The Gammaherpesvirus m2 Protein Manipulates the Fyn/Vav Pathway through a Multidocking Mechanism of Assembly

Marta Pires De Miranda,Xosé R Bustelo,J Pedro Simas,Filipa Lopes,Lénia Rodrigues,Marta Alenquer,Sofia Marques,Olivier Schwartz

doi:10.1371/journal.pone.0001654

Abstract

To establish latent infections in B-cells, gammaherpesviruses express proteins in the infected B-cells of the host that spuriously activate signalling pathways located downstream of the B-cell receptor. One such protein is M2, a murine gammaherpesvirus 68-encoded molecule that activates the Vav1/Rac1 pathway via the formation of trimolecular complexes with Scr family members. Previous reports have shown that the formation of this heteromolecular complex involves interactions between a proline rich region of M2 and the Vav1 and Fyn SH3 domains. Here, we show that the optimal association of these proteins requires a second structural motif encompassing two tyrosine residues (Tyr120 and 129). These residues are inducibly phosphorylated by Fyn in non-hematopoietic cells and constitutively phosphorylated in B-cells. We also demonstrate that the phosphorylation of Tyr120 creates specific docking sites for the SH2 domains of both Vav1 and Fyn, a condition sine qua non for the optimal association of these two signalling proteins in vivo. Interestingly, signaling experiments indicate that the expression of M2 in B-cells promotes the tyrosine phosphorylation of Vav1 and additional signaling proteins, a biological process that requires the integrity of both the M2 phosphotyrosine and proline rich region motifs. By infecting mice with viruses mutated in the m2 locus, we show that the integrity of each of these two M2 docking motifs is essential for the early steps of murine gammaherpesvirus-68 latency. Taken together, these results indicate that the M2 phosphotyrosine motif and the previously described M2 proline rich region work in a concerted manner to manipulate the signaling machinery of the host B-cell.

Highlights

Gammaherpesviruses are amongst the most prevalent of human pathogens owing to their ability to establish lifelong persistent infections within their hosts [1]
Signalling from the B cell receptor (BCR) complex is initiated when Src family kinases such as Fyn, Blk and Lyn induce the phosphorylation of immunoreceptor tyrosinebased activation motifs (ITAMs) located in the Iga and Igb cytoplasmic tails
We have previously shown that a M2 mutant protein (M2Y) containing two missense mutations on tyrosine residues 120 and 129 could not be phosphorylated by Fyn in vivo or in vitro [22], indicating that these residues were the actual Fyn targets in M2

Summary

Introduction

Gammaherpesviruses are amongst the most prevalent of human pathogens owing to their ability to establish lifelong persistent infections within their hosts [1]. Host colonisation by gammaherpesvirus involves the modulation of signalling pathways triggered upon B cell receptor (BCR) activation This strategy offers an obvious advantage to the virus since infection is not dependent on rare encounters with antigen specific naive B cells. Tyrosine-phosphorylated ITAMs recruit the cytoplasmic Syk protein tyrosine kinase, leading to its membrane translocation and the subsequent trans-phosphorylation of important B-cell signalling proteins such as Vav, Vav, phospholipase C-c2 (PLC-c2) and phosphatidylinositol-3 kinase (PI3K). These molecules promote the generation of a wide spectrum of intracellular signals and biological responses that are essential for the antigenic responses of B lymphocytes [4]

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