JVI Accepts, published online ahead of print on 30 January 2008

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J. Virol. doi:10.1128/JVI.02234-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Systematic mutagenesis of the MHV68 M2 protein identifies domains important for chronic infection

Jeremy H. Herskowitz, Andrea M. Siegel, Meagan A. Jacoby, and Samuel H. Speck^*

The Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30329

^* To whom correspondence should be addressed. Email: sspeck{at}emory.edu.

Murine gammaherpesvirus 68 (MHV68) infection of inbred mice represents a genetically tractable small-animal model for assessing the requirements for the establishment of latency, as well as reactivation from latency, within the lymphoid compartment. By day 16 post-infection, MHV68 latency in the spleen is found in B cells, dendritic cells and macrophages. However, like Epstein-Barr virus (EBV), by 3 month post-infection MHV68 latency is predominantly found in isotype switched memory B cells. The MHV68 M2 gene product is a latency-associated antigen with no discernible homology to any known cellular or viral proteins. However, depending on experimental conditions, the M2 protein has been shown to play a critical role in both the efficient establishment of latency in splenic B cells, as well as reactivation from latently infected splenic B cells. Inspection of the sequence of the M2 protein reveals several hallmarks of a signaling molecule, including multiple PXXP motifs and two potential tyrosine phosphorylation sites. Here we report the generation of a panel of recombinant MHV68 viruses harboring mutations in the M2 gene that disrupt putative functional motifs. Subsequent analyses of the panel of M2 mutant viruses revealed a functionally important cluster of PXXP motifs in the C-terminal region of M2, which have previously been implicated in binding Vav proteins (30, 43). Further characterization of two adjacent PXXP motif in near the C-terminus of the M2 protein, revealed differences in the functions of these domains in M2-driven expansion of primary murine B cells in culture. Finally, we show that tyrosine residues 120 and 129 play a critical role in both the establishment of splenic latency, as well as reactivation from latency upon explant of splenocytes into tissue culture. Taken together, these analyses will aide future studies to identify M2 interacting partners and B cell signaling pathways that are manipulated by the M2 protein.

This article has been cited by other articles:

• DeZalia, M., Speck, S. H. (2008). Identification of Closely Spaced but Distinct Transcription Initiation Sites for the Murine Gammaherpesvirus 68 Latency-Associated M2 Gene. J. Virol. 82: 7411-7421 [Abstract] [Full Text]