Rhesus Rotavirus Entry into a Polarized Epithelium Is Endocytosis Dependent and Involves Sequential VP4 Conformational Changes

Abstract

Rotavirus (RV) cell entry is an incompletely understood process, involving VP4 and VP7, the viral proteins composing the outermost layer of the nonenveloped RV triple-layered icosahedral particle (TLP), encasing VP6. VP4 can exist in three conformational states: soluble, cleaved spike, and folded back. In order to better understand the events leading to RV entry, we established a detection system to image input virus by monitoring the rhesus RV (RRV) antigens VP4, VP6, and VP7 at very early times postinfection. We provide evidence that decapsidation occurs directly after cell membrane penetration. We also demonstrate that several VP4 and VP7 conformational changes take place during entry. In particular, we detected, for the first time, the generation of folded-back VP5 in the context of the initiation of infection. Folded-back VP5 appears to be limited to the entry step. We furthermore demonstrate that RRV enters the cell cytoplasm through an endocytosis pathway. The endocytosis hypothesis is supported by the colocalization of RRV antigens with the early endosome markers Rab4 and Rab5. Finally, we provide evidence that the entry process is likely dependent on the endocytic Ca 2؉ concentration, as bafilomycin A1 treatment as well as an augmentation of the extracellular calcium reservoir using CaEGTA, which both lead to an elevated intraendosomal calcium concentration, resulted in the accumulation of intact virions in the actin network. Together, these findings suggest that internalization, decapsidation, and cell membrane penetration involve endocytosis, calcium-dependent uncoating, and VP4 conformational changes, including a fold-back.Rotaviruses (RVs) are the single most important cause of severe diarrhea requiring the hospitalization of infants and young children worldwide. Diarrheal disease caused by rotaviruses is associated with more than 500,000 deaths per year, predominantly in developing countries, and is a leading cause of pediatric hospitalizations. These viruses are also relatively common causes of disease in the elderly and the immunocompromised as well as a wide variety of animal species. Although much has been learned about various components of the viral replication cycle, the early RV entry pathway is still poorly understood. Unlike enveloped viruses that fuse to cell membranes, most nonenveloped viruses induce lysis or pore formation in the plasma or endocytic vesicle membranes in order to enter cells (43). Whether RV behaves like the other nonenveloped viruses during membrane penetration remains controversial. A direct entry of RV particles was initially proposed (26, 39), but more recent studies suggested an endocytosis step during RV entry (8,33,55). Most of the RV entry data were obtained by using the simian rhesus RV (RRV) strain and MA104 cells as a model, but different RV strains appear to use various endocytosis pathways (33). Although drugs affecting dynamin and cholesterol have been shown to impair RRV infection (33, 55), drugs and dominant negative mutants known to impair clathrin or ...

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

“…Next, to define the mechanism by which DBN1 blocks dynamin function, we preincubated WT and DBN1 −/− cells with dynasore, an inhibitor that blocks the activity of all three isoforms of dynamin (25), before addition of RV. Consistent with the previous report (22), dynasore treatment only modestly inhibited (1.5-fold) infection in WT cells (Fig. 4A).…”

Roles of VP4 and NSP1 in Determining the Distinctive Replication Capacities of Simian Rotavirus RRV and Bovine Rotavirus UK in the Mouse Biliary Tract

“…Cultured mouse cholangiocytes were seeded on 12-mm polycarbonate tissue culture inserts with 0.4-m pores (Costar transwell filters; Corning, Corning, NY) at a density of 10 5 cells/cm 2 and supplemented with fresh medium every day for 5 days. Cells were infected with the indicated trypsin-activated virus at an MOI of 200 for 1 h, washed three times with phosphate-buffered saline (PBS), and fixed with 3% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 10 min as recently described (28). Infected cells were immunostained with anti-VP6 MAb 1E11 and Alexa Fluor (AF) 488-conjugated anti-mouse IgG secondary antibody (Invitrogen) under permeabilizing conditions (PBS, 1% saponin, 1% Triton X-100, 3% bovine serum albumin) and AF 594 phalloidin (Invitrogen).…”

Relative Roles of GM1 Ganglioside, N -Acylneuraminic Acids, and α2β1 Integrin in Mediating Rotavirus Infection

“…DS-1 and RV-5 rotaviruses differ in only 3 amino acids in VP8*, so it is possible that RV-5 also uses this route. In contrast, these treatments do not affect RRV internalization, which occurs by an endocytic route that may be independent of clathrin and caveola (11,13). The Nar 3 variant of RRV is considered to utilize a clathrin-dependent pathway, possibly due to a single VP8* amino acid change from RRV (Lys187Arg) (12).…”

“…During rotavirus-cell attachment and entry, activated VP4 undergoes a major conformational change that facilitates membrane disruption by VP5* hydrophobic domains (9,10). VP4 may define rotavirus internalization, which commonly but not universally appears to involve clathrin-mediated endocytosis (11)(12)(13).…”