Abstract
RNA polyadenylation (pA) is one of the major steps in regulation of gene expression at the posttranscriptional level. In this report, a genome landscape of pA sites of viral transcripts in B lymphocytes with Kaposi sarcoma-associated herpesvirus (KSHV) infection was constructed using a modified PA-seq strategy. We identified 67 unique pA sites, of which 55 could be assigned for expression of annotated ∼90 KSHV genes. Among the assigned pA sites, twenty are for expression of individual single genes and the rest for multiple genes (average 2.7 genes per pA site) in cluster-gene loci of the genome. A few novel viral pA sites that could not be assigned to any known KSHV genes are often positioned in the antisense strand to ORF8, ORF21, ORF34, K8 and ORF50, and their associated antisense mRNAs to ORF21, ORF34 and K8 could be verified by 3′RACE. The usage of each mapped pA site correlates to its peak size, the larger (broad and wide) peak size, the more usage and thus, the higher expression of the pA site-associated gene(s). Similar to mammalian transcripts, KSHV RNA polyadenylation employs two major poly(A) signals, AAUAAA and AUUAAA, and is regulated by conservation of cis-elements flanking the mapped pA sites. Moreover, we found two or more alternative pA sites downstream of ORF54, K2 (vIL6), K9 (vIRF1), K10.5 (vIRF3), K11 (vIRF2), K12 (Kaposin A), T1.5, and PAN genes and experimentally validated the alternative polyadenylation for the expression of KSHV ORF54, K11, and T1.5 transcripts. Together, our data provide not only a comprehensive pA site landscape for understanding KSHV genome structure and gene expression, but also the first evidence of alternative polyadenylation as another layer of posttranscriptional regulation in viral gene expression.
Highlights
Kaposi sarcoma-associated herpesvirus (KSHV), referred as human herpesvirus 8 (HHV-8), is a member of gammaherpervirus subfamily [1]
Identification of KSHV pA sites by polyadenylation- sequencing (PA-seq) To elucidate a role of RNA polyadenylation in KHSV gene regulation, we performed a genome-wide analysis of viral pA sites to monitor their usage during KSHV infection
The steady-state expression level of every gene in the KSHV genome from viral latent to lytic infection was determined by PA-seq and 39RACE are shown below each agarose gel, with numbers indicating the nucleotide positions of the mapped pA sites in the KSHV genome. doi:10.1371/journal.ppat.1003749.g006
Summary
Kaposi sarcoma-associated herpesvirus (KSHV), referred as human herpesvirus 8 (HHV-8), is a member of gammaherpervirus subfamily [1]. KSHV has been linked to three malignancies including all forms of Kaposi sarcoma, a complex solid tumor of endothelial origin, and two rare B-cell lymphomas, primary effusion lymphoma (PEL or body cavity-based large cell lymphoma) and multicentric Castleman disease [2,3,4]. During latency only a small fraction of viral genes are expressed to facilitate the maintenance of viral genome, drive cell proliferation, and mediate immune invasion. KSHV has a large DNA genome (,168 kb) encoding more than 90 genes for production of viral structural and non-structural proteins, small peptides, long non-coding RNAs (lncRNAs) and small regulatory miRNAs [1,8,9,10]. Like many DNA viruses, KSHV has a complex gene organization and depends on host cell machinery for its gene expression. A full compendium of viral genome annotation is still unknown and the true nature of viral gene expression and its regulation remains to be fully understood
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