PA‐seq for Global Identification of RNA Polyadenylation Sites of Kaposi's Sarcoma–Associated Herpesvirus Transcripts

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) is a human oncovirus linked to the development of several malignancies in immunocompromised patients. Like other herpesviruses, KSHV has a large DNA genome encoding more than 100 distinct gene products. Despite being transcribed and processed by cellular machinery, the structure and organization of KSHV genes in the virus genome differs from that observed in cellular genes from the human genome. A typical feature of KSHV expression is the production of polycistronic transcripts initiated from different promoters but sharing the same polyadenylation site (pA site). This represents a challenge in determination of the 3' end of individual viral transcripts. Such information is critical for generation of a virus transcriptional map for genetic studies. Here we present PA-seq, a high-throughput method for genome-wide analysis of pA sites of KSHV transcripts in B lymphocyte with latent or lytic KSHV infection. Besides identification of all viral pA sites, PA-seq also provides quantitative information about the levels of viral transcripts associated with each pA site enabling to determine the relative expression levels of viral genes at various stages of infection. Due to the indiscriminate nature of PA-seq, the pA sites of host transcripts are also concurrently mapped in the testing samples. Therefore, this technology can simultaneously estimate the expression changes of host genes and RNA polyadenylation upon KSHV infection.

A Genome-Wide Epstein-Barr Virus Polyadenylation Map and Its Antisense RNA to EBNA

“…The most effective method for lytic EBV induction in Akata cells, under multiple tested conditions, was to treat the cells for 24 h with a 1:200 dilution of whole serum, as measured by the expression of the lytic BMRF1/2 gene. The total RNAs from both uninduced (latent) and induced (lytic) cells were harvested for construction of PA-seq libraries (46). The PA-seq libraries from JSC-1 cells were obtained from our previous study and were successfully used to determine KSHV genome-wide pA sites with high levels of accuracy and specificity (25).…”

“…We performed a genome-wide analysis of EBV polyadenylation sites using a newly developed PA-seq method (25,45,46), an important tool for further refining EBV gene annotation. The unbiased, comprehensive, and quantitative nature of PA-seq allowed us to illustrate an EBV genome landscape of pA sites and their usage during latent and lytic EBV infections in JSC-1, Raji, and Akata cells.…”

“…We subsequently verified these bicistronic/polycistronic EBV transcripts derived from several gene clusters, including LMP-1/BNLF2a/ BNLF2b, LMP-2A/LMP-2B/BNRF1, BFRF1/BFRF2/BFRF3, BORF1/BORF2, and BaRF1/ BMRF1/BMRF2, in the EBV genome. However, because our PA-seq method depends on oligo(T) priming for library construction (45,46), we were unable to define the 3= ends of those Pol III transcripts lacking a poly(A) tail. These include both latent EBER1 and EBER2 RNA transcripts, which are highly abundant, small noncoding EBV RNAs transcribed by RNA Pol III (85,86).…”

“…Subsequently, we performed a peak-calling analysis using F-Seq (25,46). Obtained peaks represent the clusters of PA-seq reads from a specific region where heterogeneity of cleavage sites exists, as normally seen in KSHV and mammalian RNA polyadenylation ), the KSHV genome (GenBank accession no.…”

“…In recent years, various efforts have been made to simultaneously map pA sites of whole transcriptomes (41)(42)(43)(44). In this report, we applied a newly developed PA-seq method (44,45) that was successfully used to map Kaposi's sarcoma-associated herpesvirus (KSHV) genome-wide pA sites (25,46) and generated a comprehensive atlas of all pA sites and their usage for EBV genome expression from latency to lytic infection in three EBV-positive (EBV ϩ ) cell lines. Analysis of the mapped pA sites in association with currently annotated genes led us to identify a new set of distinct polyadenylated transcripts antisense to various forms of EBNA.…”