Department of Microbiology and Immunology, University of Nevada, Reno, School of Medicine, Reno, Nevada, USA.
J Virol. 2021 Jun 10;95(13):e0009621. doi: 10.1128/JVI.00096-21.
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic human gammaherpesvirus and the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). During reactivation, viral genes are expressed in a temporal manner. These lytic genes encode transactivators, core replication proteins, or structural proteins. During reactivation, other viral factors that are required for lytic replication are expressed. The most abundant viral transcript is the long noncoding RNA (lncRNA) known as polyadenylated nuclear (PAN) RNA. lncRNAs have diverse functions, including the regulation of gene expression and the immune response. PAN possesses two main -acting elements, the Mta response element (MRE) and the expression and nuclear retention element (ENE). While PAN has been demonstrated to be required for efficient viral replication, the function of these elements within PAN remains unclear. Our goal was to determine if the ENE of PAN is required in the context of infection. A KSHV bacmid containing a deletion of the 79-nucleotide (nt) ENE in PAN was generated to assess the effects of the ENE during viral replication. Our studies demonstrated that the ENE is not required for viral DNA synthesis, lytic gene expression, or the production of infectious virus. Although the ENE is not required for viral replication, we found that the ENE functions to retain PAN in the nucleus, and the absence of the ENE results in an increased accumulation of PAN in the cytoplasm. Furthermore, open reading frame 59 (ORF59), LANA, ORF57, H1.4, and H2A still retain the ability to bind to PAN in the absence of the ENE. Together, our data highlight how the ENE affects the nuclear retention of PAN but ultimately does not play an essential role during lytic replication. Our data suggest that PAN may have other functional domains apart from the ENE. KSHV is an oncogenic herpesvirus that establishes latency and exhibits episodes of reactivation. KSHV disease pathologies are most often associated with the lytic replication of the virus. PAN RNA is the most abundant viral transcript during the reactivation of KSHV and is required for viral replication. Deletion and knockdown of PAN resulted in defects in viral replication and reduced virion production in the absence of PAN RNA. To better understand how the elements within PAN may contribute to its function, we investigated if the ENE of PAN was necessary for viral replication. Although the ENE had previously been extensively studied with both biochemical and approaches, this is the first study to demonstrate the role of the ENE in the context of infection and that the ENE of PAN is not required for the lytic replication of KSHV.
卡波济肉瘤相关疱疹病毒（KSHV）是一种致瘤性人类γ疱疹病毒，也是卡波济肉瘤（KS）、原发性渗出性淋巴瘤（PEL）和多中心卡斯特曼病（MCD）的病原体。在重新激活过程中，病毒基因以时间方式表达。这些裂解基因编码反式激活物、核心复制蛋白或结构蛋白。在重新激活过程中，还表达了其他需要裂解复制的病毒因子。最丰富的病毒转录物是长非编码 RNA（lncRNA），称为多聚腺苷酸化核（PAN）RNA。lncRNAs 具有多种功能，包括基因表达和免疫反应的调节。PAN 具有两个主要作用元件，即 Mta 反应元件（MRE）和表达和核保留元件（ENE）。虽然已经证明 PAN 对于病毒复制的高效性是必需的，但 PAN 内这些元件的功能仍不清楚。我们的目标是确定在感染背景下 PAN 的 ENE 是否必需。生成了包含 PAN 中 79 个核苷酸（nt）EN E 缺失的 KSHV bacmid，以评估 ENE 在病毒复制过程中的作用。我们的研究表明，EN E 对于病毒 DNA 合成、裂解基因表达或感染性病毒的产生不是必需的。尽管 EN E 对于病毒复制不是必需的，但我们发现 EN E 起保留 PAN 在核内的作用，并且没有 EN E 会导致 PAN 在细胞质中的积累增加。此外，ORF59、LANA、ORF57、H1.4 和 H2A 仍然保留在没有 EN E 的情况下与 PAN 结合的能力。总之，我们的数据突出了 EN E 如何影响 PAN 的核保留，但最终在裂解复制过程中不起关键作用。我们的数据表明，PAN 可能除了 EN E 之外还有其他功能域。KSHV 是一种致癌疱疹病毒，可建立潜伏状态并表现出发作性再激活。KSHV 疾病病理学最常与病毒的裂解复制有关。PAN RNA 是 KSHV 再激活过程中最丰富的病毒转录物，是病毒复制所必需的。在没有 PAN RNA 的情况下，PAN 的缺失和敲低导致病毒复制缺陷和病毒粒子产生减少。为了更好地了解 PAN 内的元件如何有助于其功能，我们研究了 PAN 的 EN E 是否对病毒复制是必需的。尽管 EN E 之前已经通过生化和结构方法进行了广泛的研究，但这是第一项研究表明 EN E 在感染背景下的作用，以及 PAN 的 EN E 对于 KSHV 的裂解复制不是必需的。
