Kaposi's sarcoma-associated herpesvirus K8beta is derived from a spliced intermediate of K8 pre-mRNA and antagonizes K8alpha (K-bZIP) to induce p21 and p53 and blocks K8alpha-CDK2 interaction

Kaposi's sarcoma-associated herpesvirus (KSHV) is a lymphotropic DNA tumor virus that induces Kaposi's sarcoma and AIDS-related primary effusion lymphoma. KSHV open reading frame 50 and K8 genes in early viral lytic infection express, respectively, a tricistronic and a bicistronic pre-mRNA, which undergo alternative splicing to create two major spliced mRNA isoforms, alpha and beta, by inclusion (beta) or exclusion (alpha) of an intron at nucleotides 75563 to 75645. This intron contains some suboptimal features, which cause the intron 5' splice site (ss) to interact weakly with U1 snRNA and the 3' ss to bind a U2 auxiliary factor, U2AF, with low affinity. Optimization of this intron in K8 (K8 intron 2) promoted the interaction of the 5' ss with U1 and the 3' ss with U2AF, resulting in a substantial increase in intron splicing. Splicing of K8 intron 2 has also been shown to be stimulated by the splicing of a downstream intron. This was confirmed by the insertion of a human beta-globin intron into the K8beta exon 3-exon 4 splice junction, which promoted splicing of K8beta intron 2 and conversion of the K8beta mRNA to the K8alpha mRNA that encodes a K-bZIP protein. Intron 2 contains a premature termination codon, yet the K8beta mRNA is insensitive to nonsense-mediated mRNA decay, suggesting that the truncated K8beta protein may have a biological function. Indeed, although the truncated K8beta protein is missing only a C-terminal leucine zipper domain from the K-bZIP, its expression antagonizes the ability of the K-bZIP to induce p53 and p21 and blocks K-bZIP-CDK2 interaction through interfering K8alpha mRNA production.     

Direct interactions of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50/Rta protein with the cellular protein octamer-1 and DNA are critical for specifying transactivation of a delayed-early promoter and stimulating viral reactivation

The Kaposi's sarcoma-associated herpesvirus (KSHV) delayed-early K-bZIP promoter contains an ORF50/Rta binding site whose sequence is conserved with the ORF57 promoter. Mutation of the site in the full-length K-bZIP promoter reduced Rta-mediated transactivation by greater than 80%. The K-bZIP element contains an octamer (Oct) binding site that overlaps the Rta site and is well conserved with Oct elements found in the immediate-early promoters of herpes simplex virus type 1(HSV-1). The cellular protein Oct-1, but not Oct-2, binds to the K-bZIP element in a sequence-specific fashion in vitro and in vivo and stimulates Rta binding to the promoter DNA. The coexpression of Oct-1 enhances Rta-mediated transactivation of the wild type but not the mutant K-bZIP promoter, and Oct-1 and Rta proteins bind to each other directly in vitro. Mutations of Rta within an amino acid sequence conserved with HSV-1 virion protein 16 eliminate Rta's interactions with Oct-1 and K-bZIP promoter DNA but not RBP-Jk. The binding of Rta to both Oct-1 and DNA contributes to the transactivation of the K-bZIP promoter and viral reactivation, and Rta mutants deficient for both interactions are completely debilitated. Our data suggest that the Rta/Oct-1 interaction is essential for optimal KSHV reactivation. Transfections of mouse embryo fibroblasts and an endothelial cell line suggest cell-specific differences in the requirement for Oct-1 or RBP-Jk in Rta-mediated transactivation of the K-bZIP promoter. We propose a model in which Rta transactivation of the promoter is specified by the combination of DNA binding and interactions with several cellular DNA binding proteins including Oct-1.     

Leucine zipper domain is required for Kaposi sarcoma-associated herpesvirus (KSHV) K-bZIP protein to interact with histone deacetylase and is important for KSHV replication

The Kaposi sarcoma-associated herpesvirus (KSHV; or human herpesvirus-8)-encoded protein called K-bZIP (also named K8) was found to be multifunctional. In this study, we discovered that K-bZIP interacts with histone deacetylase (HDAC) 1/2 in 12-O-tetradecanoylphorbol-13-acetate-stimulated BCBL-1 lymphocyte cells. K-bZIP appears to repress HDAC activity through this interaction, which we determined to be independent of K-bZIP SUMOylation. We dissected the domains of K-bZIP and found that the leucine zipper (LZ) domain is essential for the interaction of K-bZIP and HDAC. In addition, we constructed a KSHV bacterial artificial chromosome (BAC) with LZ domain-deleted K-bZIP (KSHVdLZ) and transfected this mutated KSHV BAC DNA into HEK 293T cells. As a result, it was consistently found that K-bZIP without its LZ domain failed to interact with HDAC2. We also showed that the interaction between K-bZIP and HDAC is necessary for the inhibition of the lytic gene promoters (ORF50 and OriLyt) of KSHV by K-bZIP. Furthermore, we found that the LZ domain is also important for the interaction of K-bZIP with the promoters of ORF50 and OriLyt. Most interestingly, although it was found to have suppressive effects on the promoters of ORF50 and OriLyt, KSHVdLZ replicates at a significantly lower level than its BAC-derived revertant (KSHVdLZRev) or KSHVWT (BAC36) in HEK 293T cells. The defectiveness of KSHVdLZ replication can be partially rescued by siRNA against HDAC2. Our results suggest that the function of K-bZIP interaction with HDAC is two-layered. 1) K-bZIP inhibits HDAC activity generally so that KSHVdLZ replicates at a lower level than does KSHVWT. 2) K-bZIP can recruit HDAC to the promoters of OriLyt and ORF50 through interaction with HDAC for K-bZIP to have a temporary repressive effect on the two promoters.     

Functional p53 signaling in Kaposi's sarcoma-associated herpesvirus lymphomas: implications for therapy

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is associated with Kaposi's sarcoma (KS) as well as primary effusion lymphomas (PEL). The expression of viral proteins capable of inactivating the p53 tumor suppressor protein has been implicated in KSHV oncogenesis. However, DNA-damaging drugs such as doxorubicin are clinically efficacious against PEL and KS, suggesting that p53 signaling remains intact despite the presence of KSHV. To investigate the functionality of p53 in PEL, we examined the response of a large number of PEL cell lines to doxorubicin. Two out of seven (29%) PEL cell lines harbored a mutant p53 allele (BCBL-1 and BCP-1) which led to doxorubicin resistance. In contrast, all other PEL containing wild-type p53 showed DNA damage-induced cell cycle arrest, p53 phosphorylation, and p53 target gene activation. These data imply that p53-mediated DNA damage signaling was intact. Supporting this finding, chemical inhibition of p53 signaling in PEL led to doxorubicin resistance, and chemical activation of p53 by the Hdm2 antagonist Nutlin-3 led to unimpaired induction of p53 target genes as well as growth inhibition and apoptosis.