Interactions of Rotavirus VP4 Spike Protein with the Endosomal Protein Rab5 and the Prenylated Rab Acceptor PRA1

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 ...

Section: Discussionsupporting

confidence: 66%

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

Wolf

Greenberg

2011

“…4E through G). Again, as shown in the merged images, VP4 did not colocalize with these compartments, despite the fact that it has been recently shown to interact with Rab5 and PRA1 in the context of cell infection (16). Interestingly, interaction of VP4 with Rab5 and PRA1 was only studied at the biochemical level in infected MA104 cells and was shown to be a transient and early event (16).…”

Section: Resultsmentioning

confidence: 99%

Rotavirus Spike Protein VP4 Binds to and Remodels Actin Bundles of the Epithelial Brush Border into Actin Bodies

Gardet

Breton

Fontanges

et al. 2006

Self Cite

We demonstrate here that VP4, a rotaviral protein, is able to specifically bind to bundled actin microfilaments that are subsequently profoundly remodeled into actin bodies. These cytoplasmic actin bodies do not localize within identified intracellular compartments. VP4-induced actin remodeling is similar to cytochalasin D effects with kinetics compatible with that of rotavirus infection. Actin bundles' remodeling occurs both in infected and in VP4-transfected cells and in various cell lines, indicating that this is a general property of the viral protein itself. Interestingly, in intestinal epithelial cells, which represent the natural target of rotavirus, VP4 is addressed to the apical membrane where it binds specifically to brush border actin bundles and elicits its remodeling, whereas cytochalasin D impaired all the filamentous actin. These observations indicate that these original properties of VP4 likely explain the previously described brush border alterations that follow rotavirus infection of enterocytes and may also participate to the mechanism of rotavirus final assembly.

“…These studies did not demonstrate, however, any physical interaction between the above-mentioned Rabs and any viral gene products. There is one description of coimmunoprecipitation of Rab5 with rotavirus VP4, but no role for this interaction in the viral life cycle was demonstrated (11). Recently, an interaction between HCV NS4B and Rab5 was described (29), and Rab5 depletion was associated with impaired HCV genome replication.…”

Section: Fig 4 Depletion Of Tbc1d20 Inhibits Hcv Infection (A) In-mentioning

confidence: 99%

A Rab-GAP TBC Domain Protein Binds Hepatitis C Virus NS5A and Mediates Viral Replication

Sklan

Staschke

Oakes

et al. 2007

Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the world's population.HCV is a positive single-stranded RNA virus whose 9.6-kb genome encodes an ϳ3,000-amino-acid polyprotein that is proteolytically processed into structural proteins (components of the mature virus) and nonstructural proteins (involved in replicating the viral genome) (15,20). The nonstructural protein 5A (NS5A) is part of the intracellular membrane-associated viral replication complex (15). Mutations in NS5A affect the rate of HCV replication (3). NS5A has generated considerable interest because of a postulated role in determining the response to interferon (21). A variety of host proteins have been reported to interact with NS5A, although for most their precise relevance in the HCV life cycle awaits definition.Like that of all positive-strand RNA viruses, HCV replication occurs in intimate association with specific intracellular membrane structures, which for HCV has been termed the membranous web (8). What host machinery is exploited to establish these sites of viral replication is unknown. Rab GTPases are small GTP-binding proteins that regulate vesicular-membrane-trafficking pathways, behaving as membraneassociated molecular switches (22). Rab proteins have been previously implicated in the life cycles of various enveloped viruses and are utilized by these viruses for endocytosis, trafficking, and sorting of their proteins (see, for example, Vonderheit and Helenius [33]). Because NS5A has been implicated in the membrane-associated replication of HCV (10), we hypothesized that among the host cell factors that associate with NS5A might be components of host cell membrane-trafficking machinery. To test this hypothesis, we used the full-length NS5A from genotype 1b as bait to screen a human liver cDNA prey library in a GAL4-based yeast two-hybrid screen. We have identified a TBC protein family member, TBC1D20, as a binding partner of HCV NS5A. TBC domain proteins contain an ϳ200-amino-acid motif initially identified in the TRE2/BUB2/ CDC16 genes (25). Like all TBC family members, TBC1D20 contains a Rab-GTPase-activating protein (GAP) homology domain required fo...