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Among the distinguishing characteristics of members of the gamma-2 herpesvirus family is the expression of a mammalian D-type cyclin homolog, termed v-cyclin. Murine gammaherpesvirus 68 (gammaHV68) is a gamma2-herpesvirus that can infect inbred and outbred strains of mice, providing a genetic system for the study of gammaherpesvirus pathogenesis. Disruption of the v-cyclin gene of gammaHV68 results in a virus that establishes latency in infected mice to wild-type levels, but is severely attenuated for virus reactivation [van Dyk, L.F., Virgin IV, H.W., Speck, S.H., 2000. J. Virol. 74:7451-7461]. Transcriptional regulation of the gammaHV68 v-cyclin has not been defined. We report here the initial characterization of the v-cyclin transcript expressed in permissive murine fibroblasts. Based on 5' mapping of the v-cyclin transcript, we identified a promoter that is involved in driving v-cyclin expression during virus replication. In addition, we determined that the promoter is responsive to the major viral lytic transactivator, Rta, encoded by orf 50. Using reporter plasmids we have analyzed both basal and Rta-induced v-cyclin promoter activity, initially identifying two regions of the v-cyclin promoter important for both basal and Rta-induced activity. Notably, only one of these regions could be shown to confer Rta responsiveness on a reporter construct containing the hsp70 TATA box. The importance of this region in regulating v-cyclin expression during virus replication was confirmed by introducing these mutations into the context of the viral genome and assessing v-cyclin expression following infection of permissive murine fibroblasts in tissue culture. In addition, we show that mutations that severely cripple Rta-induction of v-cyclin expression did not adversely impact virus reactivation from splenocytes recovered from latently infected mice, indicating that alternatively regulated v-cyclin gene expression is required for virus reactivation.

Sequence analysis of the murine gamma-herpesvirus 68 (gammaHV68) genome previously identified several open reading frames (ORFs) located at the left end of the viral genome that do not share homology with other known herpesvirus or cellular genes. Here, we show that one of these ORFs, M4, encodes a secreted glycoprotein that influences the establishment of splenic latency at early times post-infection. We generated a mutant virus containing a premature translation termination codon in the M4 ORF (M4.STOP), and demonstrated that this mutant virus replicates in vitro equivalent to wild type and marker rescue (M4.MR) viruses. M4.STOP was also capable of high-titer lytic replication in vivo, but at 16 days post-infection the establishment of latency in the spleen was significantly impaired. The defect in the establishment of splenic latency was apparent following either intranasal or intraperitoneal inoculation. In contrast, the M4.STOP mutant did not exhibit a defect in the establishment of latency in peritoneal cells. These results suggest that M4 mediates an extracellular host-pathogen interaction that impacts the establishment of latent infection in the spleen, but not the peritoneum.

All known gamma2-herpesviruses encode a cyclin homolog with significant homology to mammalian D-type cyclins. The murine gammaherpesvirus 68 (gammaHV68) viral cyclin (v-cyclin) has been shown to be oncogenic when expression is targeted to thymocytes in transgenic mice and to be critical for virus reactivation from latency. Here, we investigate the interaction of the gammaHV68 v-cyclin with cellular cyclin-dependent kinases (cdks). We show that, in contrast to the Kaposi's sarcoma-associated herpesvirus (KSHV) v-cyclin, the gammaHV68 v-cyclin preferentially interacts with cdk2 and cdc2 but does not interact with either cdk4 or cdk6. Mutation of conserved residues, predicted to be involved in cdk binding based on the gammaHV68 v-cyclin:cdk2 crystal structure, resulted in the loss of both cdk binding and the ability to mediate phosphorylation of substrates. Like the KSHV v-cyclin, the gammaHV68 v-cyclin appears to confer expanded substrate specificity to the cellular cdk binding partners. As expected, the gammaHV68 v-cyclin:cdk complexes are able to target phosphorylation of histone H1, the retinoblastoma protein (pRb), and p27(Kip1) as assessed using in vitro kinase assays. Notably, hyperphosphorylation of pRb was observed during wt gammaHV68 replication in serum-starved murine fibroblasts, but not in cells that were either mock-infected or infected with a v-cyclin null gammaHV68. In addition, infection of serum-starved murine fibroblasts also results in a v-cyclin-dependent increase in cdk2-associated kinase activity and a concomitant decrease in the levels of p27(Kip1). Taken together, the latter studies served to validate the results of the in vitro kinase assays. Finally, in vitro kinase assays revealed that the gammaHV68 v-cyclin:cdk complexes can also phosphorylate p21(Cip1), Bcl-2, and p53. The latter suggests that, at least in vitro, the gammaHV68 v-cyclin exhibits functional characteristics of both cyclin E and cyclin A.

The Rta homolog encoded by murine gammaherpesvirus 68 (gammaHV68) gene 50 is essential for virus replication and is capable of driving virus reactivation from the S11 latently infected B lymphoma cell line. Here we characterize Rta activation of gammaHV68 gene 57, which is abundantly transcribed during the early phase of virus replication. Infection of murine fibroblasts with an Rta null virus demonstrated that transcription of gene 57 is dependent on Rta expression. Analysis of the gene 57 promoter identified 2 distinct regions that are Rta responsive, either in the context of the gene 57 promoter or when cloned upstream of a heterologous promoter. Sequence analysis of these regions revealed homology to known Rta-responsive cis-elements in the closely related Kaposi's sarcoma-associated viral (KSHV) genome. In addition, two candidate binding sites for the cellular transcription factor RBP-Jkappa/CBF1 were also identified in one of the Rta-responsive regions, which may play a role in mediating Rta transactivation similar to that observed in some KSHV Rta-responsive genes. Overall, analysis of the gammaHV68 gene 57 promoter suggests that mechanisms of Rta activation are conserved among gamma2-herpesviruses.

Expression of two Epstein-Barr virus (EBV) immediate-early gene products, Zta (encoded by the BZLF1 gene) and Rta (encoded by the BRLF1 gene), are required for the switch from latent infection to virus replication. We have analyzed the regions of the BRLF1 gene promoter (Rp) that are required for Rta and Zta transactivation of Rp. Notably, significant synergy between the actions of Rta and Zta on Rp was observed in both a B cell line (DG75) and an epithelial cell line (293), suggesting that during induction of the viral lytic cycle low levels of these viral transactivators are likely sufficient to initiate the entire lytic cascade. However, while two Zta binding sites (ZREs) have been identified in Rp, the proximal ZRE was the dominant site for mediating Zta transactivation. Rta activation of Rp was diminished by mutation of the proximal Sp1 binding site, as previously reported (J. Virol. 75 (2001), 5240), but mutation of this site only had a modest impact on transactivation of Rp by Rta in the presence of Zta. Further deletion analyses of Rp failed to identify a critical site for Rta transactivation of Rp in the presence of Zta, with the exception of deleting the TATAA box of Rp, suggesting that a non-DNA binding mechanism may be involved in the observed activation of Rp by Rta. We also observed promiscuous activation of several reporter constructs by Rta, suggesting that Rta activation of gene expression may involve a general non-DNA binding mechanism. Decreasing the amount of transfected Rta expression vector reduced background Rta activation, while retaining specific activation of Rp.

Murine gammaherpesvirus 68 (gammaHV68; MHV68) infection of mice has been a useful model for characterizing the role of conserved herpesvirus genes in pathogenesis. One of the well conserved genes among gamma2-herpesvirus, gene 74, encodes a viral G-protein coupled receptor (v-GPCR). To examine the role of the gammaHV68 v-GPCR in pathogenesis we have generated a mutant virus in which 440 base pairs of the gene 74 open reading frame have been deleted (gammaHV68v-GPCRDelta440). This deletion did not affect the growth of the virus in single or multiple rounds of replication in vitro, nor acute replication in vivo as assessed by plaque assay of spleens and lungs on days 4, 7 and 9 post-infection (p.i.). The ability of the v-GPCR mutant virus to establish latency and to reactivate from latency was quantitated on days 16 and 42 p.i. While there was no detectable difference in the ability of the mutant virus to either establish latency or reactivate from latency on day 16 p.i., as compared to wild-type gammaHV68 and marker rescue virus, there was a significant decrease in the efficiency of virus reactivation by day 42 p.i. Notably, mice infected with the mutant virus lacking the v-GPCR contained a higher frequency of viral genome positive cells in the peritoneum by day 42 p.i. than mice infected with either wild type or marker rescue virus. However, analysis of virus reactivation demonstrated that approximately the same frequency of cells reactivated virus from mice infected with either the gammaHV68 v-GPCR mutant, wild-type virus, or marker rescue virus. From these experiments we conclude that the gammaHV68 v-GPCR is dispensable for acute virus replication in vivo, but does play a role in reactivation from latency.

Epstein-Barr virus (EBV) encodes multiple latency programs: a growth-transforming program (type III) latency program and restricted-latency (types I and II) programs. During type III latency, EBV expresses six nuclear antigens, all of which are encoded by a single complex transcriptional unit driven by two linked promoters, Cp and Wp, while restricted viral latency is characterized by the expression of a single nuclear antigen, EBNA1, whose expression is driven from a distinct transcription unit under the control of the Qp promoter. EBV infection of the 293 epithelial cell line frequently leads to the establishment of a type I/II latent infection. Here we report that during the initial stages of virus infection of the 293 cell line, both Cp and Wp are active. However, analysis of four established, low-passage EBV-infected 293 cell lines revealed that three of these exhibited Qp-driven transcription of the EBNA 1 gene and little or no detectable Cp and Wp activity, while the fourth cell line exhibited Cp activity. Notably, all four cell lines contained the necessary transcription factors to drive transcription initiation from Cp and Wp when transiently transfected with unmethylated reporter constructs. Furthermore, in the cell lines exhibiting restricted EBV latency the viral genomes were extensively methylated around Cp and Wp, but not Qp. In contrast, in the cell line exhibiting Cp activity the viral genomes were hypomethylated around Cp, Wp, and Qp. Taken together, these results provide evidence that the establishment of a restricted latent infection in the 293 epithelial cell line is not due to a failure to initiate the growth-transforming (type III) latency program, but rather may arise from a selection against the type III latency program. Furthermore, these results are consistent with the hypothesis that methylation of Cp and Wp is required for entry into the type I or II latency programs.

Induction of the Epstein-Barr virus lytic cycle in latently infected B cells requires the expression of the immediate-early lytic gene BZLF1. We have previously identified several cis-elements within the BZLF1 promoter that are required for induction by known inducers of the lytic cycle [E. Flemington and S. H. Speck (1990)J. Virol. 64, 1217-1226]. These include four elements termed the ZI domains (ZIA, ZIB, ZIC, and ZID) that share extensive homology and that have recently been shown to bind several cellular transcription factors [A. M. Borras, J. L. Strominger, and S. H. Speck (1996) J. Virol. 70, 3894-3901]. Here Sp1 and Sp3 are identified as the cellular factors present in crude B cell nuclear extract preparations that bind to the ZIC domain. In addition, three of the four complexes observed in electrophoretic mobility shift analyses employing probes containing either the ZIA or the ZID domains also represent Sp1 or Sp3 binding. Binding of Sp1 and Sp3 to the ZI domains was shown to be significantly weaker than binding of these factors to a consensus Sp1 site. A heterologous promoter construct containing three repeats of a consensus Sp1 site, cloned upstream of a single copy of the ZII (CREB/ AP1) element from the BZLF1 promoter linked to the beta-globin TATA box, exhibited phorbol ester inducibility. The latter observation was consistent with the functional behavior exhibited by a heterologous promoter construct containing multiple copies of the ZIC domain liked to the ZII element. However, the basal activity of the heterologous promoter construct driven by the consensus Sp1 sites was ca. 10-fold higher than that of the heterologous reporter construct containing multimerized ZIC sites. Thus, the low affinity of Sp1 binding to the ZI domains may contribute to the low-level basal activity of the BZLF1 promoter.

The Epstein-Barr virus (EBV) BZLF1 gene is expressed early upon induction of the viral lytic cycle and its protein product is unique in its ability to disrupt viral latency in some latently infected cell lines. Anti-immunoglobulin (anti-Ig) treatment of the Burkitt's lymphoma cell line Akata, which bears surface IgG, has previously been shown to synchronously induce transcription of the BZLF1 gene (K. Takada and Y. Ono, 1989, J. Virol. 63, 445-449). We have previously shown that anti-Ig induction of Akata cells activates expression of the tumor necrosis factor alpha (TNF-alpha) gene via a calcineurin-dependent mechanism (Goldfeld et al., 1992, Proc. Natl. Acad. Sci. USA 89, 12198-12201). Here, we report that anti-Ig induction of the EBV lytic cycle in Akata cells can be blocked by the immunosuppressants cyclosporin A and FK506. Furthermore, we demonstrate that synergistic induction by phorbol ester and calcium ionophore of a BZLF1 promoter-driven reporter construct in an EBV-negative BL cell line can be inhibited by addition of cyclosporin A. Thus, analogous to activation of TNF-alpha gene in Akata cells, anti-Ig induction of the BZLF1 promoter is most likely mediated by calcineurin and probably involves translocation to the nucleus of a transcription factor sequestered in the cytoplasm. As such, immunosuppressants may be useful probes for dissecting B cell activation pathways involved in regulating EBV gene transcription.

Initiation of the Epstein-Barr virus (EBV) lytic cycle is dependent on the transcription of the BZLF1 gene. The BZLF1 gene promoter (Zp) was activated by crosslinking of cell surface immunoglobulin (Ig) with anti-Ig antibody in B cells, even in the absence of other viral genes. We identified several anti-Ig response elements within Zp, which were originally defined as 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements (ZI repeats and ZII, an AP-1-like domain). Since anti-Ig crosslinking leads to activation of protein kinase C (PKC) and an increase in intracellular calcium level, Zp was tested for the response to these cellular factors. Treatment with calcium ionophore A23187 increased Zp activity. When the calcium ionophore was used in conjunction with TPA, a PKC activator, the Zp induction was synergistically enhanced. 1-(5-Isoquinolinyl sulfonyl)-2-methylpiperazine, an inhibitor of PKC, inhibited the anti-Ig inducibility of Zp. Calmodulin antagonists, compound R24571 and trifluoperazine, blocked the Zp activation with anti-Ig. These findings suggest that Zp responds directly to changes in the activity of both PKC and calcium/calmodulin-dependent protein kinase. Requirement of tyrosine kinase activation for the anti-Ig-mediated Zp activation was also demonstrated through the use of the tyrosine kinase inhibitor herbimycin. These cellular gene regulatory molecules induced with anti-Ig may cooperatively play an important part in achieving efficient EBV activation as seen with anti-Ig treatment in B cells.