The Peptide-Binding and ATPase Domains of Recombinant hsc70 Are Required To Interact with Rotavirus and Reduce Its Infectivity

Abstract

The heat shock cognate protein hsc70 has been implicated as a postattachment cell receptor for rotaviruses. Here we show that hsc70 interacts specifically with rotaviruses through its peptide-binding domain, since a recombinant full-length hsc70 protein and its peptide-binding domain, but not its ATPase domain, bound triple-layered particles in a solid-phase assay, and known ligands of hsc70 competed this binding. The peptide ligands of hsc70 were also shown to block rotavirus infectivity when added to cells before virus infection, suggesting that hsc70 on the surface of MA104 cells also interacts with the virus through its peptide-binding domain and that this interaction is important for virus entry. When purified infectious virus was incubated with soluble hsc70 in the presence of the cochaperone hsp40 and ATP and then pelleted through a sucrose cushion, the recovered virus had lost 60% of its infectivity, even though hsc70 was not detected in the pellet fraction. The hsc70-treated virus showed slightly different reactivities with monoclonal antibodies and was more susceptible to heat and basic pHs than the untreated virus, suggesting that hsc70 induces a subtle conformational change in the virus that results in a reduction of its infectivity. The relevance of the ATPase activity of hsc70 for reducing virus infectivity was demonstrated by the finding that in the presence of a nonhydrolyzable analogue of ATP, virus infectivity was not affected, and a mutant protein lacking ATPase activity failed to reduce virus infection. Altogether, these results suggest that during cell infection, the interaction of the virus with hsc70 on the surface of MA104 cells results in a conformational change of virus particles that facilitates their entry into the cell cytoplasm.Rotaviruses are the single most important cause of severe dehydrating diarrhea in young children worldwide. These nonenveloped viruses are formed by a triple-layered protein capsid which surrounds the genome, composed of 11 segments of double-stranded RNA (11). The outermost layer, which is responsible for the initial interactions of the virus with the cell surface, consists of the following two proteins: VP7, a glycoprotein that forms the smooth surface of the virion, and VP4, which forms the spikes that extend from the surface of the virus particle. Both proteins play essential roles during the early interactions of the virus with the cell surface, including receptor binding and cell penetration (11,29). To be infectious, rotaviruses depend on the proteolytic cleavage of VP4 (776 amino acids [aa]) into subunits VP8 (aa 1 to 247) and VP5 (aa 248 to 776); this cleavage does not affect cell binding but is required for entry of the virus into the cell's cytoplasm (17,25,29). Rotaviruses have a very specific cell tropism, infecting primarily the mature enterocytes at the tips of the villi of the small intestine, and the susceptibility of these cells seems to be limited to a narrow age window (26). In cell culture, rotavirus binds to a large variety of cell types...

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

“…132 However, it has been shown that Hsc70 retains its substrate binding capacity in the absence of ATPase activity; 133 the intact peptide-binding domain of sHsc70 would thus be expected to be functional as a binding protein.…”

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

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Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

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