Sarfo Akua, Starkey Jason, Mellinger Erica, Zhang Dan, Chadha Pooja, Carmichael Jillian, Wills John W
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
J Virol. 2017 Oct 13;91(21). doi: 10.1128/JVI.01161-17. Print 2017 Nov 1.
The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20 and UL24 To test whether UL21 is needed only for the A855V mutant, additional gB derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gB phenotype. This is the first example of a differential requirement for a viral protein across the four loci. UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations.
本研究的最初目标是重新审视单纯疱疹病毒1型（HSV-1）复制对UL21的需求。先前的研究表明，UL21对于在细胞培养物中的复制是可有可无的，但最近一篇关于HSV-2的报告对这些发现提出了质疑。如同在HSV-2研究中所做的那样，制备了一种UL21缺失病毒，并在互补细胞上进行传代培养，以防止选择补偿性突变。这种HSV-1突变体能够在非互补细胞中复制，即使在低感染复数（MOI）下也是如此，尽管观察到滴度有所降低。此外，在细胞质中观察到空衣壳的比例增加，这表明UL21在阻止它们从细胞核中排出方面发挥作用。令人惊讶的是，缺失突变体的传代导致合胞体（Syn）变体迅速生长。这是出乎意料的，因为UL21已被证明是Syn表型所必需的。然而，早期实验仅使用了糖蛋白B（gB）的A855V合胞体突变体，并且Syn表型也可由糖蛋白K（gK）、UL20和UL24中的替代产生。合胞体变体的测序揭示了gK基因座中的突变，但已证明UL21对于UL20和UL24是可有可无的。为了测试UL21是否仅对A855V突变体是必需的，在缺失病毒的背景下检查了其他gB衍生物，所有这些衍生物都产生了溶细胞性而非合胞体性感染位点。因此，UL21仅对gB表型是必需的。这是病毒蛋白在四个基因座上存在差异需求的第一个例子。UL21在甲型疱疹病毒中是保守的，但其作用尚不清楚。这项研究表明，HSV-1可以在没有UL21的情况下复制，尽管病毒滴度大大降低。缺失病毒在受感染细胞的细胞质中具有更大比例的空（无DNA）衣壳，这表明UL21可能在将它们保留在细胞核中发挥作用。这与显示UL21与衣壳相关并定位于受感染细胞核的报告一致。UL21似乎对于病毒膜活动也是必需的。已发现它是病毒介导的细胞融合所必需的，但仅对于在gB中具有合胞体突变的突变体（而非gK、UL20或UL24的变体）是必需的。合胞体形成所需的机制与病毒通过细胞连接直接传播所需的机制相似；这些研究表明，即使在没有合胞体突变的情况下，UL21对于细胞间传播也是必需的。
