Department of Microbiology and Immunology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA.
Microbiol Spectr. 2021 Oct 31;9(2):e0053921. doi: 10.1128/Spectrum.00539-21. Epub 2021 Sep 22.
Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is the major transactivator for viral gene expression and is required for lytic replication. In addition to transcriptional activation, IE2 is known to mediate transcriptional repression of promoters, including the major immediate-early (MIE) promoter and a bidirectional promoter within the lytic origin of replication (Lyt). The activity of IE2 is modulated by another viral protein, UL84. UL84 is multifunctional and is proposed to act as the origin-binding protein (OBP) during lytic replication. UL84 specifically interacts with IE2 to relieve IE2-mediated repression at the MIE and Lyt promoters. Originally, UL84 was thought to be indispensable for viral replication, but recent work demonstrated that some strains of HCMV (TB40E and TR) can replicate independently of UL84. This peculiarity is due to a single amino acid change of IE2 (UL122 H388D). Here, we identified that a UL84-dependent (AD169) Δ84 viral mutant had distinct IE2 localization and was unable to synthesize DNA. We also demonstrated that a TB40E Δ84 IE2 D388H mutant containing the reversed IE2 amino acid switch adopted the phenotype of AD169 Δ84. Further functional experiments, including chromatin-immunoprecipitation sequencing (ChIP-seq), suggest distinct protein interactions and transactivation function at Lyt between strains. Together, these data further highlight the complexity of initiation of HCMV viral DNA replication. Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised individuals and is also the leading viral cause of congenital birth defects. After initial infection, HCMV establishes a lifelong latent infection with periodic reactivation and lytic replication. During lytic DNA synthesis, IE2 and UL84 have been regarded as essential factors required for initiation of viral DNA replication. However, previous reports identified that some isolates of HCMV can replicate in a UL84-independent manner due to a single amino acid change in IE2 (H388D). These UL84-independent strains are an important consideration, as they may have implications for HCMV disease and research. This has prompted renewed interest into the functional roles of IE2 and UL84. The work presented here focuses on the described functions of UL84 and ascertains if those required functions are fulfilled by IE2 in UL84-independent strains.
人巨细胞病毒（HCMV）即刻早期 2（IE2）蛋白是病毒基因表达的主要转录激活因子，也是裂解复制所必需的。除了转录激活，IE2 还已知介导包括主要即刻早期（MIE）启动子和裂解复制起点内的双向启动子在内的启动子的转录抑制。IE2 的活性受另一种病毒蛋白 UL84 调节。UL84 具有多功能性，据推测在裂解复制过程中作为起始结合蛋白（OBP）发挥作用。UL84 特异性地与 IE2 相互作用，以解除 IE2 对 MIE 和 Lyt 启动子的抑制作用。最初，UL84 被认为对病毒复制是不可或缺的，但最近的工作表明，一些 HCMV 株（TB40E 和 TR）可以独立于 UL84 复制。这种特殊性归因于 IE2（UL122 H388D）的单个氨基酸变化。在这里，我们鉴定出一种依赖 UL84（AD169）Δ84 病毒突变体具有独特的 IE2 定位，并且无法合成 DNA。我们还证明了含有反向 IE2 氨基酸开关的 TB40EΔ84IE2 D388H 突变体采用了 AD169Δ84 的表型。进一步的功能实验，包括染色质免疫沉淀测序（ChIP-seq），表明在不同菌株之间 Lyt 处存在不同的蛋白相互作用和转录激活功能。总的来说，这些数据进一步强调了 HCMV 病毒 DNA 复制起始的复杂性。
人巨细胞病毒（HCMV）是免疫功能低下个体发病和死亡的重要原因，也是导致先天性出生缺陷的主要病毒原因。初次感染后，HCMV 建立终身潜伏感染，并定期重新激活和裂解复制。在裂解 DNA 合成过程中，IE2 和 UL84 一直被认为是启动病毒 DNA 复制所必需的关键因素。然而，先前的报告表明，由于 IE2 中的单个氨基酸变化（H388D），一些 HCMV 分离株可以以非依赖 UL84 的方式复制。这些不依赖 UL84 的菌株是一个重要的考虑因素，因为它们可能对 HCMV 疾病和研究具有重要意义。这促使人们重新关注 IE2 和 UL84 的功能作用。本文重点介绍了 UL84 的描述性功能，并确定了在不依赖 UL84 的菌株中是否由 IE2 发挥了这些必需的功能。
