Identification of Novel Kaposi's Sarcoma-Associated HerpesvirusOrf50Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50. RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation.IMPORTANCE Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions. KEYWORDS Kaposi's sarcoma-associated herpesvirus, RTA isoforms, alternative promoter usage, alternative splicing, transcriptional activation

Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells

“…However, due to the depth of reads in our RNA-seq data (5–100 fold more than previous studies), we detected a large number of previously unknown splice sites, which predict the existence of novel transcripts and encoded proteins throughout the KSHV genome. We specifically analyzed splicing of the ORF50 RTA and detected evidence of novel spliced transcripts containing the ORF50 coding sequence, which are derived from different upstream promoter regions, confirming and extending results from a recent study [49]. Complete characterization of these transcripts will be necessary to determine their structure and function.…”

“…RNA-seq analysis of the stranded libraries for BCBL-1, LEC-K1, BEC-K1 and TIME-K1 revealed only a low level of reads mapping antisense to ORF49 (58–112 TPM) providing minimal evidence for the unspliced transcript 50.2. No evidence for alternately spliced ORF50 transcripts encoding RTA isoforms 2 and 3, which were observed previously in TPA-induced BCBL-1 cells [49], were observed in any of the latently infected cells. …”

“…The transcript 50.5, in which intron “a” is removed, is predicted to encode a novel ORF50 isoform (isoform 5—based on the numbering scheme proposed in [49]) containing the N-terminal 136 aa of ORF44 (exon 1) spliced to the C-terminal 685 aa of ORF50 (exon 4). Only low levels of split reads identifying this transcript were detected in the BEC infections (Figure 9D).…”

“…Only low levels of split reads identifying this transcript were detected in the BEC infections (Figure 9D). The transcript 50.4, in which intron “b” is removed, has been recently identified in TPA-induced BCBL-1 cells [49] and is predicted to encode ORF50 isoform 4, with the N-terminal sequence “MPELRNM” from a region antisense to ORF47, which is spliced to the C-terminal 685 aa of ORF50. A moderate level of split reads identifying this transcript were found in the LEC-K1-3, BEC-K1-3 and TIME-K2, 3 infections (29–71 reads), but not in the TIME-K1, VERO or uninduced BCBL-1 infections (Figure 9D).…”

Alternative promoters drive human cytomegalovirus reactivation from latency

“…For example, Marek's disease virus uses promoter switching to achieve differential expression of its immediate early transactivator ICP27 during lytic and latent infections (50). Furthermore, Kaposi's sarcoma-associated herpesvirus encodes 6 splice variants of its ORF50 from 4 different promoters, resulting in multiple isoforms of the transactivator RTA, each with distinct viral transactivation potential (51). Epstein-Barr virus uses a promoter switching mechanism to maintain its distinct latency programs by driving differential expression of latency-associated genes (52)(53)(54)(55).…”