Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
J Virol. 2014 Apr;88(8):4389-402. doi: 10.1128/JVI.03457-13. Epub 2014 Feb 5.
Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention.
Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.
轮状病毒（RV）通过不同的内吞途径进入细胞。牛轮状病毒（BRV）UK 使用网格蛋白介导的内吞作用，而恒河猴轮状病毒（RRV）则采用一种不依赖网格蛋白和 caveolin 的内吞过程。鉴于这些病毒使用的细胞内化途径存在差异，我们测试了 BRV UK 的细胞内转运是否与 RRV 相同，RRV 已知可到达成熟内体（ME）以感染细胞。我们发现 BRV UK 也到达 MEs，因为其感染性取决于 Rab5 的功能、内体分选复合物必需的运输（ESCRT）和内体腔内小泡（ILVs）的形成。然而，与 RRV 不同，BRV UK 的感染性被敲低 Rab7 的表达所抑制，表明它必须转运到晚期内体（LE）才能感染细胞。Rab7 的需求也被人类和猪源的其他 RV 株共享。有趣的是，大多数到达 LEs 的 RV 株也被发现依赖 Rab9、阳离子依赖的甘露糖-6-磷酸受体（CD-M6PR）和组织蛋白酶 B、L 和 S 的活性，表明需要从高尔基体网络（TGN）运输的细胞因子通过 CD-M6PR 转运到 LEs 以促进 RV 细胞感染。此外，使用一组 UK × RRV 重组病毒，我们发现 BRV UK 对 Rab7、Rab9 和 CD-M6PR 的依赖性与 Spike 蛋白 VP4 有关。这些发现说明了 RV 进入的精心设计的途径，并揭示了一种新的过程（Rab9/CD-M6PR/组织蛋白酶），该过程可能成为药物干预的靶点。
轮状病毒是导致儿童严重胃肠炎的重要病原体。在大多数情况下，病毒通过一种内吞途径进入细胞，该途径将病毒颗粒递送至称为早期内体（EEs）的囊泡细胞器。一些病毒从 EEs 进入细胞质，在那里开始复制基因组。然而，其他病毒进入细胞更深的部位，从 EEs 运输到晚期内体（LEs）以解体并到达细胞质。在这项工作中，我们表明大多数 RV 株必须运输到 LEs，并且内体溶酶体蛋白酶从高尔基体复合物运输到 LEs，由甘露糖-6-磷酸受体介导，对于病毒离开囊泡隔间并有效启动病毒复制是必要的。我们还表明，这种深入细胞的过程与病毒的 Spike 蛋白 VP4 有关。这些发现说明了 RV 进入的精心设计的途径，可用于药物干预。
