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

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.卡波西肉瘤相关疱疹病毒（Kaposi Sarcoma-Associated Herpesvirus，KSHV）在乌干达呈地方性流行，而艾滋病病毒（Human Immunodeficiency Virus，HIV）疫情大幅提升了卡波西肉瘤（Kaposi Sarcoma，KS）的发病率。为探究KSHV在KS发生发展中的作用，本研究从乌干达境内抗逆转录病毒治疗初治（Antiretroviral Therapy-naïve，ART-naïve）的HIV阳性个体中获取KS活检组织，并通过RNA测序（RNA Sequencing，RNAseq）对肿瘤进行分析，以全局表征KSHV转录组。对23份肿瘤样本的开放阅读框75（Open Reading Frame 75，ORF75）序列进行系统发育分析，结果显示存在6个独立遗传簇，对应携带M、N或P等位基因的KSHV毒株。针对匹配特定唯一编码序列（Unique Coding Sequence，UCDS）特征的RNA读段，本研究使用此前开发的用于全局分析并定量感染内皮细胞中KSHV转录水平的基因特征文件完成定量。在所有KS肿瘤共有的KSHV潜伏区中，检测到高水平表达模式。绝大多数转录产物源自ORF72侧翼的下游潜伏转录本启动子P3(LTd)，而几乎未检测到来自P1(LTc)潜伏启动子的转录信号——该启动子在KSHV感染的淋巴瘤及组织培养细胞中呈组成型表达。RNA测序数据显示，P3(LTd)转录本存在可变编辑、剪接及终止现象，可产生多种基因产物；其中90%的P3(LTd)转录本经剪接以切除内含子区域编码的微小RNA（microRNAs，miRNAs）K1-9和K11序列。经剪接的转录本可编码卡波辛A上游的调控性上游开放阅读框（upstream Open Reading Frame，uORF），两者间重复序列的变异可产生新型或缺失型卡波辛B/C样序列。对KSHV转录本进行层次聚类及主成分分析（Principal Component Analysis，PCA），结果显示肿瘤可分为3个簇，各自具有不同的潜伏基因与裂解基因表达谱。矛盾的是，呈现潜伏表型的肿瘤总KSHV转录水平较高，而呈现裂解表型的肿瘤总KSHV转录水平较低。同一患者体内形态学特征各异的KS肿瘤，其KSHV基因表达谱却较为相似，这表明肿瘤微环境与宿主应答在感染肿瘤细胞内KSHV的激活水平中发挥关键作用。