Interaction of Rotaviruses with Hsc70 during Cell Entry Is Mediated by VP5

Abstract

Rotavirus infection seems to be a multistep process in which the viruses are required to interact with several cell surface molecules to enter the cell. The virus spike protein VP4, which is cleaved by trypsin into two subunits, VP5 and VP8, is involved in some of these interactions. We have previously shown that the neuraminidase-sensitive rotavirus strain RRV initially attaches to a sialic acid-containing cell molecule through the VP8 subunit of VP4 and subsequently interacts with integrin ␣2␤1 through VP5. After these initial contacts, the virus interacts with at least two additional proteins located at the cell surface, the integrin ␣v␤3 and the heat shock cognate protein Hsc70. In this work, we have shown that rotavirus RRV and its neuraminidase-

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

“…We have previously established that rotavirus particles interact with hsc70 through a region near the carboxy-terminal region of VP5 (45). To determine whether the interaction of TLPs with the PB domain of hsc70 was through the same region of VP5, the amino-terminal portion of VP5 (VP5-248; residues 274 to 474) or the carboxy-terminal part of this protein (VP5-474; amino acids 474 to 776) was expressed in bacteria and purified by affinity chromatography (45).…”

“…To determine whether the interaction of TLPs with the PB domain of hsc70 was through the same region of VP5, the amino-terminal portion of VP5 (VP5-248; residues 274 to 474) or the carboxy-terminal part of this protein (VP5-474; amino acids 474 to 776) was expressed in bacteria and purified by affinity chromatography (45). These recombinant polypeptides were preincubated with either the complete hsc70 protein or its PB domain, and these mixtures were then added to ELISA plates coated with TLPs.…”

“…The virus interacts with hsc70 through a domain in VP5 located between amino acid residues 642 and 658, near the carboxyl terminus of VP4 (29,45). In this work, we have shown that hsc70 interacts specifically with rotavirus through its peptide-binding domain, since (i) a recombinant full-length hsc70 protein and its peptide-binding domain, but not its ATPase domain, bound TLPs in a solid-phase assay; (ii) this binding was competed by preincubation of the protein with the carboxy-terminal region of VP5 (which contains the VP5 domain known to interact with hsc70), but not with its amino-terminal portion; and (iii) known ligands of hsc70 competed the binding of this protein to TLPs in the solid-phase assay.…”

“…After these initial contacts, RRV interacts with at least three additional proteins located at the cell surface, i.e., integrins ␣v␤3 and ␣x␤2 (19,22) and the heat shock cognate protein hsc70 (20); whether these last three interactions occur sequentially or alternatively has not been determined. The virus interaction with hsc70 is mediated by a domain in VP5 located between amino acids 642 and 659 of the protein, and this interaction takes place at a postattachment step, since a synthetic peptide that mimics this region (peptide KID) and antibodies to hsc70 block the infectivity of rotavirus but not its binding to the cell surface (20,45).hsc70 is a constitutive member of the heat shock-induced hsp70 protein family. The proteins in this family are evolutionarily conserved, but they are not functionally interchangeable; the functional diversity of these molecular chaperones may result from variations in their abilities to bind to different target proteins (15).…”

Hsp70 Negatively Controls Rotavirus Protein Bioavailability in Caco-2 Cells Infected by the Rotavirus RF Strain

“…Other heat shock proteins have already been shown to be involved in rotavirus infection. The 70-kDa heat shock cognate protein (Hsc70), whose level is not modified upon rotavirus infection, has been shown to play a role in the entry process of the virus (15,41) thanks to its well-known involvement in the clathrin-coated vesicle formation process (18). Previous works have shown that the levels of other members of the Hsp superfamily, namely, Grp78 and Grp94, also increased upon rotavirus infection of MA104 cells (8,39), but the exact function of this phenomenon has not been documented further.…”