Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Suwon, Republic of Korea.
Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany.
J Virol. 2018 Jul 17;92(15). doi: 10.1128/JVI.00462-18. Print 2018 Aug 1.
Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that can be conjugated to proteins via an enzymatic cascade involving the E1, E2, and E3 enzymes. ISG15 expression and protein ISGylation modulate viral infection; however, the viral mechanisms regulating the function of ISG15 and ISGylation are not well understood. We recently showed that ISGylation suppresses the growth of human cytomegalovirus (HCMV) at multiple steps of the virus life cycle and that the virus-encoded pUL26 protein inhibits protein ISGylation. In this study, we demonstrate that the HCMV UL50-encoded transmembrane protein, a component of the nuclear egress complex, also inhibits ISGylation. pUL50 interacted with UBE1L, an E1-activating enzyme for ISGylation, and (to a lesser extent) with ISG15, as did pUL26. However, unlike pUL26, pUL50 caused proteasomal degradation of UBE1L. The UBE1L level induced in human fibroblast cells by interferon beta treatment or virus infection was reduced by pUL50 expression. This activity of pUL50 involved the transmembrane (TM) domain within its C-terminal region, although pUL50 could interact with UBE1L in a manner independent of the TM domain. Consistently, colocalization of pUL50 with UBE1L was observed in cells treated with a proteasome inhibitor. Furthermore, we found that RNF170, an endoplasmic reticulum (ER)-associated ubiquitin E3 ligase, interacted with pUL50 and promoted pUL50-mediated UBE1L degradation via ubiquitination. Our results demonstrate a novel role for the pUL50 transmembrane protein of HCMV in the regulation of protein ISGylation. Proteins can be conjugated covalently by ubiquitin or ubiquitin-like proteins, such as SUMO and ISG15. ISG15 is highly induced in viral infection, and ISG15 conjugation, termed ISGylation, plays important regulatory roles in viral growth. Although ISGylation has been shown to negatively affect many viruses, including human cytomegalovirus (HCMV), viral countermeasures that might modulate ISGylation are not well understood. In the present study, we show that the transmembrane protein encoded by HCMV UL50 inhibits ISGylation by causing proteasomal degradation of UBE1L, an E1-activating enzyme for ISGylation. This pUL50 activity requires membrane targeting. In support of this finding, RNF170, an ER-associated ubiquitin E3 ligase, interacts with pUL50 and promotes UL50-mediated UBE1L ubiquitination and degradation. Our results provide the first evidence, to our knowledge, that viruses can regulate ISGylation by directly targeting the ISGylation E1 enzyme.
干扰素刺激基因 15（ISG15）编码一种泛素样蛋白，可通过涉及 E1、E2 和 E3 酶的酶级联反应与蛋白质结合。ISG15 的表达和蛋白质 ISGylation 调节病毒感染；然而，调节 ISG15 和 ISGylation 功能的病毒机制尚不清楚。我们最近表明，ISGylation 在病毒生命周期的多个步骤中抑制人巨细胞病毒（HCMV）的生长，并且病毒编码的 pUL26 蛋白抑制蛋白质 ISGylation。在这项研究中，我们证明 HCMV UL50 编码的跨膜蛋白是核出芽复合物的一部分，也抑制 ISGylation。pUL50 与 UBE1L（ISGylation 的 E1 激活酶）相互作用，（在较小程度上）与 ISG15 相互作用，与 pUL26 相互作用。然而，与 pUL26 不同，pUL50 导致 UBE1L 的蛋白酶体降解。干扰素-β处理或病毒感染诱导的人成纤维细胞中的 UBE1L 水平通过 pUL50 表达降低。pUL50 的这种活性涉及其 C 末端区域内的跨膜（TM）结构域，尽管 pUL50 可以以独立于 TM 结构域的方式与 UBE1L 相互作用。一致地，在用蛋白酶体抑制剂处理的细胞中观察到 pUL50 与 UBE1L 的共定位。此外，我们发现内质网（ER）相关泛素 E3 连接酶 RNF170 与 pUL50 相互作用，并通过泛素化促进 pUL50 介导的 UBE1L 降解。我们的结果表明 HCMV 的 pUL50 跨膜蛋白在调节蛋白质 ISGylation 中具有新的作用。蛋白质可以通过泛素或泛素样蛋白（如 SUMO 和 ISG15）共价结合。ISG15 在病毒感染中高度诱导，ISG15 缀合，称为 ISGylation，在病毒生长中发挥重要的调节作用。尽管已经表明 ISGylation 对许多病毒（包括人巨细胞病毒（HCMV））有负面影响，但调节 ISGylation 的病毒对策尚不清楚。在本研究中，我们表明 HCMV UL50 编码的跨膜蛋白通过导致 ISGylation 的 E1 激活酶 UBE1L 的蛋白酶体降解来抑制 ISGylation。这种 pUL50 活性需要膜靶向。支持这一发现，内质网（ER）相关泛素 E3 连接酶 RNF170 与 pUL50 相互作用，并促进 UL50 介导的 UBE1L 泛素化和降解。我们的结果提供了第一个证据，据我们所知，病毒可以通过直接针对 ISGylation E1 酶来调节 ISGylation。
