Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164–Cys164 and Cys181–Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181–Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181–Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.

中文翻译：

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病毒泛素连接酶 K5 对膜蛋白细胞外近膜茎区的可及性对底物识别的重要性

卡波氏肉瘤相关疱疹病毒 (KSHV) 是一种致癌病毒，可潜伏感染 B 细胞并在免疫功能低下的患者中引起恶性肿瘤。KSHV 在 KSHV 感染的细胞中利用两种病毒 E3 泛素连接酶 K3 和 K5 来介导参与免疫系统的几种宿主膜蛋白的多聚泛素化依赖性下调。尽管 K3 和 K5 是同一家族的成员并且具有相似的结构拓扑结构，但 K3 和 K5 具有不同的底物特异性。因此，K5可能具有与K3不同的底物识别模式；然而，底物识别的分子基础仍不清楚。在这里，我们调查了已知不是 K3 和 K5 底物的人 CD8α 未被它们识别的原因，以了解底物特异性的分子基础。CD8α 在实验条件下形成二硫键连接的同源二聚体，以评估病毒连接酶介导的下调。众所周知，每个 CD8α 单体（Cys164-Cys164 和 Cys181-Cys181）之间的茎区中的两个链间二硫键介导同源二聚化。当 Cys181–Cys181 的链间二硫键被消除时，CD8α 被具有功能性 RING 变体 (RINGv) 结构域的 K5 通过细胞质尾部的多聚泛素化下调。位于 CD8α 的茎/跨膜界面的天冬氨酸对于 K5 介导的没有 Cys181-Cys181 连接的 CD8α 突变体的下调是必不可少的。这些结果表明，靠近茎/跨膜界面的二硫键严重抑制了 K5 的底物靶向。