Analysis of the Early Steps of Herpes Simplex Virus 1 Capsid Tegumentation
Analysis of the Early Steps of Herpes Simplex Virus 1 Capsid Tegumentation
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DOI：
10.1128/jvi.03292-12
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发表时间：
2013-05-01
影响因子：
5.4
通讯作者：
Lippe, Roger
中科院分区：
文献类型：
--
作者：
Henaff, Daniel;Remillard-Labrosse, Gaudeline;Lippe, Roger
1型单纯疱疹病毒颗粒是多层结构，DNA基因组被衣壳、被膜和包膜包围。虽然成熟病毒粒子的蛋白质含量是已知的，但被膜的添加顺序和发生这种情况的细胞内隔室却存在激烈的争论。为了在最初的退出阶段探索这一过程，我们使用了两种方法：一种是体外核退出试验，它重建了核衣壳向细胞质的出口，另一种是经典的核衣壳沉降试验。正如预期的那样，体外细胞质衣壳不含U(L)34、U(L)31或病毒糖蛋白，但含有U(S)3。与先前的研究结果一致，核壳和体外衣壳均阳性表达ICP0和ICP4。出乎意料的是，核C衣壳和细胞质衣壳的U(L)36和U(L)37的检测结果也呈阳性。免疫电镜证实这些被膜蛋白与核衣壳密切相关。当胞质病毒蛋白存在于体外实验时，在衣壳上没有检测到额外的被膜蛋白。正如先前报道的那样，被皮对高盐提取敏感，但令人惊讶的是，外源蛋白稳定了被皮。最后，一些被膜蛋白似乎在输出过程中部分丢失，而其他被膜蛋白可能是在多个步骤中添加或在此过程中被修饰的。总的来说，一个新出现的图景暗示了在核阶段衣壳被多达5种被膜蛋白的早期包裹，在核出口期间一些病毒蛋白的脱落，以及在重包膜期间获得其他被膜蛋白。
Herpes simplex virus type 1 particles are multilayered structures with a DNA genome surrounded by a capsid, tegument, and envelope. While the protein content of mature virions is known, the sequence of addition of the tegument and the intracellular compartments where this occurs are intensely debated. To probe this process during the initial stages of egress, we used two approaches: an in vitro nuclear egress assay, which reconstitutes the exit of nuclear capsids to the cytoplasm, and a classical nuclear capsid sedimentation assay. As anticipated, in vitro cytoplasmic capsids did not harbor U(L)34, U(L)31, or viral glycoproteins but contained U(S)3. In agreement with previous findings, both nuclear and in vitro capsids were positive for ICP0 and ICP4. Unexpectedly, nuclear C capsids and cytoplasmic capsids produced in vitro without any cytosolic viral proteins also scored positive for U(L)36 and U(L)37. Immunoelectron microscopy confirmed that these tegument proteins were closely associated with nuclear capsids. When cytosolic viral proteins were present in the in vitro assay, no additional tegument proteins were detected on the capsids. As previously reported, the tegument was sensitive to high-salt extraction but, surprisingly, was stabilized by exogenous proteins. Finally, some tegument proteins seemed partially lost during egress, while others possibly were added at multiple steps or modified along the way. Overall, an emerging picture hints at the early coating of capsids with up to 5 tegument proteins at the nuclear stage, the shedding of some viral proteins during nuclear egress, and the acquisition of others tegument proteins during reenvelopment.