Abstract
The Kaposi’s sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman’s disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exon-intron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation.
Highlights
Kaposi’s sarcoma-associated herpesvirus (KSHV; HHV-8) is a human gammaherpesvirus and the etiological agent for Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), KSHV-inflammatory cytokine syndrome (KICS), and some cases of multicentric Castleman’s disease (MCD)[1,2,3,4]
We suggest that KSHV employs the same mechanism to stabilize intronless viral RNAs and cellular unspliced pre-mRNAs to modulate viral and host gene expression during lytic reactivation
Our previous chromatin immunoprecipitation (ChIP) study showed that ORF57 interacts with the KSHV genome near oriLyt-L [46], but ChIP assays do not distinguish between direct interactions with DNA, indirect interactions mediated through other DNA-bound proteins, or indirect interactions bridged via nascent RNA
Summary
Kaposi’s sarcoma-associated herpesvirus (KSHV; HHV-8) is a human gammaherpesvirus and the etiological agent for Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), KSHV-inflammatory cytokine syndrome (KICS), and some cases of multicentric Castleman’s disease (MCD)[1,2,3,4]. The KSHV life cycle includes both a latent and a lytic state, which require different viral gene expression programs and distinct interactions with the infected host cell [5,6,7,8,9]. A small subset of KSHV genes is expressed that allows propagation and maintenance of the KSHV genome in the absence of viral replication. During lytic reactivation KSHV orchestrates the ordered synthesis of numerous viral products that enable assembly of viral particles. The timing and amount of expression for each gene product is important for efficient production of infectious virions. During both lytic reactivation and latency, the virus manipulates the cell environment and gene expression machinery to modulate human and viral gene expression
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