Interaction of rotaviruses with Hsc70 during cell entry is mediated by VP5

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 alpha2beta1 through VP5. After these initial contacts, the virus interacts with at least two additional proteins located at the cell surface, the integrin alphavbeta3 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.     

The VP5 domain of VP4 can mediate attachment of rotaviruses to cells

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.     

Binding to sialic acids is not an essential step for the entry of animal rotaviruses to epithelial cells in culture.

The infection of target cells by animal rotaviruses requires the presence of sialic acids on the cell surface. Treatment of the cells with neuraminidases or incubation of the viruses with some sialoglycoproteins, such as glycophorin A, greatly reduces virus binding, with the consequent reduction of viral infectivity. In this work, we report the isolation of animal rotavirus variants whose infectivity is no longer dependent on the presence of sialic acids on the cell surface. In addition, although these variants bind to glycophorin A as efficiently as the wild-type virus, this interaction no longer inhibit viral infectivity. These observations indicate that the initial interaction of the mutants with the cell occurs at a site different from the sialic acid-binding site located on VP8, the smaller trypsin cleavage product of VP4. Reassortant analysis showed that the mutant phenotype segregates with the VP4 gene. Neutralizing monoclonal antibodies directed to VP4 and VP7 were tested for their ability to neutralize the variants. Antibodies to VP7 and VP5, the larger trypsin cleavage product of VP4, neutralized the mutants as efficiently as the wild-type virus. In contrast, although antibodies to VP8 were able to bind to the mutants, they showed little or no neutralizing activity. The implications of these findings in rotavirus attachment to and penetration of epithelial cells in culture are discussed.     

Integrin-using rotaviruses bind alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognize alphaXbeta2 and alphaVbeta3 by using VP7 during cell entry

Integrins alpha2beta1, alphaXbeta2, and alphaVbeta3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the alpha2beta1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the alphaXbeta2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of alpha2beta1, alphaXbeta2, and alphaVbeta3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound alpha2beta1, and VP7 interacted with alphaXbeta2 and alphaVbeta3 at a postbinding stage. DGEA inhibited rotavirus binding to alpha2beta1 and infectivity, whereas GPRP binding to alphaXbeta2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed alpha2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the alpha2 I domain. In a novel process, integrin-using viruses bind the alpha2 I domain of alpha2beta1 via DGE in VP4 and interact with alphaXbeta2 (via GPR) and alphaVbeta3 by using VP7 to facilitate cell entry and infection.     

Proteolytic enhancement of rotavirus infectivity: molecular mechanisms

The polypeptide compositions of single-shelled and double-shelled simian rotavirus particles were modified by exposure to proteolytic enzymes. Specifically, a major outer capsid polypeptide (VP3) having a molecular weight of 88,000 in double-shelled particles was cleaved by trypsin to yield two polypeptides, VP5* and VP8* (molecular weights, 60,000 and 28,000, respectively). The cleavage of VP3 by enzymes that enhanced infectivity (trypsin, elastase, and pancreatin) yielded different products compared to those detected when VP3 was cleaved by chymotrypsin, which did not enhance infectivity. The appearance of VP5* was correlated with an enhancement of infectivity. Cleavages of the major internal capsid polypeptide VP2 were also observed. The VP2 cleavage products had molecular weights similar to those of known structural and nonstructural rotavirus polypeptides. We confirmed the precursor-product relationships by comparing the peptide maps of the polypeptides generated by digestions with V-8 protease and chymotrypsin. The remaining rotavirus structural polypeptides, including the outer capsid glycoproteins (VP7 and 7a), were not altered by exposure to pancreatic enzymes. Cleavage of VP3 was not required for virus assembly, and specific cleavage of the polypeptides occurred only on assembled particles. We also discuss the role of cleavage activation in other virus-specific biological functions (e.g., hemagglutination and virulence).     

Antibodies to the trypsin cleavage peptide VP8 neutralize rotavirus by inhibiting binding of virions to target cells in culture.

Two distinct patterns of neutralization were identified by comparing the neutralization curves of monoclonal antibodies (MAbs) directed at the two surface proteins, VP4 and VP7, of rhesus rotavirus. VP7-specific MAbs were able to neutralize virus efficiently, and slight increases in antibody concentration resulted in a sharp decline in infectivity. On the other hand, MAbs to VP4 proved much less efficient at neutralizing rhesus rotavirus, and the fraction of infectious virus decreased gradually throughout a wide range of antibody concentrations. MAbs directed at VP8*, the smaller trypsin cleavage fragment of VP4, were shown to efficiently prevent binding of radiolabeled virions to MA104 cell monolayers, to an extent and at concentrations comparable to those required for neutralization of infectivity. Conversely, MAbs recognizing VP7 or the larger VP4 trypsin cleavage product, VP5*, showed little or no inhibitory effect on virus binding to cells. All MAbs studied were able to neutralize rotavirus that was already bound to the surface of cells. The MAbs directed at VP8*, but not those recognizing VP5* or VP7, were shown to mediate release of radiolabeled virus from the surface of the cells. With MAbs directed at VP7, papain digestion of virus-bound antibody molecules led to an almost complete recovery of infectivity. Neutralization could be fully restored by incubation of virus-Fab complexes with anti-mouse immunoglobulin G antiserum. Neutralization with MAbs directed at VP8* proved insensitive to digestion with papain as well as to the addition of anti-immunoglobulin antibodies.     

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis.

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. Cleavage of VP3 does not alter viral binding to cell monolayers. In previous electron microscopic studies of infected cell cultures, it has been demonstrated that rotavirus particles enter cells by both endocytosis and direct cell membrane penetration. To determine whether trypsin treatment affected rotavirus internalization, we studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Endocytosis inhibitors (sodium azide, dinitrophenol) and lysosomotropic agents (ammonium chloride, chloroquine) had a limited effect on the entry of infectious virus into cells. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 medicated 51Cr, [14C]choline, and [3H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.     

Effects on rotavirus cell binding and infection of monomeric and polymeric peptides containing alpha2beta1 and alphaxbeta2 integrin ligand sequences

Integrin-using rotaviruses bind MA104 cell surface alpha2beta1 integrin via the Asp-Gly-Glu (DGE) sequence in virus spike protein VP4 and interact with alphaxbeta2 integrin during cell entry through outer capsid protein VP7. Infection is inhibited by the alpha2beta1 ligand Asp-Gly-Glu-Ala (DGEA) and the alphaxbeta2 ligand Gly-Pro-Arg-Pro (GPRP), and virus-alpha2beta1 binding is increased by alpha2beta1 activation. In this study, we analyzed the effects of monomers and polymers containing DGEA-, GPRP-, and DGEA-related peptides on rotavirus binding and infection in intestinal (Caco-2) and kidney (MA104) cells and virus binding to recombinant alpha2beta1. Blockade of rotavirus-cell binding and infection by peptides and anti-alpha2 antibody showed that Caco-2 cell entry is dependent on virus binding to alpha2beta1 and interaction with alphaxbeta2. At up to 0.5 mM, monomeric DGEA and DGAA inhibited binding to alpha2beta1 and infection. At higher concentrations, DGEA and DGAA showed a reduced ability to inhibit virus-cell binding and infection that depended on virus binding to alpha2beta1 but occurred without alteration in cell surface expression of alpha2, beta2, or alphavbeta3 integrin. This loss of DGEA activity was abolished by genistein treatment and so was dependent on tyrosine kinase signaling. It is proposed that this signaling activated existing cell surface alpha2beta1 to increase virus-cell attachment and entry. Polymeric peptides containing DGEA and GPRP or GPRP only were inhibitory to SA11 infection at approximately 10-fold lower concentrations than peptide monomers. As polymerization can improve peptide inhibition of virus-receptor interactions, this approach could be useful in the development of inhibitors of receptor recognition by other viruses.     

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.     

The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site

Cell attachment and membrane penetration are functions of the rotavirus outer capsid spike protein, VP4. An activating tryptic cleavage of VP4 produces the N-terminal fragment, VP8*, which is the viral hemagglutinin and an important target of neutralizing antibodies. We have determined, by X-ray crystallography, the atomic structure of the VP8* core bound to sialic acid and, by NMR spectroscopy, the structure of the unliganded VP8* core. The domain has the beta-sandwich fold of the galectins, a family of sugar binding proteins. The surface corresponding to the galectin carbohydrate binding site is blocked, and rotavirus VP8* instead binds sialic acid in a shallow groove between its two beta-sheets. There appears to be a small induced fit on binding. The residues that contact sialic acid are conserved in sialic acid-dependent rotavirus strains. Neutralization escape mutations are widely distributed over the VP8* surface and cluster in four epitopes. From the fit of the VP8* core into the virion spikes, we propose that VP4 arose from the insertion of a host carbohydrate binding domain into a viral membrane interaction protein.     

Proteolysis of Monomeric Recombinant Rotavirus VP4 Yields an Oligomeric VP5* Core

Rotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of beta-sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.     

Role of VP3 in human rotavirus internalization after target cell attachment via VP7.

A cell lysate prepared from MA104 cells that had been infected with human rotavirus KUN strain (HRV-KUN) contained a 35-kilodalton protein capable of binding to MA104 cells. The binding of the 35-kilodalton protein was inhibited by a serotype 2-specific antiserum but not by antisera to other serotypes. Not only trypsin-treated, infectious HRV-KUN but also untreated, noninfectious virions effectively competed with the 35-kilodalton protein for the same cell surface binding sites. One monoclonal anti-VP7 (AH6) absorbed the 35-kilodalton protein from the HRV-KUN-infected cell lysate, whereas another monoclonal anti-VP7 (S2-2G10) inhibited the virions to compete with the 35-kilodalton protein for the cell surface binding sites. Both anti-VP7 (S2-2G10) and anti-VP3 (K-1532, K-376) monoclonal antibodies had the virus-neutralization activity, but only anti-VP7 inhibited virus adsorption. On the other hand, anti-VP3 monoclonal antibodies were capable of completely inhibiting the infection of preadsorbed HRV-KUN as long as virions were not yet internalized. Subsequent studies with [35S]methionine-labeled and purified HRV-KUN showed that not only trypsin-treated, infectious virions but also untreated, noninfectious virions were capable of efficient target cell binding and internalization. The internalization modes of these two HRV-KUN preparations were, however, quite different. Only the components of the inner capsid were internalized from trypsin-treated virions, whereas no such selective internalization was seen with untreated virions. Furthermore, anti-VP3 inhibited this selective internalization of the inner capsid from the infectious virions. From these results we conclude that VP7 is the HRV-KUN cell attachment protein and that adsorption of HRV-KUN via VP7 is independent of trypsin treatment, whereas the limited cleavage of VP3 by trypsin, which is essential for the development of HRV-KUN infectivity, is needed for the selective internalization of the inner capsid components, a process that is apparently essential for HRV-KUN infection.     

Interactions between the two surface proteins of rotavirus may alter the receptor-binding specificity of the virus.

The infection of target cells by most animal rotavirus strains requires the presence of sialic acids (SAs) on the cell surface. We recently isolated variants from simian rotavirus RRV whose infectivity is no longer dependent on SAs and showed that the mutant phenotype segregates with the gene coding for VP4, one of the two surface proteins of rotaviruses (the other one being VP7). The nucleotide sequence of the VP4 gene of four independently isolated variants showed three amino acid changes, at positions 37 (Leu to Pro), 187 (Lys to Arg), and 267 (Tyr to Cys), in all mutant VP4 proteins compared with RRV VP4. The characterization of revertant viruses from two independent mutants showed that the arginine residue at position 187 changed back to lysine, indicating that this amino acid is involved in the determination of the mutant phenotype. Surprisingly, sequence analysis of reassortant virus DS1XRRV, which depends on SAs to infect the cell, showed that its VP4 gene is identical to the VP4 gene of the variants. Since the only difference between DS1XRRV and the RRV variants is the parental origin of the VP7 gene (human rotavirus DS1 in the reassortant), these findings suggest that the receptor-binding specificity of rotaviruses, via VP4, may be influenced by the associated VP7 protein.     

Biochemical characterization of rotavirus receptors in MA104 cells

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 beta-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-beta-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.     

Heat shock cognate protein 70 is involved in rotavirus cell entry

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.     

Integrin alphavbeta8 functions as a receptor for foot-and-mouth disease virus: role of the beta-chain cytodomain in integrin-mediated infection

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three alphav integrins, alphavbeta1, alphavbeta3, and alphavbeta6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human beta8 cDNA and expression of alphavbeta8 at the cell surface. The involvement of alphavbeta8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the alphavbeta8 heterodimer or the alphav chain. Similar results were obtained with a chimeric alphavbeta8 including the beta6 cytodomain (alphavbeta8/6), showing that the beta6 cytodomain can substitute efficiently for the corresponding region of beta8. In contrast, virus binding to alphavbeta6 including the beta8 cytodomain (alphavbeta6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the alphavbeta6 chimera was recognized poorly by antibodies specific for the ectodomain of alphavbeta6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the beta6 cytodomain is important for maintaining alphavbeta6 in a conformation required for productive infection by FMDV.     

Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.     

The VP8* Domain of Neonatal Rotavirus Strain G10P[11] Binds to Type II Precursor Glycans

Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. Interaction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans, VP8* P[11] showed specific binding to glycans with the Galβ1-4GlcNAc motif, which forms the core structure of type II glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythrocytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bovine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understanding neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-restricted infectivity of neonatal viruses.     

Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum.

The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (H1 and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell. For a variant retaining only the H1 domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking H1 and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge.