Regulation of Viral and Cellular Gene Expression by Kaposi's Sarcoma-Associated Herpesvirus Polyadenylated Nuclear RNA

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma and body cavity lymphoma. In cell culture, KSHV results in a latent infection, and lytic reactivation is usually induced with the expression of K-Rta or by treatment with phorbol 12-myristate 13-acetate (TPA) and/or n-butyrate. Lytic infection is marked by the activation of the entire viral genomic transcription cascade and the production of infectious virus. KSHV-infected cells express a highly abundant, long, noncoding transcript referred to as polyadenylated nuclear RNA (PAN RNA). PAN RNA interacts with specific demethylases and physically binds to the KSHV genome to mediate activation of viral gene expression. A recombinant BACmid lacking the PAN RNA locus fails to express K-Rta and does not produce virus. We now show that the lack of PAN RNA expression results in the failure of the initiation of the entire KSHV transcription program. In addition to previous findings of an interaction with demethylases, we show that PAN RNA binds to protein components of Polycomb repression complex 2 (PRC2). RNA-Seq analysis using cell lines that express PAN RNA shows that transcription involving the expression of proteins involved in cell cycle, immune response, and inflammation is dysregulated. Expression of PAN RNA in various cell types results in an enhanced growth phenotype, higher cell densities, and increased survival compared to control cells. Also, PAN RNA expression mediates a decrease in the production of inflammatory cytokines. These data support a role for PAN RNA as a major global regulator of viral and cellular gene expression.K aposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gamma herpesvirus that is the cause of Kaposi's sarcoma (1, 2). KSHV infection in cell culture manifests mainly as a latent infection; however, there is a percentage of spontaneous lytic reactivation. Lytic infection is marked by the production of infectious virus, whereas latency is characterized by the lack of infectious virus production and the expression of a limited number of viral transcripts and proteins. One of the most abundant transcripts present in KSHV-infected cells is a long noncoding RNA, referred to as polyadenylated nuclear RNA (PAN RNA) (3, 4).We previously showed that PAN RNA interacts with cell-and virus-encoded factors and mediates the regulation of immune response gene expression (5, 6). Data from our laboratory and others indicated that PAN RNA is a major regulator of viral gene expression through a mechanism that involves a direct interaction with the viral chromosome (5, 7). PAN RNA interacts with the demethylases UTX and JMJD3 to remove the suppressive H3K23me3 mark within the KSHV viral genome. In a recent study describing the transcriptome of KSHV-infected cell lines, PAN RNA was detected in the absence of lytic-phase induction, suggesting that PAN RNA is expressed during chronic latent infection in cell culture (8). This observation suggests that PAN RNA has the potential to influence viral and cellular gene expressio...

A Lytic Viral Long Noncoding RNA Modulates the Function of a Latent Protein

“…PAN RNA knockdown during reactivation was accompanied by higher levels of episome-associated LANA and reduced viral gene expression; this is consistent with a repressive role of LANA in lytic gene expression in general (23). Of note, we also noticed that a few viral gene expression patterns did not follow the same trend in our PCR array analyses, which is consistent with a previous report (10), indicating that PAN RNA may have other functions. Importantly, similar to viral gene regulation, Affymetrix human gene expression arrays showed that PAN RNA also regulates cellular gene expression targeted by LANA (data not shown).…”

“…PAN RNA binds the transcription factor IRF4 (8), lysine demethylases UTX and JMJD3, and the lysine methyltransferase MLL2 (9), in support of the notion that, similar to cellular lncRNAs, PAN RNA interacts with transcriptional regulators and chromatin modifiers to modulate viral gene expression. Moreover, recent mapping studies have found widespread PAN RNA interaction sites on the KSHV episome as well as the host genome, and PAN RNA expression is required for optimal expression of the entire KSHV lytic gene expression program (10).…”

KSHV episomes: rugged individualists on the factory floor

“…Although not directly assessed in our study, we speculate that one function of PAN may be to trap cellular RNAPII on the KSHV genome through DNA-RNA or RNA-protein interactions. PAN has been reported to interact with several viral and cellular proteins [24][25][26][27] , and its communication network is probably incompletely defined. In addition, it is also possible that the process of transcription itself may contribute to PAN function through an enhancer-RNA-like activity to facilitate reactivation [28,29] .…”

Viral noncoding RNAs: more surprises

“…On the promoter of the transitional viral transactivator RTA/ORF50, PAN RNA interacts with demethylases JMJD3 and UTX. The ChIRP technique was extended in a global high-throughput manner and revealed ubiquitous PAN RNA binding along the entire viral genome (Rossetto et al 2013). Confirmation using a related technique, such as CHART, might clarify the specificity of PAN RNA's binding to viral chromatin.…”