Murine γ-Herpesvirus 68 Latency Protein M2 Binds to Vav Signaling Proteins and Inhibits B-cell Receptor-induced Cell Cycle Arrest and Apoptosis in WEHI-231 B Cells

Abstract

The MHV-68 latent protein, M2, does not have homology to any known viral or cellular proteins, and its function is unclear. To define the role played by M2 during MHV-68 latency as well as the molecular mechanism involved, we used M2 as bait to screen a yeast two-hybrid mouse B-cell cDNA library. Vav1 was identified as an M2-interacting protein in two independent screenings. Subsequent yeast two-hybrid interaction studies showed that M2 also binds to Vav2, but not Vav3, and that three "PXXP " motifs located at the C terminus of M2 are important for this interaction. The interactions between M2 and Vav proteins were also confirmed in vivo in 293T and WEHI-231 B-cells by co-immunoprecipitation assays. Rac1/ GST-PAK "pull-down" experiments and Western blot analysis using a phospho-Vav antibody demonstrated that expression of M2 in WEHI-231 cells enhances Vav activity. We further showed in WEHI-231 cells that M2 expression promotes proliferation and survival and is associated with enhanced cyclin D2 and repressed p27 Kip1 , p130, and Bim expression. Taken together, these experiments suggest that M2 might have an important role in disseminating the latent virus during the establishment and maintenance of latency by modulating B-cell receptor-mediated signaling events through Vav to promote B-cell activation, proliferation, and survival.

Regulating Vav1 phosphorylation by the SHP-1 tyrosine phosphatase is a fine-tuning mechanism for the negative regulation of DISC formation and Fas-mediated cell death signaling

“…Our results demonstrate a high constitutive phosphorylation of Vav, in accordance with data obtained from different B lymphocytes. 22 Further studies revealed that while reducing the amount of Vav induced more extensive cell death upon Fas activation, co-silencing of Vav with SHP-1 had no additive effect (Figure 4a), supporting the hypothesis that SHP-1 and Vav act in the same signaling pathway, downregulating DISCinduced death signaling.…”

“…These results indicate that, upon Fas stimulation, both kinases and phosphatases play complementary roles, tightly regulating the balance of phosphorylation, and SHP-1 is one of the main actors. 22 In particular, phosphorylation of Vav proteins on several tyrosine residues has been reported. 27 We hypothesized that the 95 kDa guanine nucleotide exchange factor Vav might correspond to the dephosphorylated protein of around 100 kDa, since it has been reported to be an SHP-1 substrate critical in the downregulation of NK cell killing.…”

“…21 Conflicting reports have described both activation and inhibition of cell death by Vav. 22,23 However, very little data on the functions of Vav in Fas-mediated signaling are available, except a report describing a higher level of Vav tyrosine phosphorylation in the Fas-deficient MRL/Mp-lpr/lpr mice. 24 The importance of cytoskeletal rearrangement in Fasmediated cell death is well known, while DISC formation is dependent on actin filaments.…”

Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade

“…40,50). Finally, we detected induction of genes for M6 and M9, unique MHV68 genes whose functions are unknown.…”

Rho’ing in and out of cells

“…In vivo studies showed that the interaction of M2 with Vav proteins is important for the establishment of a latent infection of MHV-68 in B cells. 109,110 Retroviruses like HIV also may establish latency. In a transcriptome study in cell lines latently infected with HIV, Cdc42 expression was found to be downregulated, which perhaps may be involved in cellular maintenance of latency.…”