Project 2-Tso

项目摘要/摘要 卡波西氏肉瘤(KS)是一种与艾滋病毒/艾滋病相关的恶性肿瘤，尽管有抗逆转录病毒药物，但它仍然很常见 治疗，在KS相关疱疹病毒(KSHV)感染高发地区。KSHV 血清阳性率在地理上是不均匀的，但在撒哈拉以南非洲(SSA)和美国部分地区很高 一些州，如南路易斯安那州，艾滋病毒-1感染率很高。在这一地区，艾滋病毒和KS 对也经历其他健康差异的有色人种的影响不成比例。因为HIV-和KSHV- 尽管HIV病毒载量很高，但感染者仍有患KS和其他KSHV相关恶性肿瘤的风险 抑制，需要对艾滋病毒携带者(PLWH)采取新的研究策略。慢性致癌病毒 感染具有产生免疫回避和病毒持久性的机制，从而导致肿瘤 成长。KSHV潜伏期基因编码少量与病毒持久性有关的病毒基因。 潜伏期相关核抗原(LANA)在维持潜伏期和帮助病毒方面起着关键作用 通过抑制MHC-1抗原递呈来逃避宿主免疫监视，而其他潜伏基因，如 因为Kaposin(K12)被报道具有致癌特性，以及可能是 肿瘤发生学。尽管KSHV潜伏期相关基因的个体功能已经被研究，但有 关于它们在潜伏期、免疫逃避和肿瘤形成中的具体作用，仍有一些悬而未决的问题。它 同样重要的是要知道，哪些宿主基因和途径与病毒基因相互作用，从而引发潜伏期程序， 并回答这种病毒和宿主的相互作用如何促进恶性转化。我们最近展示了 我们独特的CRISPR/Cas9载体，专门针对单个KSHV LANA核苷酸序列而设计， 减少约70-80%的KSHV外体拷贝，并抑制KSHV阳性细胞的生长。我们的专业 目的是利用这一新技术来干扰/消除KSHV潜伏期基因座的多个基因，并 研究它们单独和联合作用对KSHV潜伏期的影响。我们将寻求识别细胞基因和 受恶性肿瘤发展和维持的影响，并可能促进其发展和维持的途径 表型。我们假设KSHV潜伏期基因的各个组成部分分别失调表达 一组特定的细胞基因促进KSHV的持久性和协同促进生长 调节失调和肿瘤发生。我们将通过编辑多个病毒基因序列来测试这个概念 使用KS和PEL细胞培养模型与CRISPR/Cas9的潜伏期基因座。通过生物信息学分析 CRISPR/Cas9编辑的KSHV感染细胞的转录组图谱，我们将鉴定独特的宿主基因/细胞 途径及其与病毒潜伏基因的作用/相互作用在病毒潜伏、生长失调和 肿瘤发生学。

PROJECT SUMMARY/ABSTRACT Kaposi’s sarcoma (KS) is an HIV/AIDS-associated malignancy, that remains prevalent, despite antiretroviral therapy, in areas where there is high incidence of KS-associated herpesvirus (KSHV) infection. KSHV seroprevalence is geographically uneven but is high in sub-Saharan Africa (SSA) and in parts of the United States, such as Southern Louisiana, where HIV-1 infection rates are high. In this region, both HIV and KS disproportionately affect people of color who also experience other health disparities. Because HIV- and KSHV- infected individuals remain at risk to develop KS and other KSHV-associated malignances despite HIV viral load suppression, novel research strategies are needed for people living with HIV (PLWH). Chronic oncogenic viral infections possess mechanisms that engender immune avoidance and viral persistence that leads to neoplastic growth. The KSHV latency locus encodes a small number of viral genes that are involved in viral persistence. The latency-associated nuclear antigen (LANA) plays a key role in the maintenance of latency and help the virus to evade host immune surveillance by inhibiting MHC-1 antigen presentation, while other latency genes, such as kaposin (K12) was reported to have oncogenic properties, as well as microRNAs that could be essential for tumorigenesis. Although the individual function of KSHV latency-associated genes have been studied, there remain unresolved questions regarding their specific contribution to latency, immune evasion and neoplasia. It is also critical to know, which host genes and pathways interact with the viral genes to elicit the latency program, and to answer how this virus-host interplay contributes to malignant transformation. We recently demonstrated that our unique CRISPR/Cas9 vector, specifically designed to target a single KSHV LANA nucleotide sequence, reduced ~70-80% of KSHV episomal copies, and attenuated the growth of KSHV-positive cells. Our major objective here is to utilize this new technology to disrupt/eliminate multiple genes of the KSHV latency locus, and to investigate their individual and combined effects on KSHV latency. We will seek to identify cellular genes and pathways affected by, and possibly contributing to the development and maintenance of the malignant phenotype. We hypothesize that the components of the KSHV latency locus individually dysregulate expression of specific sets of cellular genes to promote KSHV persistence and synergistically to promote growth dysregulation and tumorigenesis. We will test this concept by editing multiple viral gene sequences within the latency locus with CRISPR/Cas9 using KS and PEL cell culture models. Through bioinformatics analyses of transcriptome profiles of the CRISPR/Cas9 edited KSHV infected cells, we will identify unique host genes/cellular pathway and their roles/interaction with viral latency genes in viral latency, growth dysregulation and tumorigenesis.