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The ORF46 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes uracil DNA glycosylase, an enzyme involved in DNA repair. In this study, we show that the transcriptional start site of the ORF46 gene is located at nucleotide 69,425 of the viral genome and ORF50 protein, a latent-lytic switch transactivator, activates the ORF46 promoter via RBP-Jκ protein. Three consensus RBP-Jκ-binding sites found in the ORF46 promoter are critical for the binding of RBP-Jκ protein and conferring the ORF50 responsiveness. In addition, a negative regulatory region has been determined in the ORF46 promoter, which mediates the suppression of the ORF50 responsiveness. The functional negative region of the ORF46 promoter is mainly composed of the Sp1-binding sites. Like the negative region of the ORF46 promoter, addition of Sp1-binding sequences alone in an ORF50-responsive promoter efficiently confers the suppression of the ORF50 responsiveness. Furthermore, sodium butyrate, a pleiotropic inducing agent for the KSHV lytic cycle, is able to relieve the negative regulation of the ORF46 promoter in the latently KSHV-infected cells. The identification of multiple positive and negative cis-acting regulatory elements in the viral promoters emphasizes the elaborate controls in the KSHV lytic cycle, which ensure the adequate expression of each viral lytic gene.

Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27(Kip1) protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27(Kip1) protein degradation. The induction of UBE2M and reduction of p27(Kip1) by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27(Kip1). Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27(Kip1). Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27(Kip1) expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27(Kip1) in drug resistance.

Epstein-Barr virus (EBV) BBLF1 shares 13 to 15% amino acid sequence identities with the herpes simplex virus 1 UL11 and cytomegalovirus UL99 tegument proteins, which are involved in the final envelopment during viral maturation. This study demonstrates that BBLF1 is a myristoylated and palmitoylated protein, as are UL11 and UL99. Myristoylation of BBLF1 both facilitates its membrane anchoring and stabilizes it. BBLF1 is shown to localize to the trans -Golgi network (TGN) along with gp350/220, a site where final envelopment of EBV particles takes place. The localization of BBLF1 at the TGN requires myristoylation and two acidic clusters, which interact with PACS-1, a cytosolic protein, to mediate retrograde transport from the endosomes to the TGN. Knockdown of the expression of BBLF1 during EBV lytic replication reduces the production of virus particles, demonstrating the requirement of BBLF1 to achieve optimal production of virus particles. BBLF1 is hypothesized to facilitate the budding of tegumented capsid into glycoprotein-embedded membrane during viral maturation.

The use of L(-)SddC [beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC)] for the treatment of Herpes B virus (HBV) infection is hindered by the emergence of drug-resistance associated with the L526M, L550V, and L526M/M550V mutations of the viral DNA polymerase (DP). The interactions of the anti-HBV compounds 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorode-oxycytidine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil triphosphate with HBV DP and its L(-)SddC-associated mutants have not been studied. The e antigen-negative variant of HBV associated with the G1896A mutation in the precore region has a high prevalence. Its effect on HBV DP is unclear. Because HBV DNA synthesis occurs in the nucleocapsid, we examined the kinetics of the reverse transcriptase activity from wild-type (wt) and mutated DPs with the wt or G1896A-mutated RNA template in the nucleocapsid. The effects of this template mutation on the activities of these L-nucleoside triphosphates were also examined. Results indicated that these DP mutations increased the Km values of deoxy-NTPs and decreased the efficiencies (Vmax/Km) of DPs. The additional L526M mutation increased the efficiency of the M550V-mutated DP but no more than that of the L526M-mutated DP. The G1896A mutation had impacts on the interactions between different DPs and deoxy-NTPs, except dCTP. It also had different impacts on the actions of the L-nucleoside triphosphates toward DPs. The L526M and M550V mutations caused a greater decrease in the Vmax using the wt RNA template compared with the G1896A-mutated template. The L526M, M550V, and L526M/M550V mutations caused varying degrees of resistance to the different M-nucleoside triphosphates.

Lytic cycle reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) is initiated by expression of the ORF50 gene. Here we show that YY1 protein specifically binds to the ORF50 promoter (ORF50p) region in vitro and in vivo. After treatment with chemical inducers, including sodium butyrate (SB) and TPA, the levels of YY1 protein are inversely correlated with the lytic induction of KSHV in cells. Overexpression of YY1 completely blocks the ORF50p activation in transient reporter assays, while mutation at the YY1 site in the ORF50p or knockdown of YY1 protein confers an enhancement of the ORF50p activation induced by SB and TPA. YY1 overexpression in a stable cell clone HH-B2(Dox-YY1) also inhibits expression of the ORF50 and its downstream lytic genes. On the other hand, a chimeric YY1 construct that links to its coactivator E1A can disrupt viral latency. These results imply that YY1 is involved in the regulation of KSHV reactivation.