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Direct transcriptional targets of the HHV 8/KSHV Rta Lytic Switch Protein

ABSTRACT: Lytic reactivation from latency is critical for the pathogenesis of KSHV. We previously demonstrated that the 691 amino acid KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta’s direct targets during reactivation by generating a conditionally nuclear variant of Rta. WT Rta protein is constitutively localized to cell nuclei, and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS-2; aa 516-530) was required for nuclear localization of Rta, and relocalized eGFP exclusively to cell nuclei. Analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and stimulation of viral reactivation. Fusion of Rta_NLS-1,2 to the hormone binding domain of the murine estrogen receptor generated a variant of Rta whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled post-translationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Only eight KSHV genes were activated by Rta in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different magnitudes, and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/vIL-6, ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that induction of most of the KSHV lytic cycle genes requires additional protein expression post-Rta. Keywords: Comparative transcriptome analysis by oligonucleotide microarray

OTHER RELATED OMICS DATASETS IN: PRJNA106457

ORGANISM(S): Chlorocebus aethiops Human gammaherpesvirus 8

PROVIDER: GSE11432 | GEO | 2008/08/31

SECONDARY ACCESSION(S): PRJNA106457

REPOSITORIES: GEO

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Project description:Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and isassociated with primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD) and two inflammatory diseases. KSHV-associated cancers are primarily associated with genes expressed during latency, while other pathologies are associated with lytic gene expression. The major lytic switch of the virus, RTA, interacts with cellular machinery to co-opt the host ubiquitin proteasome system to evade the immune response as well as activate the program of lytic replication. Through SILAC labeling, ubiquitin remnant enrichment and mass spectrometry, we have analyzed the RTA dependent ubiquitin-modified proteome. We identified RTA dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and naturally infected cells following lytic reactivation. We observed an enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a SUMO targeting ubiquitin ligase, may function to alleviate a SUMO dependent block to lytic reactivation. RTA targeted substrates directly through a ubiquitin ligase domain dependent mechanism as well as indirectly through cellular ubiquitin ligases, including RAUL. Our ubiquitome analysis revealed an RTA dependent mechanism of immune evasion. We provide evidence of inhibition of TAP dependent peptide transport, resulting in decreased HLA complex stability. The results of this analysis increase our understanding of mechanisms governing the latent to lytic transition in addition to the identification of a novel RTA dependent mechanism of immune evasion.Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and is associated with primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD) and two inflammatory diseases. KSHV-associated cancers are primarily associated with genes expressed during latency, while other pathologies are associated with lytic gene expression. The major lytic switch of the virus, RTA, interacts with cellular machinery to co-opt the host ubiquitin proteasome system to evade the immune response as well as activate the program of lytic replication. Through SILAC labeling, ubiquitin remnant enrichment and mass spectrometry, we have analyzed the RTA dependent ubiquitin-modified proteome. We identified RTA dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and naturally infected cells following lytic reactivation. We observed an enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a SUMO targeting ubiquitin ligase, may function to alleviate a SUMO dependent block to lytic reactivation. RTA targeted substrates directly through a ubiquitin ligase domain dependent mechanism as well as indirectly through cellular ubiquitin ligases, including RAUL. Our ubiquitome analysis revealed an RTA dependent mechanism of immune evasion. We provide evidence of inhibition of TAP dependent peptide transport, resulting in decreased HLA complex stability. The results of this analysis increase our understanding of mechanisms governing the latent to lytic transition in addition to the identification of a novel RTA dependent mechanism of immune evasion.

Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL). All PEL cell lines are infected with KSHV, and 70% are co-infected with Epstein-Barr Virus (EBV). KSHV reactivation from latency requires promoter-specific transactivation by the KSHV Rta protein through interactions with RBP-Jk (CSL), the cellular DNA binding component of the Notch signal transduction pathway. EBV transformation of primary B cells requires EBV nuclear antigen (EBNA)-2 to interact with RBP-Jk to direct the latent viral and cellular gene expression program. Although KSHV Rta and EBV EBNA-2 both require RBP-Jk for transactivation, previous studies have suggested that RBP-Jk-dependent transactivators do not function identically. We have found that the EBV latent protein LMP-1 is expressed in less than 5% of KSHV+/EBV+ PEL cells, but is induced in an Rta-dependent fashion when KSHV reactivates. KSHV Rta transactivates the EBV latency promoters in an RBP-Jk-dependent fashion and forms a ternary complex with RBP-Jk on the promoters. In B cells that are conditionally transformed by EBV alone, we show that KSHV Rta complements a short-term EBNA2 growth deficiency in an autocrine/paracrine manner. Complementaton of EBNA2-deficiency by Rta depends on RBP-Jk and LMP-1, and Rta transactivation is required for optimal growth of KSHV+/EBV+ PEL lines. Our data suggest that Rta can contribute to EBV-driven cellular growth by transactivating RBP-Jk-dependent EBV latency genes. However, our data also suggest that EBNA2 and Rta induce distinct alterations in the cellular proteomes that contribute to growth of infected cells. EREB2-5 cells were transfected and grown in the presence or absence of β-estradiol, as described. Seven days post-transfection, protein extracts were prepared, and 200 ugs. of each were analyzed using the RayBio Human Apoptosis Antibody Array Kit (RayBiotech) as per manufacturers suggestions. The membranes were exposed to autoradiography film for different times to detect the chemiluminescent signals. Images with signals in linear range were quantitated using the program ImageJ [59]. For each membrane, signals from the negative control spots were averaged, and then subtracted from each of the other spots. A signal was considered valid if its value exceeded both its average local background, and the average of all valid negative control values. Valid signals were normalized using the positive control spots (for cellular BID protein). Fold change in signals for each spot were quantitated by dividing by the valid signals for each corresponding spot on the minus β-estradiol membrane. Average fold change, and standard deviation, were calculated for each protein.

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Direct transcriptional targets of the HHV 8/KSHV Rta Lytic Switch Protein

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