Rotavirus strains differ in their need for sialic acid (SA) for initial binding to the cell surface; however, the existence of a postattachment cell receptor, common to most, if not all, rotavirus strains, has been proposed. In the present study, antibodies to the α _v and β ₃ integrin subunits, and the α _v β ₃ ligand, vitronectin, efficiently blocked the infectivity of the SA-dependent rhesus rotavirus RRV, its SA-independent variant nar3, and the neuraminidase-resistant human rotavirus strain Wa. Vitronectin and anti-β ₃ antibodies, however, did not block the binding of virus to cells, indicating that rotaviruses interact with α _v β ₃ at a postbinding step, probably penetration. This interaction was shown to be independent of the tripeptide motif arginine-glycine-aspartic acid present in the natural ligands of this integrin. Transfection of CHO cells with α _v β ₃ genes significantly increased their permissiveness to all three rotavirus strains, and the increment of virus infectivity was reverted by incubation of these cells either with antibodies to β ₃ or with vitronectin. These findings implicate α _v β ₃ integrin as a cellular receptor common to neuraminidase-sensitive and neuraminidase-resistant rotaviruses, and support the hypothesis that this integrin could determine, at least in part, the cellular susceptibility to rotaviruses.

In this work, we have identified the heat shock cognate protein (hsc70) as a receptor candidate for rotaviruses. hsc70 was shown to be present on the surface of MA104 cells, and antibodies to this protein blocked rotavirus infectivity, while not affecting the infectivity of reovirus and poliovirus. Preincubation of the hsc70 protein with the viruses also inhibited their infectivity. Triple-layered particles (mature virions), but not double-layered particles, bound hsc70 in a solid-phase assay, and this interaction was blocked by monoclonal antibodies to the virus surface proteins VP4 and VP7. Rotaviruses were shown to interact with hsc70 at a postattachment step, since antibodies to hsc70 and the protein itself did not inhibit the virus attachment to cells. We propose that the functional rotavirus receptor is a complex of several cell surface molecules that include, among others, hsc70.

Human immunodeficiency virus (HIV) infects different organs and tissues. During these infection events, subpopulations of HIV type 1 (HIV-1) develop and, if viral trafficking is restricted between subpopulations, the viruses can follow independent evolutionary histories, i.e., become compartmentalized. This phenomenon is usually detected via comparative sequence analysis and has been reported for viruses isolated from the central nervous system (CNS) and the genital tract. Several approaches have been proposed to study the compartmentalization of HIV sequences, but to date, no rigorous comparison of the most commonly employed methods has been made. In this study, we systematically compared inferences made by six different methods for detecting compartmentalization based on three data sets: (i) a sample of 45 patients with sequences gathered from the CNS, (ii) sequences from the female genital tract of 18 patients, and (iii) a set of simulated sequences. We found that different methods often reached contradictory conclusions. Methods based on the topology of a phylogenetic tree derived from clonal sequences were generally more sensitive in detecting compartmentalization than those that relied solely upon pairwise genetic distances between sequences. However, as the branching structure in a phylogenetic tree is often uncertain, especially for short, low-diversity, or recombinant sequences, tree-based approaches may need to be modified to take phylogenetic uncertainty into account. Given the frequently discordant predictions of different methods and the strengths and weaknesses of each particular methodology, we recommend that a suite of several approaches be used for reliable inference of compartmentalized population structure.

We have tested the effect of metabolic inhibitors, membrane cholesterol depletion, and detergent extraction of cell surface molecules on the susceptibility of MA104 cells to infection by rotaviruses. Treatment of cells with tunicamycin, an inhibitor of protein N glycosylation, blocked the infectivity of the SA-dependent rotavirus RRV and its SA-independent variant nar3 by about 50%, while the inhibition of O glycosylation had no effect. The inhibitor of glycolipid biosynthesis d , l -threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) blocked the infectivity of RRV, nar3, and the human rotavirus strain Wa by about 70%. Sequestration of cholesterol from the cell membrane with β-cyclodextrin reduced the infectivity of the three viruses by more than 90%. The involvement of N-glycoproteins, glycolipids, and cholesterol in rotavirus infection suggests that the virus receptor(s) might be forming part of lipid microdomains in the cell membrane. MA104 cells incubated with the nonionic detergent octyl-β-glucoside (OG) showed a ca. 60% reduction in their ability to bind rotaviruses, the same degree to which they became refractory to infection, suggesting that OG extracts the potential virus receptor(s) from the cell surface. Accordingly, when preincubated with the viruses, the OG extract inhibited the virus infectivity by more than 95%. This inhibition was abolished when the extract was treated with either proteases or heat but not when it was treated with neuraminidase, indicating the protein nature of the inhibitor. Two protein fractions of around 57 and 75 kDa were isolated from the extract, and these fractions were shown to have rotavirus-blocking activity. Also, antibodies to these fractions efficiently inhibited the infectivity of the viruses in untreated as well as in neuraminidase-treated cells. Five individual protein bands of 30, 45, 57, 75, and 110 kDa, which exhibited virus-blocking activity, were finally isolated from the OG extract. These proteins are good candidates to function as rotavirus receptors.

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 α2β1 through VP5. After these initial contacts, the virus interacts with at least two additional proteins located at the cell surface, the integrin αvβ3 and the heat shock cognate protein Hsc70. In this work, we have shown that rotavirus RRV and its neuraminidase-resistant variant nar3 interact with Hsc70 through a VP5 domain located between amino acids 642 and 658 of the protein. This conclusion is based on the observation that a recombinant protein comprising the 300 carboxy-terminal amino acids of VP5 binds specifically to Hsc70 and a synthetic peptide containing amino acids 642 to 658 competes with the binding of the RRV and nar3 viruses to the heat shock protein. The VP5 peptide also competed with the binding to Hsc70 of the recombinant VP5 protein, and an antibody to Hsc70 reduced the binding of the recombinant protein to the surface of MA104 cells. The fact that the synthetic peptide blocks the infectivity of rotaviruses RRV and nar3 but not their binding to cells indicates that the interaction of VP5 with Hsc70 most probably occurs at a postattachment step during the virus entry process.

Some animal rotaviruses require the presence of sialic acid (SA) on the cell surface to infect the cell. We have isolated variants of rhesus rotavirus (RRV) whose infectivity no longer depends on SA. Both the SA-dependent and -independent interactions of these viruses with the cell are mediated by the virus spike protein VP4, which is cleaved by trypsin into two domains, VP5 and VP8. In this work we have compared the binding characteristics of wild-type RRV and its variant nar3 to MA104 cells. In a direct nonradioactive binding assay, both viruses bound to the cells in a saturable and specific manner. When neutralizing monoclonal antibodies directed to both the VP8 and VP5 domains of VP4 were used to block virus binding, antibodies to VP8 blocked the cell attachment of wild-type RRV but not that of the variant nar3. Conversely, an antibody to VP5 inhibited the binding of nar3 but not that of RRV. These results suggest that while RRV binds to the cell through VP8, the variant does so through the VP5 domain of VP4. This observation was further sustained by the fact that recombinant VP8 and VP5 proteins, produced in bacteria as fusion products with glutathione S -transferase, were found to bind to MA104 cells in a specific and saturable manner and, when preincubated with the cell, were capable of inhibiting the binding of wild-type and variant viruses, respectively. In addition, the VP5 and VP8 recombinant proteins inhibited the infectivity of nar3 and RRV, respectively, confirming the results obtained in the binding assays. Interestingly, when the infectivity assay was performed on neuraminidase-treated cells, the VP5 fusion protein was also found to inhibit the infectivity of RRV, suggesting that RRV could bind to the cell through two sequential steps mediated by the interaction of VP8 and VP5 with SA-containing and SA-independent cell surface receptors, respectively.