Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.     

Rotavirus capsid protein VP5* permeabilizes membranes

Proteolytic cleavage of the VP4 outer capsid spike protein into VP8* and VP5* proteins is required for rotavirus infectivity and for rotavirus-induced membrane permeability. In this study we addressed the function of the VP5* cleavage fragment in permeabilizing membranes. Expressed VP5* and truncated VP5* proteins were purified by nickel affinity chromatography and assayed for their ability to permeabilize large unilamellar vesicles (LUVs) preloaded with carboxyfluorescein (CF). VP5* and VP5* truncations, but not VP4 or VP8*, permeabilized LUVs as measured by fluorescence dequenching of released CF. Similar to virus-induced CF release, VP5*-induced CF release was concentration and temperature dependent, with a pH optimum of 7.35 at 37 degrees C, but independent of the presence of divalent cations or cholesterol. VP5*-induced permeability was completely inhibited by VP5*-specific neutralizing monoclonal antibodies (2G4, M2, or M7) which recognize conformational epitopes on VP5* but was not inhibited by VP8*-specific neutralizing antibodies. In addition, N-terminal and C-terminal VP5* truncations including residues 265 to 474 are capable of permeabilizing LUVs. These findings demonstrate that VP5* permeabilizes membranes in the absence of other rotavirus proteins and that membrane-permeabilizing VP5* truncations contain the putative fusion region within predicted virion surface domains. The ability of recombinant expressed VP5* to permeabilize membranes should permit us to functionally define requirements for VP5*-membrane interactions. These findings indicate that VP5* is a specific membrane-permeabilizing capsid protein which is likely to play a role in the cellular entry of rotaviruses.     

Membrane vesicles containing the Sendai virus binding glycoprotein, but not the viral fusion protein, fuse with phosphatidylserine liposomes at low pH

Membrane vesicles containing the Sendai virus hemagglutinin/neuraminidase (HN) glycoprotein were able to induce carboxyfluorescein (CF) release from loaded phosphatidylserine (PS) but not loaded phosphatidylcholine (PC) liposomes. Similarly, fluorescence dequenching was observed only when HN vesicles, bearing self-quenched N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE), were incubated with PS but not PC liposomes. Thus, fusion between Sendai virus HN glycoprotein vesicles and the negatively charged PS liposomes is suggested. Induction of CF release and fluorescence dequenching were not observed when Pronase-treated HN vesicles were incubated with the PS liposomes. On the other hand, the fusogenic activity of the HN vesicles was not inhibited by treatment with dithiothreitol (DTT) or phenylmethanesulfonyl fluoride (PMSF), both of which are known to inhibit the Sendai virus fusogenic activity. Fusion was highly dependent on the pH of the medium, being maximal after an incubation of 60-90 s at pH 4.0. Electron microscopy studies showed that incubation at pH 4.0 of the HN vesicles with PS liposomes, both of which are of an average diameter of 150 nm, resulted in the formation of large unilamellar vesicles, the average diameter of which reached 450 nm. The relevance of these observations to the mechanism of liposome-membrane and virus-membrane fusion is discussed.     

Assembly of highly infectious rotavirus particles recoated with recombinant outer capsid proteins

Assembly of the rotavirus outer capsid is the final step of a complex pathway. In vivo, the later steps include a maturational membrane penetration that is dependent on the scaffolding activity of a viral nonstructural protein. In vitro, simply adding the recombinant outer capsid proteins VP4 and VP7 to authentic double-layered rotavirus subviral particles (DLPs) in the presence of calcium and acidic pH increases infectivity by a factor of up to 10(7), yielding particles as infectious as authentic purified virions. VP4 must be added before VP7 for high-level infectivity. Steep dependence of infectious recoating on VP4 concentration suggests that VP4-VP4 interactions, probably oligomerization, precede VP4 binding to particles. Trypsin sensitivity analysis identifies two populations of VP4 associated with recoated particles: properly mounted VP4 that can be specifically primed by trypsin, and nonspecifically associated VP4 that is degraded by trypsin. A full complement of properly assembled VP4 is not required for efficient infectivity. Minimal dependence of recoating on VP7 concentration suggests that VP7 binds DLPs with high affinity. The parameters for efficient recoating and the characterization of recoated particles suggest a model in which, after a relatively weak interaction between oligomeric VP4 and DLPs, VP7 binds the particles and locks VP4 in place. Recoating will allow the use of infectious modified rotavirus particles to explore rotavirus assembly and cell entry and could lead to practical applications in novel immunization strategies.     

Active function of membrane receptors for enveloped viruses : I. Specific requirement for liposome-associated sialoglycolipids, but not sialoglycoproteins, to allow lysis of phospholipid vesicles by reconstituted Sendai viral envelopes

Phospholipid liposomes composed of phosphatidylcholine (PC) and cholesterol (chol), bearing the sialoglycoprotein glycophorin (GP), are able to effectively bind Sendai virus particles, but not to be lysed by them. Incorporation of gangliosides (gangl) into the above phospholipid vesicles (yielding liposomes composed of PC/chol/gangl/GP), although not increasing their ability to interact with Sendai virions, rendered them susceptible to the viral lytic activity. This was inferred from the ability of the virus to induce release of carboxyfluorescein (CF) upon interaction at 37 degrees C with liposomes composed of PC/chol/gangl/GP. Lysis of liposomes required the presence of the two viral envelope glycoproteins, namely the hemagglutinin/neuraminidase (HN) and the fusion (F) polypeptides, and was inhibited by phenylmethyl sulfonylfluoride (PMSF), dithiothreitol (DTT) and trypsin, showing that virus-induced lysis of PC/chol/gangl/GP liposomes reflects the fusogenic activity of the virus. Incubation of Sendai virus particles with liposomes containing the acidic phospholipid dicetylphosphate (DCP) but lacking sialic acid containing receptors, also resulted in release of the liposome content. Lysis of these liposomes was due to the activity of the viral HN glycoprotein, therefore not reflecting the natural viral fusogenic activity. Fluorescence dequenching studies, using fluorescently labeled reconstituted Sendai virus envelopes (RSVE), have shown that the viral envelopes are able to fuse with neutral, almost to the same extent, as with negatively charged liposomes. However, fusion with negatively charged liposomes, as opposed to fusion with neutral liposomes, was mediated by the viral HN glycoprotein and not by the viral fusion polypeptide.     

Heterogeneity of Raft-type membrane microdomains associated with VP4, the rotavirus spike protein, in Caco-2 and MA 104 cells

Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-beta-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.     

Dissecting rotavirus particle-raft interaction with small interfering RNAs: insights into rotavirus transit through the secretory pathway

Studies of rotavirus morphogenesis, transport, and release have shown that although these viruses are released from the apical surface of polarized intestinal cells before cellular lysis, they do not follow the classic exocytic pathway. Furthermore, increasing evidence suggests that lipid rafts actively participate in the exit of rotavirus from the infected cell. In this study, we silenced the expression of VP4, VP7, and NSP4 by using small interfering RNAs (siRNAs) and evaluated the effect of shutting down the expression of these proteins on rotavirus-raft interactions. Silencing of VP4 and NSP4 reduced the association of rotavirus particles with rafts; in contrast, inhibition of VP7 synthesis slightly affected the migration of virions into rafts. We found that inhibition of rotavirus migration into lipid rafts, by either siRNAs or tunicamycin, also specifically blocked the targeting of VP4 to rafts, suggesting that the association of VP4 with rafts is mostly mediated by the formation of viral particles in the endoplasmic reticulum (ER). We showed that two populations of VP4 exist, one small population that is independently targeted to rafts and a second large pool of VP4 whose association with rafts is mediated by particle formation in the ER. We also present evidence to support the hypothesis that assembly of VP4 into mature virions takes place in the late stages of transit through the ER. Finally, we analyzed the progression of rotavirus proteins in the exocytic pathway and found that VP4 and virion-assembled VP7 colocalized with ERGIC-53, suggesting that rotavirus particles transit through the intermediate compartment between the ER and the Golgi complex.     

Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.     

Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells.

Rotaviruses are triple-layered particles that contain four major capsid proteins, VP2, VP4, VP6, and VP7, and two minor proteins, VP1 and VP3. We have cloned each of the rotavirus genes coding for a major capsid protein into the baculovirus expression system and expressed each protein in insect cells. Coexpression of different combinations of the rotavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The coexpression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similar to double-layered rotavirus particles. Coexpression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of nonneutralizing and neutralizing epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs. The production of VP2/4/6 particles indicated that VP4 interacts with VP6. Cell binding assays performed with each of the VLPs indicated that VP4 is the viral attachment protein. Chimeric particles containing VP7 from two different G serotypes also were obtained. The ability to express individual proteins or to coexpress different subsets of proteins provides a system with which to examine the interactions of the rotavirus structural proteins, the role of individual proteins in virus morphogenesis, and the feasibility of a subunit vaccine.     

Temperature-dependent kinetics of the activities of influenza virus

The temperature dependence of membrane interactions between PR8 influenza virus and virus receptor (GD1a)-containing liposomes was studied. For quantitation, the octadecylrhodamine B chloride (R18) membrane marker was incorporated into liposomes at quenched concentrations. Upon interaction with target membranes, the marker gets diluted, and dequenching can be measured in a fluorescence spectrophotometer. Rate constants were calculated from the dequenching curves under low pH conditions, which allow for fusion, and at neutral pH, where no specific fusion occurs. Activation energies were determined from Arrhenius plots. The results were compared with the temperature dependence of other viral activities like infectivity, hemolysis, and fusion with erythrocytes. For the slow reaction at pH 7.4, where only non-specific lipid transfer takes place, the activation energy was about 24 kcal/mole between 15 degrees C and 45 degrees C. For the fast, hemagglutinin (HA)-specific fusion reaction (pH 5.3), a very low activation energy (approximately 7 kcal/mole) was found between 25 degrees C and 37 degrees C, whereas below 25 degrees C it was much higher (approximately 34 kcal/mole). The temperature range with low activation energy coincides with the one for optimal infectivity, hemolysis, and fusion with erythrocytes. Furthermore, it is the same range in which the conformational change of HA takes place, which in the absence of a partner membrane leads to an irreversible inactivation of the fusion protein.     

Characterization of early steps in the poliovirus infection process: receptor-decorated liposomes induce conversion of the virus to membrane-anchored entry-intermediate particles

The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptor-decorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry.     

Rotavirus VP4 and VP7-Derived Synthetic Peptides as Potential Substrates of Protein Disulfide Isomerase Lead to Inhibition of Rotavirus Infection

Rotavirus infection of MA104 cells has been shown to be inhibited by cell membrane-impermeant thiol/disulfide exchange inhibitors and anti-PDI antibodies. To characterise the amino acid sequences of rotavirus structural proteins potentially mediating cell surface PDI?Csubstrate interactions, rotavirus-derived peptides from VP4 and VP7 (RRV) and VP7 (Wa), and their modified versions containing serine instead of cysteine were synthesized. Cysteine-containing VP7 peptides corresponding to residues 189?C210 or 243?C263 caused an infectivity inhibitory effect of about 64 and 85?%, respectively, when added to cells. Changing cysteine to serine significantly decreased the inhibitory effect. A cysteine-containing peptide corresponding to VP4 residues 200?C219 and its scrambled version reduced infectivity by 92 and 80?%, respectively. A cysteine to serine change in the original VP4 200?C219 peptide did not affect its inhibitory effect. Non-rotavirus related sequences containing cysteine residues efficiently inhibited rotavirus infectivity. Antibodies against VP7 residues 189?C210 or 243?C263 significantly inhibited rotavirus infectivity only after virus attachment to cells had occurred, whereas those against VP4 200?C219 peptide inhibited infectivity irrespective of whether virus or cell-attached virus was antibody-treated. A direct PDI?Cpeptide interaction was shown by ELISA for cysteine-containing VP7 and VP4 peptides. Virus?Ccell attachment was unaffected by the peptides inhibiting virus infectivity. The results showed that even though cysteine residues in the peptides tested are important in both virus infectivity inhibition and in vitro PDI?Cpeptide interaction, the accompanying amino acid sequence also plays some role. As a whole, our findings further support our hypothesis that cell surface PDI from MA104 cells might be contributing to rotavirus entry at a post-attachment step.     

Kinetics of fusion and lipid transfer between virus receptor containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay

Octadecylrhodamine B chloride (R18) and ganglioside GD1a (virus receptor) were incorporated into small unilamellar liposomes [Hoekstra et al. (1984) Biochemistry 23, 5675-5681]. Upon interaction of these liposomes with PR8 influenza viruses without prebinding, two types of dequenching were observed at 37 degrees C, both second-order processes: a fast reaction at pH 5.3, 2k = 17.53 x 10(-3) (Q.s)-1, and a slow reaction at pH 7.4, 2k = 0.335 x 10(-3) (Q.s)-1. The maximal level of dequenching was the same for both. Upon prebinding of liposomes to PR8 viruses (30 min, 0 degrees C, pH 7.4) at high concentrations, a very fast dequenching occurred when the prebinding mixture was diluted into prewarmed (37 degrees C) 10 mM PBS, pH 5.3. For the initial phase, a first-order rate constant of 0.5 s-1 could be extrapolated. After a quick drop in velocity during the first 30 s, the reaction was kinetically indistinguishable from the one found without prebinding. A second-order process with 2k = 16.52 x 10(-3) (Q.s)-1 became rate-limiting. The fast reactions at pH 5.3 can be abolished by inactivation or removal of the virus hemagglutinin. We conclude that the reaction at pH 5.3 reflects the hemagglutinin-dependent fusion process known to occur between influenza viruses and partner membranes at low pH; however, second-order kinetics indicate that specific binding rather than fusion is the rate-limiting step. For the slow dequenching, which is not affected by prebinding, the rate constant is 20 times lower than for the fast reaction, and the process is independent of viral hemagglutinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Antibodies to rotavirus outer capsid glycoprotein VP7 neutralize infectivity by inhibiting virion decapsidation

The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90 degrees light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound antibody under conditions that generated Fab fragments but not under conditions that generated F(ab')(2) fragments. Electron microscopy showed that RRV virions reacted with an anti-VP7 MAb stayed as triple-layered particles in the presence of excess EDTA. Furthermore, the infectivity of rotavirus neutralized via VP8*, but not that of rotavirus neutralized via VP7, could be recovered by lipofection of neutralized particles into MA-104 cells. These data are consistent with the notion that antibodies directed at VP8* neutralize by inhibiting binding of virus to the cell. They also indicate that antibodies directed at VP7 neutralize by inhibiting virus decapsidation, in a manner that is dependent on the bivalent binding of the antibody.     

pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization

The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.     

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.     

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.     

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.