卡波西肉瘤相关疱疹病毒(KSHV)属于γ-疱疹病毒家族,是一种众所周知的人类致癌病毒。在受感染的细胞中,165kbp的病毒基因组是包裹在染色质中的环状DNA。基因表达的严格控制是潜伏期的关键,过渡到裂解阶段,以及病毒相关恶性肿瘤的发展。远端顺式调节元件,如增强器和消音器,可以以位置和方向无关的方式调节基因表达。开放染色质是增强剂的另一个特征。为了系统地寻找增强剂,我们克隆了KSHV基因组中萤光素酶基因下游的所有开放染色质区域,并测试了它们在感染和未感染细胞中的增强子活性.在潜伏期相关的核抗原启动子的上游检测到沉默子。在K12p-OriLyt-R和ORF29内含子区域中鉴定出两个组成型增强子,其中ORF29内含子是一种组织特异性增强子。以下发起人:OriLyt-L,PANp,ALTp,和末端重复序列(TR)充当裂解诱导的增强子。复制和转录激活因子(RTA)的表达,裂解周期的主调节器,足以在未感染的细胞中诱导裂解增强剂的活性。我们建议跨越约24kbp区域的TR充当“病毒超增强子”,将潜伏期相关核抗原(LANA)的抑制作用与RTA的激活作用相结合。利用CRISPR激活和干扰技术,我们确定了这些增强子与其调节基因之间的联系。这里描述的沉默子和增强子为疱疹病毒的复杂基因调控提供了额外的一层。重要性在这项研究中,我们进行了系统的功能测定,以鉴定致癌疱疹病毒基因组中的顺式调节元件,卡波西肉瘤相关疱疹病毒(KSHV)。类似于其他疱疹病毒,KSHV同时呈现潜伏期和裂解期。因此,我们的检测是在未感染的细胞中进行的,在潜伏感染期间,在裂解条件下。我们确定了两种组成型增强剂,其中一个似乎是组织特异性增强剂。此外,四种裂解诱导的增强剂,它们都对复制和转录激活因子(RTA)有反应,已确定。此外,在主要潜伏期启动子和裂解基因基因座之间鉴定出沉默子。利用CRISPR激活和干扰技术,我们确定了这些增强子与其调节基因之间的联系。终端重复,跨越约24kbp的区域,似乎是一种“病毒超级增强子”,它将潜伏期相关核抗原(LANA)的抑制作用与RTA的激活作用结合在一起,以调节潜伏期到裂解过渡。
Kaposi\'s sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (
RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a \"viral super-enhancer\" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of
RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi\'s sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (
RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a \"viral super-enhancer\" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of
RTA to regulate latency to lytic transition.