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.     

Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4.

Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.     

A synthetic peptide corresponding to the cleavage region of VP3 from rotavirus SA11 induces neutralizing antibodies.

Antibodies were elicited in rabbits by immunization with the synthetic tetradecapeptide Gln-Asn-Thr-Arg-Asn-Ile-Val-Pro-Val-Ser-Ile-Val-Ser-Arg, corresponding to amino acids 228 to 241 of SA11-VP3. Protein specificity of the antipeptide serum is demonstrated. The antipeptide serum revealed neutralizing activity directed against SA11 in a neutralization assay. Human rotavirus strains Wa, S2, and Hochi and bovine strains NCDV and UK were not neutralized, demonstrating the strain-specific neutralizing activity of the raised antipeptide serum. Upon immune electron microscopy, aggregation of SA11 particles was observed.     

Specificity and affinity of sialic acid binding by the rhesus rotavirus VP8* core

Nuclear magnetic resonance spectroscopy demonstrates that the rhesus rotavirus hemagglutinin specifically binds alpha-anomeric N-acetylneuraminic acid with a K(d) of 1.2 mM. The hemagglutinin requires no additional carbohydrate moieties for binding, does not distinguish 3' from 6' sialyllactose, and has approximately tenfold lower affinity for N-glycolylneuraminic than for N-acetylneuraminic acid. The broad specificity and low affinity of sialic acid binding by the rotavirus hemagglutinin are consistent with this interaction mediating initial cell attachment prior to the interactions that determine host range and cell type specificity.     

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.     

Engineered RNase P ribozymes increase their cleavage activities and efficacies in inhibiting viral gene expression in cells by enhancing the rate of cleavage and binding of the target mRNA

Engineered RNase P ribozymes are promising gene-targeting agents that can be used in both basic research and clinical applications. We have previously selected ribozyme variants for their activity in cleaving an mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). The variant exhibited enhanced cleavage and substrate binding and was at least 30 times more efficient in cleaving TK mRNA in vitro than the ribozyme derived from the wild type sequence. Our results provide the first direct evidence to suggest that a point mutation at nucleotide 95 of RNase P catalytic RNA from Escherichia coli (G(95) --> U(95)) increases the rate of cleavage, whereas another mutation at nucleotide 200 (A(200) --> C(200)) enhances substrate binding of the ribozyme. A reduction of about 99% in TK expression was observed in cells expressing the variant, whereas a 70% reduction was found in cells expressing the ribozyme derived from the wild type sequence. Thus, the RNase P ribozyme variant is highly effective in inhibiting HSV-1 gene expression. Our study demonstrates that ribozyme variants increase their cleavage activity and efficacy in blocking gene expression in cells through enhanced substrate binding and rate of cleavage. These results also provide insights into the mechanism of how RNase P ribozymes efficiently cleave an mRNA substrate and, furthermore, facilitate the development of highly active RNase P ribozymes for gene-targeting applications.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

Serotype-specific epitope(s) present on the VP8 subunit of rotavirus VP4 protein.

cDNA clones representing the VP8 and VP5 subunits of VP4 of symptomatic human rotavirus strain KU (VP7 serotype 1 and VP4 serotype 1A) or DS-1 (VP7 serotype 2 and VP4 serotype 1B) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2 and VP4 serotype 2) were constructed and inserted into the pGEMEX-1 plasmid and expressed in Escherichia coli. Immunization of guinea pigs with the VP8 or VP5 protein of each strain induced antibodies that neutralized the rotavirus from which the VP4 subunits were derived. In a previous study (M. Gorziglia, G. Larralde, A.Z. Kapikian, and R. M. Chanock, Proc. Natl. Acad. Sci. USA 87:7155-7159, 1990), three distinct serotypes and one subtype of VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. The results obtained by cross-immunoprecipitation and by neutralization assay with antisera to the VP8- and VP5-expressed proteins suggest that the VP8 subunit of VP4 contains the major antigenic site(s) responsible for serotype-specific neutralization of rotavirus via VP4, whereas the VP5 subunit of VP4 is responsible for much of the cross-reactivity observed among strains that belong to different VP4 serotypes.     

Secretion of the rotavirus VP8* protein in Lactococcus lactis

Secretion of the VP8* subunit of the VP4 capsid protein of rotavirus by Lactococcus lactis has been achieved. For this purpose, a secretion vector has been constructed with the lactococcal signal sequence AL9 and the VP8*-encoding gene fragment. The amount of VP8* secreted by L. lactis in the culture supernatant was quantified and visualised by Western blot. Furthermore, it was shown to retain its hemagglutination capability, indicating that the conformation of the secreted peptide may be retaining its biological activity.     

STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core

The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*-disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide's methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me, Neu5,9Ac(2)alpha2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection.     

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments.

In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.     

Heterotypic passive protection induced by synthetic peptides corresponding to VP7 and VP4 of bovine rotavirus.

We have evaluated the potential of two peptides derived from highly conserved regions of rotavirus outer capsid proteins (VP7 and VP4) to act as a rotavirus vaccine. The capacity of peptides coupled to rotavirus VP6 spherical particles to provide passive protection in a murine model was compared with the protection induced by peptide-keyhole limpet hemocyanin (KLH) conjugates. Female mice were immunized a total of three times before and during pregnancy. Suckling mouse pups were challenged at 7 days of age with either homologous or heterologous rotavirus serotypes. The efficacy of vaccination was determined by analyzing the clinical symptoms and measuring xylose adsorption in the intestine. In this model the VP4 peptide-VP6 conjugate provided protection equal to that obtained using bovine rotavirus (BRV) as the immunogen. The VP7 peptide-VP6 conjugate provided slightly less protection than the VP4 peptide-VP6 conjugate. A mixture of the VP4 peptide-VP6 and VP7 peptide-VP6 conjugates provided better heterologous protection than immunization with BRV. In contrast, KLH-conjugated peptides provided only partial protection. The significance of a synthetic-peptide-based rotavirus vaccine in the prevention of rotavirus infections is discussed.     

Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.     

VP26 of white spot syndrome virus functions as a linker protein between the envelope and nucleocapsid of virions by binding with VP51

The envelopment of the nucleocapsid is an important step in white spot syndrome virus (WSSV) assembly. Previous studies showed that VP26, a major envelope protein of WSSV, can interact with viral nucleocapsid. In this study, using the biotin label transfer technique, we found that the biotin label was transferred from Bio-rVP26 to the viral capsid protein VP51 or from Bio-MBP-VP51 to VP26. Far-Western analyses provided further evidence for direct interaction between VP26 and VP51. Therefore, we conclude that VP26 functions as a matrix-like linker protein between the viral envelope and nucleocapsid, which suggests that VP26 is a key factor in the envelopment of WSSV virion.     

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate‐binding domain (VP8*)

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate‐binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus‐cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*_64‐224 of canine rotavirus strain K9 interacts with N‐acetylneuraminic and N‐glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase‐sensitive animal rotaviruses from pigs (CRW‐8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N‐glycolyl‐ over the N‐acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.     

Photorhabdus luminescens Tc toxin is inhibited by the protease inhibitor MG132 and activated by protease cleavage resulting in increased binding to target cells

Photorhabdus luminescens Tc toxins consist of the cell‐binding component TcA, the linker component TcB, and the enzyme component TcC. TccC3, a specific isoform of TcC, ADP‐ribosylates actin and causes redistribution of the actin cytoskeleton. TccC5, another isoform of TcC, ADP‐ribosylates and activates Rho proteins. Here, we report that the proteasome inhibitor MG132 blocks the intoxication of cells by Tc toxin. The inhibitory effect of MG132 was not observed, when the ADP‐ribosyltransferase domain of the TcC component was introduced into target cells by protective antigen, which is the binding and delivery component of anthrax toxin. Additionally, MG132 affected neither pore formation by TcA in artificial membranes nor binding of the toxin to cells. Furthermore, the in vitro ADP‐ribosylation of actin by the enzyme domain of TccC3 was not affected by MG132. Similar to MG132, several calpain inhibitors blocked the action of the Tc toxin. Proteolytic cleavage of the binding component TcA induced by P. luminescens protease PrtA1 or by collagenase largely increased the toxicity of the Tc toxin. MG132 exhibited no inhibitory effect on the cleaved TcA component. Moreover, binding of TcA to target cells was largely increased after cleavage. The data indicate that Tc toxin is activated by proteolytic processing of the TcA component, resulting in increased receptor binding. Toxin processing is probably inhibited by MG132.     

A novel method to demonstrate parathyroid hormone binding on unfixed living target cells in culture

We present a rapid and specific procedure that enables one to identify living target cells of peptide hormones within heterogeneous cell populations by means of morphological demonstration of ligand-receptor binding. This is exemplified using a biotinylated parathyroid hormone (PTH) analogue (biotinyl-b-PTH 1-84) which reacts with avidin-fluorescein (avidin-FITC) as a histochemical marker. The experiments revealed a fine dot-like distribution pattern of binding after 1-5 min of incubation at room temperature, changing to a more clustered pattern after 10 min of incubation. Competition of labeled and unlabeled PTH exhibited lack of staining if unlabeled PTH was applied in excess. The results suggest that the demonstrated binding sites represent specific receptors for PTH on living target cells.     

13C NMR studies of the binding of soybean trypsin inhibitor to trypsin.

NMR studies of the complex between trypsin and soybean trypsin inhibitor with 1-¹³C-arginine and modified inhibitor with 1-¹³C-lysine show that these complexes involve almost exclusively non-covalent binding of the inhibitor to the enzyme for trypsin/¹³C-Lys-inhibitor at pH 6.5 and 8.1 and for trypsin/¹³C-Arg-inhibitor at pH 5.0. At pH 7.1 for trypsin/¹³C-Arg-inhibitor both non-covalent and acyl enzyme forms are observed. Under no conditions did we observe evidence for a tetrahedral adduct between enzyme and inhibitor.