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.
A genome-wide polyadenylation landscape in the expression of human herpesviruses has not been reported. In this study, we provide the first genome landscape of viral RNA polyadenylation sites in B cells from KSHV latent to lytic infection by using a modified PA-seq protocol and selectively validated by 3′ RACE. We found that KSHV genome contains 67 active pA sites for the expression of its ∼90 genes and a few antisense transcripts. Among the mapped pA sites, a large fraction of them are for the expression of cluster genes and the production of bicistronic or polycistronic transcripts from KSHV genome and only one-third are used for the expression of single genes. We found that the size of individual PA peaks is positively correlated with the usage of corresponding pA site, which is determined by the number of reads within the PA peak from latent to lytic KSHV infection, and the strength of cis-elements surrounding KSHV pA site determines the expression level of viral genes. Lastly, we identified and experimentally validated alternative polyadenylation of KSHV ORF54, T1.5, and K11 during viral lytic infection. To our knowledge, this is the first report on alternative polyadenylation events in KSHV infection.