Identification of the Physiological Gene Targets of the Essential Lytic Replicative Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein

Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 gene product is essential for lytic KSHV replication and virion production. Recombinant ORF57-null mutants fail to accumulate several lytic cycle mRNAs at wild-type levels, leading to decreased production of lytic proteins necessary for efficient replication. Several mechanisms by which ORF57 may enhance expression of lytic KSHV mRNAs have been proposed, including mRNA stabilization, mRNA nuclear export, increased polyadenylation, and transcriptional activation. ORF57 activity is also gene specific, with some genes being highly dependent on ORF57, whereas others are relatively independent. Most experiments have utilized transfection models for ORF57 and have not systematically examined the gene specificity and potential mechanisms of action of ORF57 in the context of KSHV-infected cells. In this study, the KSHV genes that are most highly upregulated by ORF57 during KSHV lytic replication were identified by a combination of high-throughput deep RNA sequencing, quantitative PCR, Northern blotting, and rapid amplification of cDNA ends methods. Comparison of gene expression from a ⌬ORF57 KSHV recombinant, a rescued ⌬ORF57 KSHV recombinant, and wild-type KSHV revealed that two clusters of lytic genes are most highly dependent on ORF57 for efficient expression. Despite contiguous location in the genome and shared polyadenylation of several of the ORF57-dependent genes, ORF57 regulation was promoter and polyadenylation signal independent, suggesting that the mRNAs are stabilized by ORF57. The eight genes identified to critically require ORF57 belong to both early and late lytic temporal classes, and seven are involved in DNA replication, virion assembly, or viral infectivity, explaining the essential role of ORF57 in infectious KSHV production. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus involved in the causation of several human cancers. The KSHV ORF57 protein is required for KSHV to replicate and produce infectious virus. We have identified several KSHV genes whose expression is highly dependent on ORF57 and shown that ORF57 increases expression of these genes specifically. These genes code for proteins that are required for the virus to replicate its DNA and to infect other cells. Identifying the targets and mechanism of action of ORF57 provides further approaches to discover antiviral therapy. K aposi's sarcoma associated herpesvirus (KSHV; human herpesvirus 8) is a human gammaherpesvirus associated with the development of Kaposi's sarcoma and several pathogenic lymphoproliferative syndromes (for a review, see reference 1). Lytic KSHV replication plays a role in pathogenesis by maintaining the infected cell reservoir, and lytic-phase KSHV proteins contribute to cell proliferation, inflammation, and immune evasion (2, 3). Understanding the mechanism of action of proteins required for lytic herpesvirus replication is therefore critical for further understanding of oncogenic mechanisms and the development of new antivi...

Identification and Characterization of the Physiological Gene Targets of the Essential Lytic Replicative Epstein-Barr Virus SM Protein

“…This is in contrast to the gene profile of the KSHV ORF57-dependent genes. In KSHV, while some important late genes belong to the highly ORF57-dependent set, the majority are genes essential for lytic KSHV DNA replication (32). Thus, SM is essential for later steps in the cascade of lytic virus production than ORF57 in KSHV, where the proximate block in ORF57 KO mutants occurs at DNA replication and prior to late gene transcription (32).…”

“…Interestingly, all SM-dependent RNAs were late lytic cycle RNAs. Thus, the effect of EBV SM on EBV gene regulation is different from that of its homolog Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57, since ORF57 increases both early and late lytic RNA accumulation as recently shown (32). Since EBV late gene transcription is dependent on DNA replication, this raised the possibility that SM effects on these genes were primarily due to enhanced DNA replication in the presence of SM, although no early genes required for DNA replication were particularly SM dependent ( Fig.…”

New insights into the expression and functions of the Kaposi's sarcoma-associated herpesvirus long noncoding PAN RNA

“…If these pathways are indeed redundant, PAN RNA will only be completely destabilized by deletion of the ENE, knockout of ORF57, and inactivation of PABPC1, which will be difficult to establish empirically. Consistent with this idea, PAN RNA levels are only ~1.5–2.5-fold lower in ORF57-deleted virus (Verma et al, 2015; Majerciak et al, 2007) suggesting that PABPC1 overexpression and/or the ENE compensate for ORF57 deletion in the context of viral infection. In contrast, no studies using a virus specifically deleted for the ENE have been reported, so its effects on steady-state levels during viral replication have not been verified.…”

Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms

“…Kaposi sarcoma-associated herpesvirus (KSHV, human herpesvirus 8 or HHV8) open reading frame (ORF) 57 is a posttranscriptional regulator of viral gene expression [for review see (Majerciak & Zheng, 2015)] essential for virus gene expression and replication (Majerciak, Pripuzova et al, 2007;Han & Swaminathan, 2006;Verma, Li et al, 2015). A full-length ORF57 protein, naturally occurring as a homodimer (Majerciak, Pripuzova et al, 2015), is composed of 455 amino acid (aa) residues.…”