Rhesus Rotavirus Trafficking during Entry into MA104 Cells Is Restricted to the Early Endosome Compartment

Abstract

Endocytosis has recently been implicated in rotavirus (RV) entry. We examined the role of Rabs, which regulate endosomal trafficking, during RV entry. Several structural proteins of neuraminidase-sensitive and -insensitive RVs colocalized with Rab5, an early endosome marker, but not Rab7, a late endosome marker. Dominant-negative and constitutively active mutants demonstrated that Rab5 but not Rab4 or Rab7 affects rhesus RV (RRV) infectivity. These data suggest that early RRV trafficking is confined to the early endosome compartment and requires Rab5. R otavirus (RV), a nonenveloped member of the Reoviridae family, is the single most important cause of severe diarrhea globally and is a leading cause of death in children under the age of 5 (17). The virus particle encloses 11 double-stranded RNA segments within a triple-layered icosahedral capsid. The outermost layer is composed of the glycoprotein VP7 and protruding spikes of trimeric VP4. Two major domains comprise VP4. VP5* forms the foot of the spike, whereas VP8* is the head (5, 6). While the mechanisms governing RV cellular entry are not fully understood, both VP7 and VP4 are required for binding and penetration (15,21). Like fusion mechanisms in enveloped viruses, VP5* is believed to rearrange upon uncoating and membrane interaction, resulting in a folded-back trimeric structure which is proposed to mediate membrane penetration (4,5,26,28,29). Although RV entry was initially thought to occur via direct plasma membrane penetration (7, 13), recent studies indicate that RV traffics through the endosomal pathway (1, 10, 22, 27). As different RV strains, which vary in neuraminidase (NA) sensitivity of cell binding, may use distinct endocytic pathways, a definitive model of RV entry does not currently exist (10,22,27). We recently demonstrated that rhesus RV (RRV) enters a polarized epithelium using the endosomal route, as evidenced by colocalization of trimeric VP7 and VP5* with endosomal markers Rab4 and Rab5 (27). Furthermore, specific pharmacological interventions reduced RRV infectivity (27). Rabs, small cellular GTPases important in endosomal trafficking, are powerful tools for studying endocytosis due to their unique expression in specific endocytic compartments and fine regulation of vesicular trafficking (24). Dominantnegative (DN) and constitutively active (CA) Rab mutants have been widely used to identify the entry trafficking steps required by various viral infections (11,14,16,19,23). Rab5, which is present at the plasma membrane but is preferentially associated with early endosome (EE) vacuoles (23), functions in internalization, transport of newly formed vesicles to the EE, fusion, and trafficking to the late endosome (LE). Rab4 localizes primarily to the EE and regulates recycling to the plasma membrane. Rab7, present on the LE, governs transfer to lysosomes. Rab5 and Rab7 are proposed to be present on the maturing endosome, an intermediary vesicle between the EE and LE (16).Here, we examined the roles of Rab proteins during RRV entry. Rab5 has p...

Structural Correlates of Rotavirus Cell Entry

“…Our finding, that in BSC-1 cells, RRV does not enter from Rab5 endosomes, appears at first to be at variance with published conclusions that the virus traffics to Rab5 comparments in MDCK cells [35] and MA104 cells [36]. In the former study, overexpressing the same Rab5 mutants used in our experiments gave only a twofold increase for constitutively active Rab5 and a twofold decrease for inactive Rab5; in the latter study, inactive Rab5 had roughly a fourfold effect, while constitutively active Rab5 had no effect at all.…”

Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry

“…Two subunits of the endosomal vacuolar H + -ATPase (v-ATPase; V0 subunit C and V1 subunit B1) were also detected in agreement with previous results that showed that bafilomycin, an inhibitor of the v-ATPase, affects RV infection (8,10,21). Coatomer complex elements (COPB1, COPA) and the GTPase ARF1-whose depletion has been associated, among other phenotypes, with defects in the function of early endosomes (EE) (22)-were tightly clustered in our data set.…”

“…Analysis of the data by functional clustering showed that the genes that scored as positives are implicated in a broad array of cellular functions and also suggested several statistically enriched biological pathways that could be involved in the virus life cycle (Table S2). Among the pathways identified are several that have been reported to be relevant for, and in some cases highly regulated during, RV infection, including tight junction (TJ) function (16), endocytosis (8,10), and calcium signaling (17), as well as the protein-ubiquitination pathway (18,19).…”

“…It has also been reported that RV cell entry depends on dynamin and cholesterol (8), although contradicting results were recently reported in Madin Darby canine kidney cells (9). RRV infection of monkey kidney MA104 cells has been shown to depend on the small GTPase RAB5, but not on RAB4 or RAB7 (10). The interaction of the RV spike protein VP4 with surface receptors determines the endocytic pathway used by RVs to enter cells (11).…”

The Spike Protein VP4 Defines the Endocytic Pathway Used by Rotavirus To Enter MA104 Cells

“…In the case of the NA-sensitive rhesus rotavirus (RRV), its NA-resistant variant Nar3 has been shown to attach to cells directly by interacting with integrin ␣2␤1 (15,16). Ultimately, the interactions described are believed to lead to rotavirus internalization by endocytosis (19,(32)(33)(34). Rotaviruses can be internalized into MA104 cells using different endocytic pathways depending on the virus strain.…”