Avian and 1918 Spanish Influenza A Virus NS1 Proteins Bind to Crk/CrkL Src Homology 3 Domains to Activate Host Cell Signaling

Krister Melén,Kalle Saksela,Ralf Wagner,Leena S. Heikkinen,Arunas Kazlauskas,Thedi Ziegler,Ilkka Julkunen

doi:10.1074/jbc.m707195200

Abstract

NS1 (nonstructural protein 1) is an important virulence factor of the influenza A virus. We observed that NS1 proteins of the 1918 pandemic virus (A/Brevig Mission/1/18) and many avian influenza A viruses contain a consensus Src homology 3 (SH3) domain-binding motif. Screening of a comprehensive human SH3 phage library revealed the N-terminal SH3 of Crk and CrkL as the preferred binding partners. Studies with recombinant proteins confirmed avid binding of NS1 proteins of the 1918 virus and a representative avian H7N3 strain to Crk/CrkL SH3 but not to other SH3 domains tested, including p85alpha and p85beta. Endogenous CrkL readily co-precipitated NS1 from cells infected with the H7N3 virus. In transfected cells association with CrkL was observed for NS1 of the 1918 and H7N3 viruses but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence motif. SH3 binding was dispensable for suppression of interferon-induced gene expression by NS1 but was associated with enhanced phosphatidylinositol 3-kinase signaling, as evidenced by increased Akt phosphorylation. Thus, the Spanish Flu virus resembles avian influenza A viruses in its ability to recruit Crk/CrkL to modulate host cell signaling.

Highlights

Influenza A virus belongs to the Orthomyxoviridae family of enveloped viruses
Analysis of NS1 protein sequences from other strains of influenza A revealed that this sequence motif is very common among avian influenza A viruses but only rarely found in viruses isolated from humans (Fig. 1)
Role of Src homology 3 (SH3) Binding in Cellular Functions of NS1—To study the functional role and relevance of Crk/CrkL binding by NS1, we tested the activities of A/Brevig and A/Mallard NS1 proteins and their SH3 binding-deficient derivatives in two different cellular functions that have been reported for NS1, namely inhibition of interferon-induced gene expression and activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway

Summary

Introduction

Influenza A virus belongs to the Orthomyxoviridae family of enveloped viruses. Its genome is organized into eight singlestranded, negative-sense RNA segments that code for 11 iden-. In good agreement with the recombinant protein data involving isolated Crk/CrkL SH3 domains, in transfected cells full-length Crk/CrkL proteins bound well to NS1 proteins containing a class II SH3-binding consensus site but not detectably to NS1 proteins lacking this motif.

Results

Conclusion