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*

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 p27Kip1, 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.

␥-Herpesviruses comprise a subfamily of animal double-stranded DNA viruses widespread in nature. There are two known human ␥-herpesviruses, namely Epstein-Barr virus (EBV) 5 and human herpesvirus-8 (HHV-8) also known as Kaposi's sarcoma herpesvirus, which are largely disseminated in the population. Infections by the ␥-herpesviruses have been implicated in the development of lymphoproliferative diseases and in some cases, tumorigenesis (1,2). One of the characteristic biological features of the ␥-herpesviruses is their ability to establish a life-long latent infection in lymphocytes, during which only a few viral genes are expressed. One hypothesis is that these viral genes play a major role in the establishment and maintenance of viral latency. Understanding the molecular mechanisms whereby latency-associated proteins alter the regulation of the cellular pathways of the infected host cell, as well as evade immune surveillance will provide essential information for devising therapeutic strategies against these viruses. ␥-Herpesviruses employ similar strategies to subvert the infected host cell, encoding for viral proteins, which often interfere with cellular processes in the host cell, in particular targeting the transcriptional machinery and signaling pathways. In EBV, the EBNA-1, EBNA-2, EBN-3A, -B, -C, and EBNA-LP latency-associated proteins are involved in the altered transcriptional regulation of the host's genes (3,4), whereas the LMP-1 and LMP-2A proteins are involved in the altered regulation of endogenous cellular signaling pathways. In the case of LMP-1, it functions as a constitutively active CD40 receptor. This viral protein binds and activates TRAF-2 and -3 proteins leading to NF-B activation and consequently cell proliferation (5,6). It has been demonstrated that LMP-2A associates with the cellular tyrosine kinases Fyn, Lyn, and Syk, modulating their interactions with the B-cell receptor (7,8), thereby acting in the same manner as a constitutively active B-cell receptor. In B-cell receptor-negative B-cells, LMP-2A activates signals that are usually generated by the B-cell receptor to inhibit apoptosis of infected cells (9). LMP-2A has been shown to activate the phosphatidylinositol 3-kinase/Akt (also called PKB) signaling cascade, which has a major role in cell proliferation and survival (10,11). During latency, HHV-8 expresses viral proteins, such as the D-type cyclin homologue (v-cyclin) (12,13), the transcription regulators LANA-1 and LANA-2 (vIRF-3) (14,15), kaposin A (16), and vFLIP (17) to manipulate cellular signaling pathways. Another HHV-8 latent protein, LAMP, has been shown to inhibit B-cell receptordependent signal transduction and to associate with the cellular proteins TRAF-1, -2, and -3, suggesting that it might also mimic the function of the CD40 receptor (18,19).
It is evident that the biological functions of some of these ␥-herpesviruses latent proteins are to promote lymphocyte proliferation and survival to preserve and propagate the viral genomes during latent infection. Mammalian cell proliferation requires successful transition through cell cycle phases (G 1 , S, G 2 , and M) (20,21). The expression of D-type cyclins (cyclins D1, D2, and D3) is predominantly regulated by extracellular mitogenic (or inhibitory) signals and are rate-limiting for transition through G 1 . Once cells have reached late G 1 , the E-type cyclins (E1 and E2) accumulate and allow S phase initiation, followed by induction of cyclin A in S phase. The D-type cyclins preferentially associated with and activate CDK-4 and -6, whereas cyclin E/A with CDK2. The primary physiological substrate for the CDKs is the retinoblastoma (pRb) family of pocket proteins (pRb, p107, and p130). When hypophosphorylated, the pocket proteins bind to and inactivate the E2F family of transcription factors that are required for the transcription of genes that are necessary for entry into S-phase. The activities of the CDKs are negatively regulated by two families of CDK inhibitors, namely the Cip/ Kip family (comprising p21 Cip1 , p27 Kip1 , and p57 Kip2 ) and the INK4 family. The Cip/Kip family inhibits a broad range of cyclin-CDK complexes, whereas the INK4 family members specifically inhibit cyclin D-CDK4/6 (21). The HHV-8 v-cyclin D has been shown to be refractory to the inhibitory effects of the Cip/Kip family of CKIs, and has been shown to increase the rates of proliferation of the infected cells, thereby propagating the viral genome (22). Another mechanism whereby the viral genome is propagated during latency is via inhibition of apoptosis.
The Bcl-2 family of proteins is central to regulating the commitment of cells to apoptosis (programmed cell death) (23,24). The Bcl-2 family of proteins contains both anti-and pro-apoptotic members and can be divided into three distinct classes: the anti-apoptotic sub-group (e.g. Bcl-2 and Bcl-XL); the pro-apoptotic, multi-Bcl-2 homology (BH) domain sub-group (e.g. Bak and Bax); and the pro-apoptotic, BH3 domain-only sub-group (e.g. Bad, Bik, Bid, Bim, PUMA, and Bmf). The pro-apoptotic BH3-only member of the Bcl-2 family, Bim, plays a crucial role in regulating apoptosis in lymphocytes during negative selection. Bim, like other BH3-only Bcl-2 proteins, mediates apoptosis through inducing mitochondrial cytochrome c release, which in turn activates caspase-9, caspase-3, and eventually the downstream cell death machinery (23,25). Bim is believed to promote apoptosis by neutralizing pro-survival Bcl-2 proteins in a mechanism that requires members of the multi-BH-domain pro-apoptotic subgroup.
The murine ␥-herpesvirus 68 (MHV-68) is closely related to the human HHV-8 and EBV. Because ␥-herpesviruses often share similar mechanisms to manipulate the host cellular signaling cascades, MHV-68 is often used as a rodent model of ␥-herpesviral infection. M2 is a unique latency-associated protein of MHV-68. It has no known cellular or viral homologues and does not contain any previously defined conserved domains. This protein is expressed during both the establishment and long term maintenance of latency, indicating that it is likely to play a key role in viral persistence (26,27). To study the functional role of this viral protein we sought to identify cellular targets of M2. Yeast-two-hybrid screening of a cDNA library derived from MHV-68 latently infected B lymphocytes demonstrated M2 interaction with both the Vav1 and Vav2 guanine nucleotide exchange factors. In immature B-cell line WEHI-231, the interaction between M2 and Vav promotes cell proliferation and survival.

Construction of a Splenic B Cell cDNA Library from Latently Infected BALB/c Mice
Splenic B220 ϩ cells from three MHV-68 latently infected mice were purified at 15, 18, and 21 days post infection by the MACS Streptavidin kit (Miltenyl Biotec, Surrey, UK). The percentage of B220ϩ cells over total cell number determined by fluorescence-activated cell sorting analysis was of 95%. mRNA was extracted by the mRNA Fast Track 2.0 mRNA isolation system (Invitrogen), and the cDNA was synthesized by the TimeSaver cDNA Synthesis kit (Amersham Biosciences). cDNAs were ligated to the oligonucleotide adaptors 5Ј-pCCG GTG CTT CCG G-3Ј and 5Ј-pCGA GGC CAC GAA GGC C-3Ј, which allowed the insertion into the XhoI site of ACT2 vector (Clontech, Basingstoke, UK). Clones containing the cDNA molecules were released from ACT2 by cre-lox recombination in BNN132 Escherichia coli cells. A cDNA library containing 2 ϫ 10 5 independent clones with an average insert size of 0.9 kb was obtained.

Plasmids
Yeast Two-hybrid System Plasmids-The following genes were PCRamplified from the MHV-68 genome and cloned into the MCS of pGBT9 ( tech) MCS by digestion with the same restriction enzymes used for cloning into pCMV-Myc, and these plasmids were designated pCMV-HA-M2, pCMV-HA-M2⌬P1, and pCMV-HA-M2⌬P2, respectively. vav2 gene was subcloned from pACT2-Vav2 into pCMV-HA and pCMV-Myc MCS, in-frame with the HA and Myc tags, into the EcoRI sites, and these plasmids were designated by pCMV-HA-Vav2 and pCMV-Myc-Vav2, respectively.

Yeast Two-hybrid System
All experiments were performed in the yeast reporter strain Y190. The yeast vector pGBT9 is a Gal4 DNA-binding domain (Gal4BD) encoding vector that allows fusion of desired protein to the Gal4BD. The yeast vector pACT2 is a Gal4 activation domain (Gal4AD) encoding vector that allows fusion of desired protein to the Gal4AD. The yeast two-hybrid library screen and protocols were as recommended (Clontech).

Immunoprecipitation and Immunoblotting
For each immunoprecipitation, Cells were washed in phosphatebuffered saline and lysed with 1 ml Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris, pH 7.4, 10 mM sodium molybdate, 1 mM sodium orthovanadate, 5 mM iodoacetamide, 1 mM sodium fluoride, and Complete Mini protease inhibitors (Roche Applied Science)) for 15 min on ice. 293T cell lysates (500 g) were pre-cleared for 1 h with protein G-Sepharose, then incubated with the antibody of interest for 1 h, and then with protein G-Sepharose for 1 h. Beads were washed four times with 1 ml of lysis buffer and resuspended in 50 l of 2ϫ SDS-PAGE loading buffer. Immunoprecipitates were then Western blotted as before.

Cell Cycle Analysis
Cell cycle analyses were performed by propidium iodide staining as described previously (29). Briefly, cells were washed with phosphatebuffered saline and fixed in 90% ethanol, 10% phosphate-buffered saline. Following fixation, cells were washed again and then incubated with 500 g/ml DNase-free RNase A (Sigma) and 20 g/ml propidium iodide (Sigma) for 30 min at 37°C prior to analysis using a FACScan flow cytometer (BD Biosciences).

M2 Interacts with Vav1 and Vav2
Guanine Nucleotide Exchange Factors in the Yeast Two-hybrid Screens-M2 is a unique viral protein with no known mammalian or viral domain homology. To determine its function and cellular targets, we performed a yeast two-hybrid screen using M2 as bait. Full-length M2 protein was fused in-frame with the DNA binding domain of the GAL4 (GAL4-BD) yeast transcription fac-

M2-Vav Inhibits Cell Cycle Arrest and Apoptosis
tor and used as a bait to screen a cDNA library derived from MHV-68 latently infected mouse splenic B-lymphocytes fused with the activation domain of GAL4 (GAL4-AD) (TABLE ONE). A total of 8.7 ϫ 10 7 transformants was analyzed in two independent rounds of screening. Of the seven positive clones selected for sequence analysis, four were found to contain the C-terminal region of Vav1 (amino acids 532-845), encoding for a fragment of the zinc finger (ZF) region and the entire SH3-SH2-SH3 domains, following comparison with the nucleotide data base at the National Library of Medicine using the BLAST algorithm (TABLE  ONE). The other three clones did not match any of the published cDNAs, and their identities remained undefined. The Vav family of guanine nucleotide exchange factors is composed of three highly homologous proteins, namely Vav1, -2, and -3 (31). Additional interaction experiments using the yeast two-hybrid system showed that M2 is also able to bind to the C-terminal region of Vav2 (amino acids 536 -868) encoding for the SH3-SH2-SH3 domains but not to Vav3.
The PXXP Motifs Located at the C Terminus of the Viral Protein Are Important for M2-Vav Interaction-After confirming interaction of Vav1 and -2 with M2, we examined the amino acid sequence of M2 required for binding to Vav. We observed that M2 protein contains 9 PXXP motifs, where P represents a proline residue and X any amino acid. These loosely defined motifs have been proposed to have a role in binding to signaling molecules through their SH3 domains (32). Given that the C terminus region of Vav proteins contain two SH3 domains, it is possible these moieties are mediating the M2 binding. To determine if these PXXP motifs were important for binding to Vav, we constructed several M2 deletion mutants lacking specific PXXP motifs. These mutants were cloned in-frame with the binding domain of GAL4 and used in a yeast two-hybrid experiment to study for their interactions with Vav1 and Vav2 SH3-SH2-SH3 regions. These results showed that amino acids 160 -179 of M2 are essential for Vav1 and Vav2 SH3-SH2-SH3 binding (Fig. 1). This region of M2 contains three PXXP motifs, at amino acid residues 162-165, 167-170, and 174 -176. To further investigate the importance of these motifs in Vav binding, we generated two M2 mutants in which the proline residues at positions 167 and 170 (M2⌬P1 mutant) and 165, 167, 170, and 174 (M2⌬P2 mutant) were replaced by alanine residues. As shown in Fig. 1, the M2⌬P1 and M2⌬P2 mutations partially or totally abrogated the binding of M2 to Vav1 and Vav2, respectively. These results demonstrated that the three PXXP motifs located at the C terminus of M2 are essential for binding to the SH3-SH2-SH3 region of both Vav1 and Vav2 proteins.

M2 Interacts with Vav1 and Vav2
Proteins in Vivo-We next studied the in vivo interaction between M2 and Vav proteins in mammalian cells by co-immunoprecipitation (Fig. 2, A and B). To this end, 293T cells were transiently transfected with Vav mammalian expression vectors either alone or in combination with M2, M2⌬P1, or M2⌬P2. 48 h after transfection, cell lysates were immunoprecipitated with either an anti-M2 serum or with the pre-immune serum. The immunoprecipitates were then resolved by SDS-PAGE and immunoblotted with anti-Vav antibodies. As shown in Fig. 2 (A and B), M2 co-immunoprecipitated with both Vav1 and Vav2 proteins in vivo, respectively. These results also showed that the binding of M2⌬P1 and M2⌬P2 to Vav proteins was partially abrogated, consistent with previous results obtained using the yeast two-hybrid system. Nevertheless, the yeast two-hybrid experiments have shown complete abrogation of binding of M2⌬P2 to Vav1 and Vav2 SH3-SH2-SH3 domains. One possible explanation for the co-immunoprecipitation results is that the full-length Vav protein might bind to another domain in M2. Another explanation could be that, although direct interaction between Vav proteins and M2 has been abrogated in the M2⌬P2 mutant, Vav is able to interact with other proteins (e.g. endogenous Vav proteins) that reside in the complex with M2. Accounting for this second hypothesis is the fact that M2 has 9 PXXP motifs that can potentially interact with other signaling proteins.
Because B-lymphocytes constitute the main reservoir of MHV-68 during long term latency, we reasoned that it would be important to study M2 interaction with Vav proteins in B-cells. To this end, we sought to generate WEHI-231 and WEHI-bcl-XL cell lines expressing M2 protein (29). WEHI-231 and WEHI-bcl-XL were infected with

Screening of an MHV-68 latently infected B-cell cDNA library with the latency-associated M2 protein
Yeast cells (Y190) sequentially transformed with M2 in-frame with Gal4BD in pGBT9 (pGBT9-M2) and a spleen B-cell cDNA library in pACT2 (pACT2-cDNA/B220ϩ) were selected on ϪTrp, ϪLeu, ϪHis medium containing 50 mM 3-AT. Colonies growing in the selection medium were further selected by the expression of ␤-galactosidase by the filter lift assay as described by the manufacturer (Clontech). Clones were sequenced by a 377 DNA sequencer (ABI Prism).

M2-Vav Inhibits Cell Cycle Arrest and Apoptosis
We next performed co-immunoprecipitation assays to examine M2-Vav interaction in B-cells. Because binding of M2 to Vav proteins in B-lymphocytes could be dependent on B-cell receptor activation, cells were either unstimulated or stimulated with anti-IgM for 10 min before harvesting. Because the WEHI-231 cells were infected with an M2 containing a Myc tag, the B-cell lysates were immunoprecipitated using an anti-Myc antibody, resolved by SDS-PAGE, and immunoblotted using anti-Vav antibodies. The results showed that M2 interacts with Vav2 in B-lymphocytes (Fig. 3B). However, we failed to show interaction between M2 and Vav1 (data not shown). This might be explained by the fact that Vav2 is the predominant Vav family member in B-lymphocytes and plays a main role in cell proliferation, survival, and development, whereas Vav1 is more important for T-lymphocyte signaling. That M2 would at least preferentially interact with Vav2 in B-lymphocytes is supported by the observation that these cells are long term reservoirs for MHV-68 viruses during latent infection. We further observed that B-cell receptor activation did not have any effect on M2-Vav2 co-immunoprecipitation, indicating that this interaction is not dependent upon B-lymphocyte activation.
M2 Promotes Vav Activation in B Cells-Because M2 interacts with Vav1 and -2 proteins it is therefore possible that M2 can modulate Vav activity. To investigate this possibility, we examined Vav activity using a GST-PAK pull-down assay. Rac is a Vav substrate and can only bind to its own substrate, PAK, when converted by Vav into its active GTPassociated form. Thus, quantification of the amount of Rac pulled down by GST-PAK would allow us to indirectly gauge the activity of Vav. Equivalent amounts of WEHI-231, WEHI-231-M2, WEHI-231-M2⌬P1, and WEHI-231-M2⌬P2 cell lysates were incubated with GST-PAK and pulled down using glutathione-agarose beads. The results showed that expression of M2 leads to an ϳ2-fold increase in the amount of activated Rac compared with WEHI-231 cells not expressing M2. This induction of Rac activity was not detected in WEHI-231 cells expressing M2⌬P1 or M2⌬P2 mutants, indicating that interaction between M2 and Vav is essential for the observed increase in Rac activity (Fig. 4A).
Because Vav proteins are the only known guanine nucleotide exchange factors regulated by tyrosine kinases, we next studied if inhibition of tyrosine kinase activity would interfere with the M2-induced up-regulation of Rac activity. To this end, we incubated WEHI-231 and WEHI-231-M2 cells with 0, 100, 200, and 400 M genistein, a known tyrosine kinase inhibitor, prior to performing the GST-PAK pull-down. The results showed that increasing amounts of genistein caused reduction in the amount of activated Rac in WEHI-231-M2 cells (Fig. 4B), indicating further that Vav proteins are responsible for the increase in the amount of activated Rac observed in M2-expressing cells.
To further confirm that M2 enhances Vav activity, we prepared cell lysates from WEHI-231 cells transfected with M2 or the control empty expression vector, and probed them with a phospho-Vav (Tyr-174) antibody, which recognizes Vav in its activated form. Because Vav activity is modulated by B-cell receptor-mediated signaling, the WEHI-231   (Fig. 4C). Because Vav activates phosphatidylinositol 3-kinase/Akt activity in B-cells, we also examined the activity of Akt using an active phosphorylation-specific antibody. Consistent with the earlier results, we also detected higher levels of Akt phosphorylation in the WEHI-231 cells expressing M2.

M2 Inhibits Apoptosis/Cell Cycle Arrest of Immature WEHI-231 B-lymphocytes, Induced by B-cell Receptor Activation-We have previously demonstrated that Vav proteins play an important role in medi-
ating the B-cell receptor-induced signaling cascade, which is essential for B-cell proliferation and survival (39,41). To further delineate the molecular mechanisms of M2 we investigated the modulation of cell cycle arrest and apoptosis in the murine immature WEHI-231 B-cells activated stimulation of B-cell receptor with anti-IgM. To study the effects of M2 on B-cell receptor-induced cell cycle arrest independently of apoptosis, a WEHI-231 cell line overexpressing bcl-XL (WEHI-bcl-XL), which arrests at G 0 /G 1 following B-cell receptor activation but do not undergo apoptosis was also used (29). WEHI-231, WEHI-231-M2, WEHI-bcl-XL, and WEHI-bcl-XL-M2 cells were cultured with a monoclonal antibody directed against anti-IgM and collected at 0, 6, 12, 24, 48, and 72 h after treatment. The cell cycle distribution profiles of propidium iodide-stained cells were measured by flow cytometry. The results showed that M2 expression inhibits G 0 /G 1 cell cycle arrest and apoptosis induced by B-cell receptor-stimulation (Fig. 5A). It was evident that the number of apoptotic WEHI-231 cells at 24 and 48 h after anti-IgM treatment was approximately twice that of the M2-expressing WEHI-231 cells. The fraction of WEHI-231-M2 and WEHI-bcl-XL-M2 cells going through the S and G 2 /M phase at 24 and 48 h after anti-IgM treatment was also approximately three and two times higher when compared with WEHI-231 and WEHI-bcl-XL cells, respectively.
To confirm the cell cycle results, we performed an analysis of cell number of the four WEHI-231 cell lines in the presence or absence of anti-IgM (Fig. 5B). The result showed that anti-IgM treatment caused a decrease in cell number of the WEHI-231 over the time course of 72 h. The anti-IgM treated WEHI-231 expressing M2 continued to increase in number, albeit at a slower rate compared with the untreated cells. In the presence of anti-IgM, the number of WEHI-bcl-XL cells remained constant for 48 h, whereas the WEHI-bcl-XL cells expressing M2 continued to proliferate throughout the time course. The number of WEHI-bcl-XL cells increased marginally at 72 h, and this probably reflected the fact that the anti-IgM became inactive over time. These results confirmed the earlier cell cycle analysis results as well as the pro-survival and pro-proliferative effects of M2.
To establish the molecular mechanisms whereby M2 controls cell proliferation and survival, we investigated the expression of proteins important in B-cell proliferation and survival in WEHI-231 and WEHIbcl-XL with or without M2 following anti-IgM treatment (Fig. 6). Due to high levels of apoptosis in the WEHI-231 and WEHI-231-M2 cells after anti-IgM treatment, their protein expression was only followed up to 24 h. Consistent with previous results (29), we observed that anti-IgM treatment up-regulated p130 and the CKI p27 Kip1 and down-regulated cyclin D2 expression in both WEHI-231 and WEHI-bcl-XL cells. Upon anti-IgM stimulation, down-regulation of cyclin D2 expression was detectable as early as 6 h in both WEHI-231 and WEHI-231-M2 cells. However, the level of cyclin D2 continued to decline after 6 h of anti-IgM treatment in WEHI-231 cells, whereas further cyclin D2 downregulation was not observed in the WEHI-231 cells expressing M2. The use of an antibody specific for the CDK4/6 phosphorylation site of pRB, showed a down-regulation of pRB-directed CDK4/6 activity 24 h after anti-IgM in WEHI-231 but not in the WEHI-231-M2 cells. In WEHI-231 cells, the pocket protein p130 and the CKI p27 Kip1 were both induced at 24 h after anti-IgM, concomitant with cycle arrest. In contrast, no induction of p130 and p27 Kip1 were detected in the WEHI-231 cells expressing M2. The results also showed that there was no appreciable difference in the expression of pRB and p107, cyclin A and -E, and CDK2, -4, and -6 between WEHI-231 and WEHI-231-M2 cells.
In WEHI-bcl-XL cells, the expression of cyclin D2 was down-regulated at 6 h after anti-IgM treatment, reaching a nadir at 24 h, before recovering at 48 and 72 h. In comparison, the level of cyclin D2 remained constant throughout the time course in the WEHI-bcl-XL expressing M2. Similarly, induction of p27 Kip1 and p130 expression was detected at 24 h in WEHI-bcl-XL but not in WEHI-bcl-XL-M2 cells. In WEHI-bcl-XL, the initial down-regulation of cyclin D2 expression correlated with a decline of CDK4/6-dependent pRB phosphorylation, but no recovery of CDK4/6 kinase activity was detected at 48 and 72 h. This is probably due to the accumulation of the CKI p27 Kip1 at 48 and 72 h after anti-IgM in these cells. In the WEHI-bcl-XL cells expressing M2, the CDK4/6 kinase activity declined after anti-IgM treatment, but remained at a higher level compared with WEHI-bcl-XL cells. It was notable that the expression of cyclin A was also down-regulated at 48 and 72 h following anti-IgM treatment in the WEHI-bcl-XL but not in the WEHI-bcl-XL-M2 cells. This down-regulation of cyclin A expression is likely to be the consequence of the down-regulation of cyclin D2 and induction of p130 and p27 Kip1 expressions. The expression levels and patterns of other cell cycle regulators, including p107, cyclin E, CDK2, -4, -6, and E2F1 were comparable between the two lines, WEHIbcl-XL and WEHI-bcl-XL-M2.
To study the potential mechanism employed by M2 to inhibit apoptosis, we also examined the expression patterns of the Bcl-2 family of apoptosis regulators. We observed that anti-IgM treatment caused the down-regulation of the anti-apoptotic Bcl-2 protein Bcl-XL in WEHI-231 cells, thereby confirming previous results. We also found that the expression levels of the BH-3 only protein Bim was lower in B-cells ectopically expressing M2 than controls, suggesting that increases in the ratio of Bcl-XL to Bim caused by M2 render cells less susceptible to anti-IgM-induced apoptosis.

DISCUSSION
The MHV-68 viral protein, M2, is expressed during the establishment and long term maintenance of viral latency, indicating that it plays a vital role in viral persistence (26,33). Infection experiments using an M2-deficient MHV-68 virus in mouse models have shown that M2 is essential for the establishment and maintenance of latent infection in vivo (34). The initial establishment of MHV-68 latency involves a transient expansion of germinal center GC B-cells in the spleen, whereas the long term latency is maintained primarily in memory B-cells as well as germinal center B-cells (27). It is believed that like other ␥-herpesvi- M2 is a unique latency-associated protein of MHV-68, with no known cellular or viral homologues, or conserved domains. Little information is available on its mode of action in the establishment of latent infection by MHV-68. To delineate the role played by M2 during MHV-68 latent infection and identify the precise molecular mechanisms 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 screens. Subsequent yeast two-hybrid interaction studies showed that M2 binds to Vav1 and Vav2, but not Vav3, and that the proline residues 165, 167, 170, and 174 located at the C terminus of M2 are important for this interaction. These proline residues are integrated into "PXXP" motifs, which are known to bind to SH3 domains present at the C-terminal region of both Vav proteins (32,36,37). These interactions between M2 and Vav proteins were also confirmed in vivo in 293T and WEHI-231 cells by co-immunoprecipitation assays.
The Vav family of proteins functions as guanine-nucleotide exchange factors for the Rho GTPases, Rac1, Rac2, and RhoG, and these proteins are crucial components of the B-cell antigen receptor signal transduction machinery (31). Vav is rapidly phosphorylated following stimulation of the antigen receptors in B lymphocytes and integrates signals from the B-cell receptor and CD19 co-receptor with downstream signaling pathways (31). Analyses of Vav-deficient mice have demonstrated a key role for Vav in B-lymphocyte development and proliferation. In Vav1 and Vav2 double knock-out mice, the immature B-cells are present in normal numbers within the bone marrow, but fail to develop efficiently into mature B-cells. There is an 80% decrease in the number of splenic B-cells in Vav1/Vav2 knock-out mice, as compared with wild-type mice. Moreover, Vav1 and -2 null B-cells also fail to proliferate in response to B-cell receptor stimulation (38).
Because B-lymphocytes are the main reservoir for MHV-68 during its latent infection, we investigated the functional significance of M2-Vav interaction using the murine B-cell line WEHI-231, a commonly used cell model for studying B-cell receptor-mediated signal transduction, cell cycle arrest, and apoptosis (29). Using WEHI-231 cells, we found that M2 expression augments Vav activity. GST-PAK pull-down experiments showed that expression of M2 protein leads to increased activity of Vav substrate, Rac, in WEHI-231 B-cells. This increase in Rac activity is not observed in cells expressing M2 mutants, M2⌬P1 or M2⌬P2 that are compromised in Vav-binding, indicating that the M2-Vav interaction is essential for the induction of Vav activity. This result was further confirmed using a phospho-specific antibody, which specifically recognizes the activated form of Vav proteins. Studies of Vav1-and -2-deficient B-cells showed that phosphatidylinositol 3-kinase/Akt signaling pathway is defective (39), indicating that Vav proteins integrate B-cell receptor signals with the phosphatidylinositol 3-kinase/Akt signaling pathway. Consistent with this, we also detected a comparatively higher basal Akt activity (as revealed by the phospho-Akt antibody) in M2-expressing B-cells. The observation that the increase in Vav/Rac1 activity by M2 can be blocked by the addition of the tyrosine kinase inhibitor, genistein, suggests that tyrosine kinase activity is involved. Because M2 has no obvious tyrosine kinase catalytic domain, it is most likely that this viral protein enhances Vav activity through bringing Vav into close proximity with cellular tyrosine kinases. The fact that M2 has nine PXXP motifs, which are known to have a main role in recruiting proteins in signaling cascades through its interaction with SH3 domains (32), further supports this hypothesis.
For successful colonization of the host, ␥-herpesviruses have to amplify their viral genomes in latently infected host B lymphocytes, while at the same time evade the immune surveillance of the host. The proliferation and survival of the latently infected lymphocytes requires the expression of latent viral antigens. We and others have previously shown that Vav protein is a component of the B-cell receptor-mediated signaling cascade, which is important for proliferation, survival, and development of B-lymphocytes. Our present data show that ectopic expression of M2 in WEHI-231 cells can overcome the B-cell receptormediated cell cycle arrest and apoptosis, suggesting that the latent protein M2 can promote B-cell proliferation and survival through the upregulation of Vav activity. Similar strategies have been employed by other ␥-herpesviruses to manipulate B-cell signaling events to induce cell activation, proliferation, survival, and differentiation. For example, the LMP-2A protein of EBV imitates a constitutively activated B-cell receptor (7,8), whereas another oncogenic EBV protein LMP-1 mimics a deregulated CD40 receptor, which is a co-receptor of the B-cell receptor (5,6,40). Although EBV manipulates the B-cell-signaling events at receptor levels via LMP-1 and LMP-2A, MHV-68 influences the signaling molecule downstream of the B-cell receptor via M2, which functions as an activator of Vav. Our previous data shows that Vav has an important role in setting the threshold for antigen receptor-mediated activation of B lymphocytes, which is likely due to its regulation of receptor clustering (41). M2 expression is likely to modulate this threshold to promote cell proliferation, survival, and development through enhancing Vav activity. Consistent with this idea is the observation that the expression levels of the pro-apoptotic Bcl-2 family protein Bim and the cell cycle inhibitors p27 Kip1 and p130 are lower in the B-cells expressing M2 compared with their non-M2-expressing counterparts (42). Moreover, cyclin D2 is also expressed at higher and more sustainable levels in WEHI-231 cells expressing M2 following anti-IgM treatment (42). The  NOVEMBER 11, 2005 • VOLUME 280 • NUMBER 45 induction of cyclin D2 expression by M2 could have a similar functional significance as the expression of the cyclin D-related v-cyclin by HHV-8 during latent infection (12,13). We have shown previously that cyclin D2 is critical for B-cell receptor-mediated proliferation using B-cells derived from cyclin D2-knock-out mice (43,44). Similarly, the molecular basis for the proliferative defect observed in vav Ϫ/Ϫ B-cells is their failure to express cyclin D2 (41). These results taken together with our current observations suggest that M2 targets cell cycle regulators, such as cyclin D2 through augmenting Vav activity to promote B-cell proliferation during latent infection. Further supporting this idea, Vav proteins have also been shown recently to have a role in promoting B-cell survival (45). However, constitutively activated B lymphocytes, which are constantly proliferating and have an extended-life span, are predisposed to acquiring mutations in proto-oncogenes or tumor suppressor genes that could result in the development of lymphoproliferative diseases, including lymphoma and leukemia. Indeed, latent EBV infection has been linked with a number of human malignancies, including Burkitt lymphoma, Hodgkin disease, and lymphoproliferative disease (46), whereas 10% of the MHV-68 latently infected mice develop lymphoproliferative disease identified as lymphomas of B-cell origin (47).

M2-Vav Inhibits Cell Cycle Arrest and Apoptosis
In summary, this study has identified the Vav proteins as the cellular targets of the MHV-68 ␥-herpesvirus latency-associated M2 protein.
Our data also show that binding of M2 to Vav proteins promotes Vav activity as well as B-cell activation, proliferation, and survival. This functional interaction between M2 and Vav could have a key role in the establishment and maintenance of viral genomes during MHV-68 ␥-herpesvirus latent infection.