Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
J Virol. 2012 Nov;86(21):11886-98. doi: 10.1128/JVI.01879-12. Epub 2012 Aug 22.
It is well known that proteins in the tegument (located between the viral capsid and envelope proteins) play critical roles in the assembly and budding of herpesviruses. Tegument proteins UL16 and UL11 of herpes simplex virus (HSV) are conserved among all the Herpesviridae. Although these proteins directly interact in vitro, UL16 was found to colocalize poorly with UL11 in cotransfected cells. To explain this discrepancy, we hypothesized that UL16 is initially made in an inactive form and is artificially transformed to the binding-competent state when cells are disrupted. Consistent with a regulated interaction, UL16 was able to fully colocalize with UL11 when a large C-terminal segment of UL16 was removed, creating mutant UL16(1-155). Moreover, membrane flotation assays revealed a massive movement of this mutant to the top of sucrose gradients in the presence of UL11, whereas both the full-length UL16 and the C-terminal fragment (residues 156 to 373) remained at the bottom. Further evidence for the presence of a C-terminal regulatory domain was provided by single-amino-acid substitutions at conserved cysteines (C269S, C271S, and C357S), which enabled the efficient interaction of full-length UL16 with UL11. Lastly, the binding site for UL11 was further mapped to residues 81 to 155, and to our surprise, the 5 Cys residues within UL16(1-155) are not required, even though the modification of free cysteines in UL16 with N-ethylmaleimide does in fact prevent binding. Collectively, these results reveal a regulatory function within the C-terminal region of UL16 that controls an N-terminal UL11-binding activity.
众所周知，衣壳（位于病毒衣壳和包膜蛋白之间）中的蛋白在疱疹病毒的组装和出芽中起着关键作用。单纯疱疹病毒（HSV）的衣壳蛋白 UL16 和 UL11 在疱疹病毒科中是保守的。尽管这些蛋白在体外直接相互作用，但在共转染的细胞中发现 UL16 与 UL11 共定位不佳。为了解释这种差异，我们假设 UL16 最初以非活性形式存在，当细胞被破坏时，它被人为地转化为具有结合能力的状态。与受调控的相互作用一致，当去除 UL16 的大 C 端片段时，UL16 能够与 UL11 完全共定位，产生突变体 UL16(1-155)。此外，膜浮选测定显示，在存在 UL11 的情况下，该突变体大量移动到蔗糖梯度的顶部，而全长 UL16 和 C 端片段（残基 156 至 373）仍留在底部。通过保守半胱氨酸（C269S、C271S 和 C357S）的单个氨基酸取代提供了存在 C 端调节结构域的进一步证据，这使得全长 UL16 能够与 UL11 有效相互作用。最后，进一步确定了 UL11 的结合位点位于残基 81 至 155，令我们惊讶的是，UL16(1-155) 中的 5 个半胱氨酸残基不是必需的，尽管用 N-乙基马来酰亚胺修饰 UL16 中的游离半胱氨酸实际上确实阻止了结合。总的来说，这些结果揭示了 UL16 的 C 端区域内的调节功能，该功能控制 N 端 UL11 结合活性。
