Selective switch between latency and lytic replication of Kaposi's sarcoma herpesvirus and Epstein-Barr virus in dually infected body cavity lymphoma cells.

The BC-1 cell line, derived from a body cavity-based, B-cell lymphoma, is dually infected with Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In these studies, the relationships between these two gammaherpesviruses and BC-1 cells were characterized and compared. Single-cell cloning experiments suggested that all BC-1 cells contain both genomes. In more than 98% of cells, both viruses were latent. The two viruses could be differentially induced into their lytic cycles by chemicals. EBV was activated into DNA replication and late-gene expression by the phorbol ester tetradecanoyl phorbol acetate (TPA). KSHV was induced into DNA replication and late-gene expression by n-butyrate. Amplification of both EBV and KSHV DNAs was inhibited by phosphonoacetic acid. Induction of the KSHV lytic cycle by n-butyrate was accompanied by the disappearance of host-cell beta-actin mRNA. Induction of EBV by TPA was not accompanied by such an effect on host-cell gene expression. Induction of the KSHV lytic cycle by n-butyrate was associated with the expression of several novel polypeptides. Recognition of one of these, p40, served as the basis of development of an assay for antibodies to KSHV in the sera of infected patients. BC-1 cells released infectious EBV; however, there was no evidence for the release of encapsidated KSHV genomes by BC-1 cells, even though n-butyrate-treated cells contained numerous intranuclear nucleocapsids. The differential inducibility of these two herpesviruses in the same cell line points to the importance of viral factors in the switch from latency to lytic cycle.     

The corepressor mSin3a interacts with the proline-rich domain of p53 and protects p53 from proteasome-mediated degradation

While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14(ARF), Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage.     

Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells.

The pattern of Epstein-Barr virus (EBV) RNAs expressed in Raji cells superinfected with P3HR1 EBV was examined. RNAs whose expression was of an immediate-early type (resistant to treatment of the cells with anisomycin) were identified. These RNAs, encoding the EBV reading frames BZLF1 and BRLF1, were probably expressed from defective virus within the P3HR1 preparation, and some of them were responsible for the induction of the EBV productive cycle in the Raji cells. The structures of the B95-8 RNAs equivalent to the anisomycin-resistant RNAs were determined. The RNA encoding the BZLF1 reading frame contained two splices which extended and modified the reading frame from that previously described.     

The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination

Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.     

Contribution of C/EBP proteins to Epstein-Barr virus lytic gene expression and replication in epithelial cells

The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPbeta and had limited C/EBPalpha expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPalpha and C/EBPbeta. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPbeta expression and a prolonged increase in C/EBPalpha expression. In AGS/BX1 cells, endogenous C/EBPalpha and C/EBPbeta proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPalpha and C/EBPbeta proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPbeta and C/EBPalpha expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.     

Human herpesvirus 8 viral interleukin-6 interacts with splice variant 2 of vitamin K epoxide reductase complex subunit 1

Viral interleukin-6 (vIL-6) specified by human herpesvirus 8 is, unlike its cellular counterpart, secreted very inefficiently and can signal via vIL-6(2):gp130(2) signaling complexes from the endoplasmic reticulum (ER) compartment. Intracellular, autocrine activities of vIL-6 are important for proproliferative and prosurvival activities of the viral cytokine in latently infected primary effusion lymphoma (PEL) cells. However, the molecular determinants of vIL-6 ER localization and function are unclear. Using yeast two-hybrid analysis, we identified the database-documented but uncharacterized splice variant of vitamin K epoxide reductase complex subunit 1 (VKORC1), termed VKORC1 variant 2 (VKORC1v2), as a potential interaction partner of vIL-6. In transfected cells, epitope-tagged VKORC1v2 was found to localize to the ER, to adopt a single-transmembrane (TM) topology placing the C tail in the ER lumen, and to bind vIL-6 via these sequences. Deletion mutagenesis and coprecipitation assays mapped the vIL-6-binding domain (vBD) of VKORC1v2 to TM-proximal residues 31 to 39. However, while sufficient to confer vIL-6 binding to a heterologous protein, vBD was unable to induce vIL-6 secretion when fused to (secreted) hIL-6, suggesting a VKORC1v2-independent mechanism of vIL-6 ER retention. In functional assays, overexpression of ER-directed vBD led to suppression of PEL cell proliferation and viability, effects also mediated by VKORC1v2 depletion and, as reported previously, by vIL-6 suppression. The growth-inhibitory and proapoptotic effects of VKORC1v2 depletion could be rescued by transduced wild-type VKORC1v2 but not by a vIL-6-refractory vBD-altered variant, indicating the functional relevance of the vIL-6-VKORC1v2 interaction. Notably, gp130 signaling was unaffected by VKORC1v2 or vBD overexpression or by VKORC1v2 depletion, suggesting an alternative pathway of vIL-6 activity via VKORC1v2. Combined, our data identify a novel and functionally significant interaction partner of vIL-6 that could potentially be targeted for therapeutic benefit.     

Differential regulation of Epstein-Barr virus (EBV) latent gene expression in Burkitt lymphoma cells infected with a recombinant EBV strain

Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.