Rotaviruses Reach Late Endosomes and Require the Cation-Dependent Mannose-6-Phosphate Receptor and the Activity of Cathepsin Proteases To Enter the Cell

Abstract

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. IMPORTANCERotavirus 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.

Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition

“…One possibility is that high concentrations of pDGE-RGD could interfere with virus-cell attachment, even after it took place, by mediating detachment of part of the viral population from the cell surface. It is also possible that integrin-bound pDGE-RGD is endocytosed and inhibits virion conversion into the transcriptionally active, double-layered particle, which for Wa has been proposed to occur during exit from late endosomes (56). Interestingly, Wa VP8* binds the GM1 ganglioside, which can associate with integrins such as ␣5␤1 and ␣2␤1 in membrane microdomains (57,58) and has been proposed to classify cargo for uptake and trafficking in late endosomes (59).…”

Solar and Temperature Treatments Affect the Ability of Human Rotavirus Wa To Bind to Host Cells and Synthesize Viral RNA

“…Recently, several studies have probed the life cycle of rotavirus infection (43)(44)(45). Interestingly, some rotavirus strains (including human rotavirus Wa) undergo internalization via clathrin-mediated endocytosis to enter an endosomal compartment and then a late endosome, while other rotavirus strains can enter the host cell via a clathrin-independent process (43).…”

“…Interestingly, some rotavirus strains (including human rotavirus Wa) undergo internalization via clathrin-mediated endocytosis to enter an endosomal compartment and then a late endosome, while other rotavirus strains can enter the host cell via a clathrin-independent process (43). This internalization process is VP4 dependent.…”

Trafficking of Bluetongue Virus Visualized by Recovery of Tetracysteine-Tagged Virion Particles

“…While the bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, the rhesus rotavirus strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent (47,50). Recently, an in-depth study on rotavirus entry has further shown that although both bovine and rhesus strains reach maturing endosomes to establish virus infection, bovine rotavirus, unlike the rhesus strain, has to traffic to late endosomes (54). This requirement for the late endosomes was also shared by other rotavirus strains of human and porcine origin.…”