KSHV Infection Mimics the Hypoxic Response Based on Next-Generation Sequencing [mRNA-Seq]

Purpose: Kaposi’s sarcoma associated-herpesvirus (KSHV) causes several hyperproliferative disorders, including Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. KSHV encodes for a number of microRNAs (miRNAs), and KSHV infection can affect the expression of cellular miRNAs. Hypoxia has been shown to induce KSHV reactivation, directly induce several KSHV lytic genes, and also induce the most abundant latent viral protein, LANA. Also, several KSHV proteins can stabilize and increase the cellular levels of hypoxia-inducible factor (HIF-1a). However, the degree to which hypoxic pathways are utilized by KSHV has yet to be determined. Methods: We investigated the interplay between hypoxia and KSHV infection by comparing the 31effects of hypoxia and KSHV infection on miRNA and mRNA expression, and by examining the 32effects of hypoxia on uninfected and KSHV-infected cells. This was accomplished using next-33generation sequencing (NGS), qRT-PCR, Taqman assays, and pathway analysis. Results: NGS analysis of human mRNAs revealed striking similarities (~34%) between the transcriptomic response to hypoxia and the transcriptomic response to KSHV infection. Additionally, hsa-miR-210, a key hypoxia-inducible miRNA with pro-angiogenic and anti-apoptotic properties, was found significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Finally, KSHV infected cells differed somewhat in their response to hypoxia compared to KSHV-uninfected controls. Conclusions: These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and induces miR-210 up-regulation. The understanding of how these miRNAs, genes and pathways are regulated by HIF-1a and KSHV infection are essential to a better understanding of the biology of KSHV-associated diseases. Overall design: 6 samples analyzed. Two experimental conditions: hypoxic uninfected cells (SLK cells) and hypoxic chronically KSHV-infected cells (SLKK cells) (n=3). Two sequencing platforms: microRNA-Seq and mRNA-Seq.

研究目的：卡波西肉瘤相关疱疹病毒（Kaposi’s sarcoma associated-herpesvirus, KSHV）可引发多种增殖异常性疾病，包括卡波西肉瘤、原发性渗出性淋巴瘤及多中心Castleman病。KSHV编码多种微小RNA（microRNA, miRNA），且KSHV感染会调控细胞内miRNA的表达水平。已有研究证实，缺氧可诱导KSHV裂解激活，直接上调多个KSHV裂解期基因，同时还能诱导丰度最高的潜伏期病毒蛋白LANA（潜伏相关核抗原，Latency-associated nuclear antigen）。此外，多种KSHV蛋白可稳定并提高细胞内缺氧诱导因子1α（hypoxia-inducible factor 1α, HIF-1α）的表达水平。但目前尚不明确KSHV对缺氧通路的利用程度。实验方法：本研究通过对比缺氧与KSHV感染对miRNA及mRNA表达的影响，并探究缺氧对未感染及KSHV感染细胞的调控作用，以此解析缺氧与KSHV感染之间的相互作用。实验采用二代测序（next-generation sequencing, NGS）、定量逆转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction, qRT-PCR）、Taqman检测法及通路分析完成相关实验。实验结果：对人类mRNA的二代测序分析显示，缺氧与KSHV感染所引发的转录组响应存在显著相似性（约34%）。此外，通过Taqman检测发现，hsa-miR-210——一种兼具促血管生成与抗凋亡特性的关键缺氧诱导miRNA——在KSHV感染与缺氧处理后均显著上调。最后，与未感染KSHV的对照组相比，KSHV感染细胞对缺氧的响应存在一定差异。研究结论：本研究结果表明，KSHV可利用部分缺氧细胞应答通路并诱导miR-210上调。阐明这些miRNA、基因及通路受HIF-1α与KSHV感染的调控机制，对于深入理解KSHV相关疾病的生物学特征至关重要。整体实验设计：共分析6份样本，包含2种实验分组：缺氧处理的未感染SLK细胞，以及缺氧处理的慢性KSHV感染SLKK细胞，每组各3份生物学重复。实验采用两种测序平台：microRNA测序（microRNA-Seq）与mRNA测序（mRNA-Seq）。