Quantitative RNAseq analysis of Ugandan KS tumors reveals KSHV gene expression dominated by transcription from the LTd downstream latency promoter

Abstract

KSHV is endemic in Uganda and the HIV epidemic has dramatically increased the incidence of Kaposi sarcoma (KS). To investigate the role of KSHV in the development of KS, we obtained KS biopsies from ART-naïve, HIV-positive individuals in Uganda and analyzed the tumors using RNAseq to globally characterize the KSHV transcriptome. Phylogenetic analysis of ORF75 sequences from 23 tumors revealed 6 distinct genetic clusters with KSHV strains exhibiting M, N or P alleles. RNA reads mapping to specific unique coding sequence (UCDS) features were quantitated using a gene feature file previously developed to globally analyze and quantitate KSHV transcription in infected endothelial cells. A pattern of high level expression was detected in the KSHV latency region that was common to all KS tumors. The clear majority of transcription was derived from the downstream latency transcript promoter P3(LTd) flanking ORF72, with little evidence of transcription from the P1(LTc) latency promoter, which is constitutive in KSHV-infected lymphomas and tissue-culture cells. RNAseq data provided evidence of alternate P3(LTd) transcript editing, splicing and termination resulting in multiple gene products, with 90% of the P3(LTd) transcripts spliced to release the intronic source of the microRNAs K1-9 and 11. The spliced transcripts encode a regulatory uORF upstream of Kaposin A with alterations in intervening repeat sequences yielding novel or deleted Kaposin B/C-like sequences. Hierarchical clustering and PCA analysis of KSHV transcripts revealed three clusters of tumors with different latent and lytic gene expression profiles. Paradoxically, tumors with a latent phenotype had high levels of total KSHV transcription, while tumors with a lytic phenotype had low levels of total KSHV transcription. Morphologically distinct KS tumors from the same individual showed similar KSHV gene expression profiles suggesting that the tumor microenvironment and host response play important roles in the activation level of KSHV within the infected tumor cells.

Tumor-specific changes in Kaposi sarcoma-associated herpesvirus genomes in Ugandan adults with Kaposi sarcoma

“…In a parallel study of viral transcriptomes [80], abundant expression of a chimeric Kaposin transcript fused to the 14.8-kb segment was found in tumor U008-B, consistent with the viral genome structure we observed. Another tumor from the same participant, U008-D ( Fig 5B ), had 100% nucleotide identity and was confirmed to have the same duplication and breakpoint junctions ( Fig 5F ).…”

Oncogenic herpesvirus engages the endothelial transcription factors SOX18 and PROX1 to increase viral genome copies and virus production

“…We further show that the late lytic protein K8.1 is expressed in the majority of the studied KS biopsies, suggesting that, similar to KLECs in culture, lytic protein expression is likely a common feature of KS tumours. This is supported by detection of the lytic proteins K5 and K15 in KS biopsies 37, 38 and a recent RNA-seq study on African-KS lesions where KSHV lytic transcripts were detected in the tumours analysed 15 . It is not known whether KS-SCs, like the KLEC cultures, constitutively produce and release infectious virus.…”

“…Notably, SOX18, PROX1 and COUPTF2, all followed the distribution of LANA and K8.1, indicating that these TFs are expressed in SCs together with the late lytic protein K8.1 (Fig.1a,b, Supplementary Fig.1a,b). K8.1 and other lytic transcripts have been recently detected in KS lesions by RNA-Seq 15 . Here, we observed a cytoplasmic K8.1 staining, consistent with the predicted distribution of the protein.…”

Transcription factor AP‐4 (TFAP4)‐upstream ORF coding 66 aa inhibits the malignant behaviors of glioma cells by suppressing the TFAP4/long noncoding RNA 00520/microRNA‐520f‐3p feedback loop

“…The main function of uORF is to regulate gene expression during gene translation . There is evidence that uORF has a vital regulatory role in some diseases including cancer, endocrine and metabolic diseases, and neurological diseases …”

“…6 There is evidence that uORF has a vital regulatory role in some diseases including cancer, endocrine and metabolic diseases, and neurological diseases. 7,8 Transcription factor AP-4 is a member of the basic helix-loop-helix-zipper family, which contains a basic DNA binding domain.…”