Department of Microbiology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea.
Department of Microbiology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
J Virol. 2020 Jun 16;94(13). doi: 10.1128/JVI.00110-20.
The human cytomegalovirus (HCMV) UL50 gene encodes a transmembrane protein, pUL50, which acts as a core component of the nuclear egress complex (NEC) for nucleocapsids. Recently, pUL50 has been shown to have NEC-independent activities: downregulation of IRE1 to repress the unfolded protein response and degradation of UBE1L to inhibit the protein ISG15 modification pathway. Here, we demonstrate that a 26-kDa N-terminal truncated isoform of pUL50 (UL50-p26) is expressed from an internal methionine at amino acid position 199 and regulates the activity of pUL50 to induce the loss of valosin-containing protein (VCP/p97). A UL50(M199V) mutant virus expressing pUL50(M199V) but not UL50-p26 showed delayed growth at a low multiplicity of infection. There was also delayed accumulation of the viral immediate early 2 (IE2) protein in the mutant virus, and this correlated with the reduced expression of VCP/p97, which promotes IE2 expression. Infection with mutant virus did not significantly alter ISGylation levels. In transient expression assays, pUL50 induced VCP/p97 loss posttranscriptionally, and this was dependent on the presence of its transmembrane domain. In contrast, UL50-p26 did not destabilize VCP/p97 but, rather, inhibited pUL50-mediated VCP/p97 loss and the associated major IE gene suppression. Both pUL50 and UL50-p26 interacted with VCP/p97, although UL50-p26 did so more weakly than pUL50. UL50-p26 interacted with pUL50, and this interaction was much stronger than the pUL50 self-interaction. Furthermore, UL50-p26 was able to interfere with the pUL50-VCP/p97 interaction. Our study newly identifies UL50-p26 expression during HCMV infection and suggests a regulatory role for UL50-p26 in blocking pUL50-mediated VCP/p97 loss by associating with pUL50. Targeting the endoplasmic reticulum (ER) by viral proteins may affect ER-associated protein homeostasis. During human cytomegalovirus (HCMV) infection, pUL50 targets the ER through its transmembrane domain and moves to the inner nuclear membrane (INM) to form the nuclear egress complex (NEC), which facilitates capsid transport from the nucleus to the cytoplasm. Here, we demonstrate that pUL50 induces the loss of valosin-containing protein (VCP/p97), which promotes the expression of viral major immediate early gene products, in a manner dependent on its membrane targeting but that a small isoform of pUL50 is expressed to negatively regulate this pUL50 activity. This study reports a new NEC-independent function of pUL50 and highlights the fine regulation of pUL50 activity by a smaller isoform for efficient viral growth.
人巨细胞病毒（HCMV）UL50 基因编码一种跨膜蛋白，pUL50，它作为核出核复合物（NEC）的核心成分，用于核衣壳。最近，pUL50 已被证明具有 NEC 非依赖性活性：下调 IRE1 以抑制未折叠蛋白反应和降解 UBE1L 以抑制蛋白质 ISG15 修饰途径。在这里，我们证明了从氨基酸位置 199 的内部甲硫氨酸表达的 26kDa N 端截断同工型 pUL50（UL50-p26），并调节 pUL50 的活性以诱导包含 valosin 的蛋白（VCP/p97）的丢失。表达 pUL50（M199V）但不表达 UL50-p26 的 UL50（M199V）突变病毒在低感染复数下表现出生长延迟。突变病毒中病毒即刻早期 2（IE2）蛋白的积累也延迟，这与 VCP/p97 的表达减少相关，VCP/p97 促进 IE2 表达。突变病毒感染并未显著改变 ISGylation 水平。在瞬时表达试验中，pUL50 转录后诱导 VCP/p97 丢失，这依赖于其跨膜结构域的存在。相比之下，UL50-p26 不会使 VCP/p97 不稳定，而是抑制 pUL50 介导的 VCP/p97 丢失和相关的主要 IE 基因抑制。pUL50 和 UL50-p26 均与 VCP/p97 相互作用，尽管 UL50-p26 的相互作用弱于 pUL50。UL50-p26 与 pUL50 相互作用，并且这种相互作用比 pUL50 自身相互作用强得多。此外，UL50-p26 能够干扰 pUL50-VCP/p97 相互作用。我们的研究在 HCMV 感染期间新鉴定了 UL50-p26 的表达，并表明 UL50-p26 在通过与 pUL50 相关联来阻断 pUL50 介导的 VCP/p97 丢失方面发挥调节作用。病毒蛋白靶向内质网（ER）可能会影响 ER 相关蛋白的稳态。在人巨细胞病毒（HCMV）感染期间，pUL50 通过其跨膜结构域靶向 ER，并移动到核内膜（INM）以形成核出核复合物（NEC），从而促进衣壳从细胞核向细胞质的运输。在这里，我们证明 pUL50 以依赖其膜靶向的方式诱导包含 valosin 的蛋白（VCP/p97）的丢失，这促进了病毒主要即刻早期基因产物的表达，但表达了 pUL50 的小同工型，以负调控这种 pUL50 活性。本研究报告了 pUL50 的一种新的 NEC 非依赖性功能，并强调了较小同工型对 pUL50 活性的精细调节，以实现有效的病毒生长。
