Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures
Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures
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DOI：
10.1128/jvi.02351-15
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发表时间：
2016-02-01
影响因子：
5.4
通讯作者：
Peeples, Mark E.
中科院分区：
文献类型：
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作者：
Corry, Jacqueline;Johnson, Sara M.;Peeples, Mark E.
所有已进入临床试验阶段的减毒呼吸道合胞病毒（RSV）活疫苗都是在Vero细胞中生产的。在这些细胞中产生的附着糖蛋白(G)比在其他永生化细胞中产生的糖蛋白要小。这些病毒粒子在原代分化良好的人气道上皮（HAE）细胞培养中传染性低5倍。由于HAE细胞直接从人气道中分离出来，因此Vero细胞生长的疫苗病毒在接种疫苗后很可能在启动鼻上皮感染方面同样无效，因此，需要更大的接种量才能有效接种。我们假设含有完整G蛋白的Vero细胞衍生病毒对HAE细胞培养具有更强的传染性。使用特异性越来越强的蛋白酶抑制剂，我们确定了组织蛋白酶L是负责切割的蛋白酶。我们的证据表明，在内吞循环过程中，分裂发生在内核体或溶酶体的后期。Vero细胞的组织蛋白酶L活性是HeLa细胞的100倍。此外，组织蛋白酶L能够切割Vero细胞生长的病毒粒子中的G蛋白，而不能切割HeLa细胞生长的病毒粒子，这表明两种细胞系中G蛋白的翻译后修饰存在差异。我们通过诱变确定了对裂解重要的氨基酸，这些氨基酸包括一个可能的组织蛋白酶L裂解位点。含有在Vero细胞中产生的修饰的、不可切割的G蛋白的病毒在培养的HAE细胞中具有5倍的传染性，这证实了我们的假设，并表明在未来的RSV减毒活疫苗中包含这种突变的价值。
All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines.