Department of Molecular Biology and Biophysics and the Molecular Biology and Biochemistry Graduate Program, University of Connecticut Health Center, Farmington, Connecticut, USA.
MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, United Kingdom.
J Virol. 2015 Jan;89(1):443-53. doi: 10.1128/JVI.01913-14. Epub 2014 Oct 15.
During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.
Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.
在 DNA 包装过程中，单纯疱疹病毒 1（HSV-1）原衣壳通过涉及病毒蛋白酶激活、支架蛋白排出以及病毒 DNA 摄取的过程转化为含有 DNA 的衣壳。包装需要六种次要衣壳蛋白（UL6、UL15、UL17、UL25、UL28 和 UL33）和一种病毒蛋白 UL32，后者未被发现与衣壳有关。尽管已经为每种次要衣壳蛋白分配了功能，但 UL32 在包装中的作用仍然是一个谜。使用含有功能性血凝素标记 UL32 的 HSV-1 变体，我们证明 UL32 以真正的晚期动力学合成，并表现出以前未被识别的定位模式。在感染后 6 至 9 小时（hpi），UL32 在宿主细胞核内的病毒复制隔室中积累，而在 24 hpi 时，它还存在于细胞质中。新生成的 UL32 缺失突变体用于确认，尽管包含野生型衣壳蛋白水平的 B 衣壳被合成，但这些原衣壳无法启动包装过程。此外，我们表明 UL32 对氧化还原敏感，并鉴定了两个高度保守的氧化还原酶样 C-X-X-C 基序，这些基序对蛋白质功能至关重要。此外，在没有 UL32 的情况下，病毒蛋白 UL6、UL25 和 VP19C 和病毒蛋白酶 VP24 的二硫键图谱发生改变，表明 UL32 可能在原衣壳组装和成熟过程中调节二硫键形成。
尽管已经为 HSV 的七种必需包装蛋白中的六种分配了功能，但 UL32 在包装中的作用仍然是个谜。UL32 是一种富含半胱氨酸的病毒蛋白，含有类似于参与调节二硫键形成的蛋白中的 C-X-X-C 基序。我们之前已经证明，二硫键对于病毒衣壳的形成和稳定性以及 UL6 门环的形成和稳定性都是必需的。在本报告中，我们证明在感染新分离的 UL32 缺失突变病毒的细胞中，病毒蛋白 UL6、UL25 和 VP19C 和病毒蛋白酶 VP24 的二硫键图谱发生改变，表明 UL32 作为一种伴侣蛋白能够调节二硫键形成。此外，这些结果表明，正确调节二硫键对于启动包装至关重要。
