with the nuclear pore complex. NS1 localizes to the nucleolus and contains a NoLS involving key basic residues Arg-224 and Arg-229 [4,8], the role of NS1 nucleolar localization and its contribution to viral replication is unknown

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Conclusion