Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

Chen Zhao,Ran Chen,Darren P Baker,Robert M Krug,Haripriya Sridharan,Shanshan Wang

doi:10.1038/ncomms12754

Abstract

The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP.

Highlights

The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon
ISG15 is an ubiquitin-like protein (Ubl) that is highly induced by the interferon (IFN)-a/b produced during infection of human cells by viral and bacterial pathogens[1]
Because our results show that viral proteins are primary ISGylation targets in influenza B virus-infected cells, the defect(s) in the replication of the 67 mutant virus is likely attributable to the actions of the ISGylated NP, M1 and/or non-structural protein 1 of influenza B virus (NS1B) proteins that are not sequestered a kD 170 130

Summary

Introduction

The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. ISGylation inhibits influenza A virus replication in human tissue culture cells[12,13,14] This inhibition results from the ISGylation of one or two lysines (Ks) of the viral NS1A protein, leading to the loss of NS1A function[13,14]. A subsequent study of ISG15-deficient patients and cells derived from these patients showed that intracellular free ISG15 in human cells downregulates the IFN-a/b response by binding and stabilizing USP18, a protein with two activities, removal of ISG15 from ISGylated proteins and inhibition of IFN signalling[18,19]. Unlike the wt NS1B protein, the mutant NS1B protein did not inhibit IFN-b-induced ISGylation as assayed in transfection experiments[23]

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Results

Conclusion