Rhesus Monkey Rhadinovirus ORF57 Induces gH and gL Glycoprotein Expression through Posttranscriptional Accumulation of Target mRNAs

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is expressed early during lytic KSHV replication, enhances expression of many KSHV genes, and is essential for virus production. ORF57 is a member of a family of proteins conserved among all human and many animal herpesviruses that are multifunctional regulators of gene expression and act posttranscriptionally to increase accumulation of their target mRNAs. The mechanism of ORF57 action is complex and may involve effects on mRNA transcription, stability, and export. ORF57 directly binds to REF/Aly, a cellular RNA-binding protein component of the TREX complex that mediates RNA transcription and export. We analyzed the effects of an ORF57 mutation known to abrogate REF/Aly binding and demonstrate that the REF-binding mutant is impaired in activation of viral mRNAs and noncoding RNAs confined to the nucleus. Although the inability to bind REF leads to decreased ORF57 activity in enhancing gene expression, there is no demonstrable effect on nuclear export of viral mRNA or the ability of ORF57 to support KSHV replication and virus production. These data indicate that REF/Aly-ORF57 interaction is not essential for KSHV lytic replication but may contribute to target RNA stability independent of effects on RNA export, suggesting a novel role for REF/Aly in viral RNA metabolism.

Section: Discussioncontrasting

confidence: 56%

Binding of Cellular Export Factor REF/Aly by Kaposi's Sarcoma-Associated Herpesvirus (KSHV) ORF57 Protein Is Not Required for Efficient KSHV Lytic Replication

Verma²,

Swaminathan

2012

“…1D and C). Importantly, it has also been shown in a previous study on ORF57 from rhesus monkey rhadinovirus (RRV) that insertion of a beta-globin intron in ORF57-dependent genes does not rescue their weak expression (61). Thus, the detrimental sequence bias of K15 is a dominant feature and requires ORF57 for efficient expression.…”

Section: Discussionmentioning

confidence: 93%

ORF57 Overcomes the Detrimental Sequence Bias of Kaposi's Sarcoma-Associated Herpesvirus Lytic Genes

Vogt¹,

Hackmann²,

Rabner³

et al. 2015

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which enhances the expression of intronless KSHV genes on multiple posttranscriptional levels. However, it remains elusive how ORF57 recognizes viral RNAs. Here, we demonstrate that ORF57 also increases the expression of the multiple intron-containing K15 gene. The nucleotide bias of the K15 cDNA revealed an unusual high AT content. Thus, we optimized the K15 cDNA by raising the frequency of GC nucleotides, yielding an ORF57-independent version. To further prove the importance of the sequence bias of ORF57-dependent RNAs, we grouped KSHV mRNAs according to their AT content and found a correlation between AT-richness and ORF57 dependency. More importantly, latent genes, which have to be expressed in the absence of ORF57, have a low AT content and are indeed ORF57 independent. The nucleotide composition of K15 resembles that of HIV gag, which cannot be expressed unless RNA export is facilitated by the HIV Rev protein. Interestingly, ORF57 can partially rescue HIV Gag expression. Thus, the KSHV target RNAs of ORF57 and HIV gag RNA may share certain motifs based on the nucleotide bias. A bioinformatic comparison between wild-type and sequenceoptimized K15 revealed a higher density for hnRNP-binding motifs in the former. We speculate that binding of particular hnRNPs to KSHV lytic transcripts is the prerequisite for ORF57 to enhance their expression. IMPORTANCEThe mostly intronless genes of KSHV are only expressed in the presence of the viral regulator protein ORF57, but how ORF57 recognizes viral RNAs remains elusive. We focused on the multiple intron-containing KSHV gene K15 and revealed that its expression is also increased by ORF57. Moreover, sequences in the K15 cDNA mediate this enhancement. The quest for a target sequence or a response element for ORF57 in the lytic genes was not successful. Instead, we found the nucleotide bias to be the critical determinant of ORF57 dependency. Based on the fact that ORF57 has only a weak affinity for nucleic acids, we speculate that a cellular RNA-binding protein provides the sequence preference for ORF57. This study provides evidence that herpesviral RNA regulator proteins use the sequence bias of lytic genes and the resulting composition of the viral mRNP to distinguish between viral and cellular mRNAs. K aposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus that was identified in KS tissues from AIDS patients (1). It is also associated with two lymphoproliferative disorders, the primary effusion lymphoma and the plasma cell variant of multicentric Castleman's disease. KSHVassociated malignancies are rare in immunocompetent people but more common in the context of immunosuppression, especially in HIV patients or transplant recipients. The viral life cycle is divided into a latent and a lytic phase. In contrast to other herpesviruses, tumorigenesis is also a consequence of the lytic cycle (2-5).Herpesviruses replicate in the nucleus, and viral gene expression is dependent on the host ...

“…Atypical codon usage in rhesus rhadinovirus (RRV) has been correlated with differences in mRNA stability and dependence on ORF57, the RRV homolog of SM (35,36). In order to assess codon usage in SM-dependent versus SM-independent genes, we used the CAIcal toolset (37) and calculated relative synonymous codon usage (RSCU) for each coding sequence of SM-dependent genes and the comparison set of SM-independent genes.…”

Section: Identification Of Ebv Sm-dependent Rna Expression During Ebvmentioning

confidence: 99%

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

Thompson

Verma

et al. 2016

Epstein-Barr virus (EBV) SM protein is an essential lytic cycle protein with multiple posttranscriptional mechanisms of action. SM binds RNA and increases accumulation of specific EBV transcripts. Previous studies using microarrays and PCR have shown that SM-null mutants fail to accumulate several lytic cycle mRNAs and proteins at wild-type levels. However, the complete effect of SM on the EBV transcriptome has been incompletely characterized. Here we precisely identify the effects of SM on all EBV transcripts by high-throughput RNA sequencing, quantitative PCR (qPCR), and Northern blotting. The effect of SM on EBV mRNAs was highly skewed and was most evident on 13 late genes, demonstrating why SM is essential for infectious EBV production. EBV DNA replication was also partially impaired in SM mutants, suggesting additional roles for SM in EBV DNA replication. While it has been suggested that SM specificity is based on recognition of either RNA sequence motifs or other sequence properties, no such unifying property of SM-responsive targets was discernible. The binding affinity of mRNAs for SM also did not correlate with SM responsiveness. These data suggest that while target RNA binding by SM may be required for its effect, specific activation by SM is due to differences in inherent properties of individual transcripts. We therefore propose a new model for the mechanism of action and specificity of SM and its homologs in other herpesviruses: that they bind many RNAs but only enhance accumulation of those that are intrinsically unstable and poorly expressed. IMPORTANCEThis study examines the mechanism of action of EBV SM protein, which is essential for EBV replication and infectious virus production. Since SM protein is not similar to any cellular protein and has homologs in all other human herpesviruses, it has potential importance as a therapeutic target. Here we establish which EBV RNAs are most highly upregulated by SM, allowing us to understand why it is essential for EBV replication. By comparing and characterizing these RNA transcripts, we conclude that the mechanism of specific activity is unlikely to be based simply on preferential recognition of a target motif. Rather, SM binding to its target RNA may be necessary but not sufficient for enhancing accumulation of the RNA. Preferential effects of SM on its most responsive RNA targets may depend on other inherent characteristics of these specific mRNAs that require SM for efficient expression, such as RNA stability. E pstein-Barr virus (EBV) SM protein is an essential regulatory protein expressed by EBV during the lytic phase of replication (for reviews, see references 1 and 2). SM protein is detectable after immediate early gene expression and prior to expression of other early gene products (3, 4). When the SM gene is insertionally inactivated in recombinant EBV genomes, the resultant virus is incapable of completing the lytic cycle and producing infectious virions (5, 6). The exact reasons that SM is essential for lytic replication and virus pro...