Different Rotavirus Strains Enter MA104 Cells through Different Endocytic Pathways: the Role of Clathrin-Mediated Endocytosis

Abstract

Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins ␣2␤1 and ␣v␤3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.

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

“…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 caveolin-dependent endocytosis have no inhibitory effects on RRV replication (33, 55). Moreover, conflicting observations have been made about the effects of drugs that block endosomal acidification (8,15,33). There is thus no definitive model of the RV entry process at this time (2,42,54).…”

“…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).…”

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).…”

“…Rotavirus usage of GM1 shows a possible association with internalization by clathrin-mediated endocytosis (Tables 1 and 2). MA104 cell entry by Wa, DS-1, UK, and TFR-41 is blocked by treatments that inhibit clathrin-mediated endocytosis and alter endosomal pH, suggesting that they utilize a pathway that includes clathrinmediated endocytosis (11,12). UK VP4 is sufficient to direct rotavirus internalization by this pathway (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).…”