Comprehensive Mapping and Analysis of Kaposi's Sarcoma-Associated Herpesvirus 3′ UTRs Identify Differential Posttranscriptional Control of Gene Expression in Lytic versus Latent Infection

Abstract

b 3= untranslated regions (UTRs) are known to play an important role in posttranscriptional regulation of gene expression. Here we map the 3= UTRs of Kaposi's sarcoma-associated herpesvirus (KSHV) using next-generation RNA sequencing, 3= rapid amplification of cDNA ends (RACE), and tiled microarray analyses. Chimeric reporters containing the KSHV 3= UTRs show a general trend toward reduced gene expression under conditions of latent infection. Those 3= UTRs with a higher GC content are more likely to be associated with reduced gene expression. KSHV transcripts display an extensive use of shared polyadenylation sites allowing for partially overlapping 3= UTRs and regulatory activities. In addition, a subset of KSHV 3= UTRs is sufficient to convey increased gene expression under conditions of lytic infection. These results suggest a role for viral 3= UTRs in contributing to differential gene expression during latent versus lytic infection. The 3= untranslated regions (UTRs), defined as the portion of an mRNA transcript extending from the stop codon to the polyadenylated tail, are known to play an important role in posttranscriptional regulation of gene expression (1). 3= UTRs can alter transcript stability, subcellular localization, and translation efficiency (2-5). Recent studies have demonstrated global alterations in host transcript 3= UTR compositions under different biological conditions related to cell growth and transformation (6, 7). These findings imply a possible role for 3= UTRs expressed by tumor viruses during infection.Although well studied in some RNA viruses (8), the role of viral 3= UTRs remains understudied in DNA viruses. KSHV is a large, double-stranded DNA virus associated with various hyperproliferative disorders, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (9, 10). Like all herpesviruses, KSHV is characterized by a long-term latent infection, during which the virus effectively evades the host immune system. KSHV harbors approximately 85 well-established protein-encoding genes, at least 5 of which are expressed during latent infection (9, 11). A temporal cascade of gene expression occurs during lytic infection that culminates with the production and dissemination of virions (12-14). To date, only 38 of the ϳ85 KSHV genes have a published, experimentally determined 3= UTR. To our knowledge, no studies have yet examined differences in composition or changes in regulatory activity of DNA virus 3= UTRs in latent (persistent) infection versus lytic (productive) infection.Here, using high-throughput RNA deep sequencing (RNAseq) analysis, we map a majority of the predominant 3= UTRs from most of the known mRNA transcripts (n ϭ 84). We confirmed the accuracy of 66 of these 3= UTRs using traditional rapid amplification of cDNA ends (RACE) analysis and of 5 additional 3= UTRs using tiled microarray analysis. Furthermore, using chimeric reporter assays, we tested their functionality in different cellular contexts. This work unveils a likely role for some KS...

Expression of the Antisense-to-Latency Transcript Long Noncoding RNA in Kaposi's Sarcoma-Associated Herpesvirus

“…These results were obtained from RNA-seq analysis of ALT-enriched RNA, but similar percentages were observed at the same position in libraries from non-ASO-enriched RNA (all raw reads are available in the NCBI Sequence Read Archive under accession code SRP082699). As the two genome-encoded adenine residues upstream of position 130873 interfere with determining the exact start of this poly(A) tract, this result is consistent with the published TTS at positions corresponding to 130870 to 130872 in the JSC1 genome (13,19,21,59,60).…”

“…From these studies at least 16 potential lncRNAs have been reported (13,(19)(20)(21), although there are discrepancies between the different approaches (Table 1). Seven potential lncRNAs found by tiling microarray (19,20) were not detected by subsequent RNA-seq studies (13,21,59,60). We sought to integrate these and prior studies (1, 13, 19-21, 50, 59-61) into an updated, comprehensive map of KSHV transcription annotations (Fig.…”

“…More than half of all KSHV ORFs are carried on polycistronic transcripts, Ͼ25% of all ORFs are carried by spliced mRNAs, and a few transcripts share promoters and/or pA signals (13,21,59,60,78,79). The ALT lncRNA is coterminal with the bicistronic mRNA K14-ORF74 and includes the K14 and ORF74 CDS, but it is expected that K14 and ORF74 proteins are not translated from ALT, as these ORFs are Ͼ8 kb downstream of the ALT TSS.…”

“…Some human miRNAs can downregulate the 3= UTRs for K14 and ORF74 in latent PEL cells (60), and it is possible that these microRNAs have the same effect on ALT. It is speculated that grouping specific genes into bi-or polycistronic transcripts allows KSHV to maximize its coding capacity and level of gene regulation (78,79).…”

Kaposi's Sarcoma-Associated Herpesvirus K3 and K5 Ubiquitin E3 Ligases Have Stage-Specific Immune Evasion Roles during Lytic Replication

“…In latently infected B cells, the K3 promoter is free of RNA polymerase (RNAP) II, while the K5 promoter is occupied by stalled RNAP II, showing that the K5 gene is poised for expression and is expressed independently of RTA during latency (87,88). Recent studies have begun to characterize the 3= untranslated regions (UTRs) of KSHV protein-encoding genes (89,90): while the K5 3= UTR carries a modest stabilizing effect, the K3 3= UTR exhibits a destabilizing activity (89), miR-K3 has a small inhibitory effect on the a K3 3= UTR reporter activity (90), and the 3= UTR of K3 is among the shortest of all the 3= UTRs in the KSHV genome (89,90). Finally, while vIRF1 has been shown to bind to a region adjacent to the K3 coding sequence (91), its binding region is 1 kb downstream of the transcription start site (TSS) of K3 (40,56), raising questions about the mechanism by which vIRF1 might regulate K3 gene expression.…”