Institute of Medical Virology, Charité Universitätsmedizin Berlin, Berlin, Germany.
J Virol. 2012 Mar;86(6):3370-82. doi: 10.1128/JVI.06556-11. Epub 2011 Dec 28.
The product of the human cytomegalovirus (HCMV) UL71 gene is conserved throughout the herpesvirus family. During HCMV infection, protein pUL71 is required for efficient virion egress and is involved in the final steps of secondary envelopment leading to infectious viral particles. We found strong indications for oligomerization of pUL71 under native conditions when recombinant pUL71 was negatively stained and analyzed by electron microscopy. Oligomerization of pUL71 during infection was further verified by native and reducing polyacrylamide gel electrophoresis (PAGE). By in silico analyses of the pUL71 sequence, we noticed a basic leucine zipper (bZIP)-like domain, which might serve as an oligomerization domain. We demonstrated the requirement of the bZIP-like domain for pUL71 oligomerization by coimmunoprecipitation and bimolecular fluorescence complementation using a panel of pUL71 mutants. These studies revealed that the mutation of two leucine residues is sufficient to abrogate oligomerization but that intracellular localization of pUL71 was unaffected. To investigate the relevance of the bZIP domain in the viral context, recombinant viruses carrying mutations identical to those in the panel of pUL71 mutants were generated. bZIP-defective viral mutants showed impaired viral growth, a small-plaque phenotype, and an ultrastructural phenotype similar to that of the previously described UL71 stop mutant virus. The majority of virus particles within the viral assembly compartment exhibited various stages of incomplete envelopment, which is consistent with the growth defect for the bZIP mutants. From these data we conclude that the bZIP-like domain is required for oligomerization of pUL71, which seems to be essential for correct envelopment of HCMV.
人巨细胞病毒 (HCMV) UL71 基因的产物在疱疹病毒家族中是保守的。在 HCMV 感染过程中，蛋白 pUL71 是病毒粒子有效出芽所必需的，并且参与导致感染性病毒颗粒的二次包膜的最后步骤。当重组 pUL71 被负染并通过电子显微镜分析时，我们发现 pUL71 在天然条件下存在强烈的寡聚化迹象。感染过程中的 pUL71 寡聚化通过天然和还原聚丙烯酰胺凝胶电泳（PAGE）进一步得到证实。通过对 pUL71 序列的计算机分析，我们注意到一个碱性亮氨酸拉链（bZIP）样结构域，它可能作为一个寡聚化结构域。我们通过使用一系列 pUL71 突变体的共免疫沉淀和双分子荧光互补实验证明了 bZIP 样结构域对 pUL71 寡聚化的要求。这些研究表明，两个亮氨酸残基的突变足以消除寡聚化，但 pUL71 的细胞内定位不受影响。为了研究 bZIP 结构域在病毒环境中的相关性，我们生成了携带与 pUL71 突变体面板中相同突变的重组病毒。bZIP 缺陷型病毒突变体显示出病毒生长受损、小斑块表型和与先前描述的 UL71 终止突变病毒相似的超微结构表型。在病毒组装隔室中的大多数病毒粒子表现出不同阶段的不完全包膜，这与 bZIP 突变体的生长缺陷一致。从这些数据中我们得出结论，bZIP 样结构域是 pUL71 寡聚化所必需的，这似乎对于 HCMV 的正确包膜至关重要。
