Receptor activity of rotavirus nonstructural glycoprotein NS28.

Rotavirus morphogenesis involves the budding of subviral particles through the rough endoplasmic reticulum (RER) membrane of infected cells. During this process, particles acquire the outer capsid proteins and a transient envelope. Previous immunocytochemical and biochemical studies have suggested that a rotavirus nonstructural glycoprotein, NS28, encoded by genome segment 10, is a transmembrane RER protein and that about 10,000 Mr of its carboxy terminus is exposed on the cytoplasmic side of the RER. We have used in vitro binding experiments to examine whether NS28 serves as a receptor that binds subviral particles and mediates the budding process. Specific binding was observed between purified simian rotavirus SA11 single-shelled particles and RER membranes from SA11-infected monkey kidney cells and from SA11 gene 10 baculovirus recombinant-infected insect cells. Membranes from insect cells synthesizing VP1, VP4, NS53, VP6, VP7, or NS26 did not possess binding activity. Comparison of the binding of single-shelled particles to microsomes from infected monkey kidney cells and from insect cells indicated that a membrane-associated component(s) from SA11-infected monkey kidney cells interfered with binding. Direct evidence showing the interaction of NS28 and its nonglycosylated 20,000-Mr precursor expressed in rabbit reticulocyte lysates and single-shelled particles was obtained by cosedimentation of preformed receptor-ligand complexes through sucrose gradients. The domain on NS28 responsible for binding also was characterized. Reduced binding of single-shelled particles to membranes was seen with membranes treated with (i) a monoclonal antibody previously shown to interact with the C terminus of NS28, (ii) proteases known to cleave the C terminus of NS28, and (iii) the Enzymobead reagent. VP6 on single-shelled particles was suggested to interact with NS28 because (i) a monoclonal antibody to the subgroup I epitope on VP6 reduced particle binding, (ii) a purified polyclonal antiserum raised against recombinant baculovirus-produced VP6 reduced ligand binding, and (iii) a monoclonal antibody to a conserved epitope on VP6 augmented ligand binding. These experimental data provide support for the hypothesized receptor role of NS28 before the budding stage of rotavirus morphogenesis.     

Serine protein kinase activity associated with rotavirus phosphoprotein NSP5.

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [gamma-32P]ATP resulted in 32P labeling of 28- to 35-kDa NSP5. The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.     

A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells

Previous studies have shown that the nonstructural glycoprotein NSP4 plays a role in rotavirus pathogenesis by functioning as an enterotoxin. One prediction of the mechanism of action of this enterotoxin was that it is secreted from virus-infected cells. In this study, the media of cultured (i) insect cells infected with a recombinant baculovirus expressing NSP4, (ii) monkey kidney (MA104) cells infected with the simian (SA11) or porcine attenuated (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western blotting, immunoprecipitation and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparent molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 aa112-175). The secretion of NSP4 aa112-175 was not affected by treatment of cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network. A partial gene fragment coding for NSP4 aa112-175 was cloned and expressed using the baculovirus-insect cell system. Purified NSP4 aa112-175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expressed endogenously, similarly to full-length NSP4. NSP4 aa112-175 caused diarrhea in neonatal mice, as did full-length NSP4. These results indicate that NSP4 aa112-175 is a functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.     

Analysis of Rotavirus Nonstructural Protein NSP5 Phosphorylation

The rotavirus nonstructural phosphoprotein NSP5 is encoded by a gene in RNA segment 11. Immunofluorescence analysis of fixed cells showed that NSP5 polypeptides remained confined to viroplasms even at a late stage when provirions migrated from these structures. When NSP5 was expressed in COS-7 cells in the absence of other viral proteins, it was uniformly distributed in the cytoplasm. Under these conditions, the 26-kDa polypeptide predominated. In the presence of the protein phosphatase inhibitor okadaic acid, the highly phosphorylated 28- and 32- to 35-kDa polypeptides were formed. Also, the fully phosphorylated protein had a homogeneous cytoplasmic distribution in transfected cells. In rotavirus SA11-infected cells, NSP5 synthesis was detectable at 2 h postinfection. However, the newly formed 26-kDa NSP5 was not converted to the 28- to 35-kDa forms until approximately 2 h later. Also, the protein kinase activity of isolated NSP5 was not detectable until the 28- and 30- to 35-kDa NSP5 forms had been formed. NSP5 immunoprecipitated from extracts of transfected COS-7 cells was active in autophosphorylation in vitro, demonstrating that other viral proteins were not required for this function. Treatment of NSP5-expressing cells with staurosporine, a broad-range protein kinase inhibitor, had only a limited negative effect on the phosphorylation of the viral polypeptide. Staurosporine did not inhibit autophosphorylation of NSP5 in vitro. Together, the data support the idea that NSP5 has an autophosphorylation activity that is positively regulated by addition of phosphate residues at some positions.     

Cellular proteins associated with simian virus 40 early gene products in newly infected cells.

Immunoprecipitates of extracts of simian virus 40-infected permissive monkey kidney cells contained two proteins with molecular weights of 56,000 and 32,000 (52K and 32K) in addition to the known viral early gene products. Immunoprecipitates of cells infected with the 0.54-0.59 deletion mutants that lack the viral 17K gene product did not contain thhe 56K and 32K proteins. The additional proteins appeared in immunoprecipitates of deletion mutant extracts if unlabeled extracts of wild-type-infected cells were added before addition of antiserum. The proteins can also be identified in uninfected cells by co-precipitation with unlabeled viral proteins. Thus, it appears that the 56K and 32K proteins are cellular products that associate with the viral proteins, the 17K in particular, and are indirectly immunoprecipitated by anti-tumor serum.     

Development of a method for detection of human rotavirus in water and sewage.

The simian rotavirus SA11 was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA11 adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter-bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SA11 was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SA11 serum which would detect the simian, human bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SA11. The buoyant density of the sewage isolates in CsCl gradients was 1.36 g/cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 +/- 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology.     

Development of a method for detection of human rotavirus in water and sewage

The simian rotavirus SA11 was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA11 adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter-bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SA11 was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SA11 serum which would detect the simian, human bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SA11. The buoyant density of the sewage isolates in CsCl gradients was 1.36 g/cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 +/- 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology.     

Coding assignments of double-stranded RNA segments of SA 11 rotavirus established by in vitro translation

The segmented double-stranded (ds) RNA genome of the simian rotavirus SA 11, after denaturation, can be translated in a cell-free protein synthesizing system. Of the 11 genome segments, 9 can be resolved on polyacrylamide gels and thus could be individually isolated and translated, providing a means of identifying the polypeptide encoded by each segment. On the basis of electrophoretic mobility of products in sodium dodecyl sulfate-polyacrylamide gels, the probable gene-coding assignments of dsRNA segments 1 to 6 were determined. RNA segments 1 to 4 code for polypeptides I1, I2, I3, and I4, respectively; segment 5 codes for a polypeptide very similar in mobility to a minor polypeptide present in SA 11-infected cells, O1A; and segment 6 codes for the major inner-capsid polypeptide I5.     

Completion of the gene coding assignments of SA11 rotavirus: gene products of segments 7, 8, and 9.

Improved fractionation of double-stranded RNA segments 7, 8, and 9 of simian rotavirus SA11 has permitted their isolation and individual translation in vitro. Segment 7 codes for p31 (NS2), segment 8 codes for p33 (NS1), and the segment 9 gene product resembles the gp34 precursor observed in SA11 virus-infected cells. In vitro glycosylation of translation products of segments 5 and 10 was also observed.     

Demonstration of an immunodominant neutralization site by analysis of antigenic variants of SA11 rotavirus

Serotype-specific monoclonal antibodies were used to select mutants of SA11 rotavirus that were resistant to neutralization. The antigenic characteristics of these mutants were studied with with a panel of monoclonal antibodies. We isolated one type of mutant which showed a dramatic increase (greater than 10-fold) in resistance to neutralization by hyperimmune antiserum, and this together with other data indicates the presence on the rotavirus major outer shell glycoprotein of an immunodominant antigenic site involved in virus neutralization. The mutants were also useful in classifying neutralizing monoclonal antibodies.     

The nonstructural glycoprotein of rotavirus affects intracellular calcium levels.

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirus-infected cells.     

Cloning and sequence of UK bovine rotavirus gene segment 7: marked sequence homology with simian rotavirus gene segment 8.

The genome of the UK bovine rotavirus, which consists of eleven segments of dsRNA was polyadenylated and reverse-transcribed into cDNA. Complementary cDNA strands were annealed and the termini of the duplexes completed using DNA polymerase I. Full-length DNA copies of RNA segments 7, 8 and 9 were cloned into the Pst I site of pBR322 and a clone containing the entire gene 7 was identified and sequenced. Gene 7 is 1059 nucleotides in length and contains a single long open reading frame capable of coding for a protein of 317 amino-acids. The known gene product of segment 7 is a protein with an estimated molecular weight of 33,000 daltons. When the UK bovine rotavirus gene 7 sequence was compared with the published data for the homologous gene (segment 8) of the simian rotavirus SA11, it was found to be identical to it in size and the arrangement of the proposed coding and non-coding regions, and very similar in nucleotide sequence (88% homology). Most of the base changes are silent and the predicted amino-acid sequences are almost identical (96% homology).     

Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system.

Rotaviruses are the major pathogens that cause life-threatening diarrhea in young children and animals. We inserted a simian rotavirus SA11 gene 6 cDNA into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. The major capsid antigen (VP6) was expressed in high yields (20 to 150 micrograms/10(6) cells) when Spodoptera frugiperda cells were infected with baculovirus recombinants containing SA11 gene 6 inserts. Reactivity with monospecific polyclonal and monoclonal antibodies suggested that VP6, expressed intracellularly or found in the media, maintained native antigenic determinants. VP6 purified from the media from infected cells also possessed a native oligomeric structure, was immunogenic in guinea pigs, and was able to spontaneously assemble into morphologic subunits. Antisera from immunized guinea pigs failed to neutralize virus in plaque reduction assays, but detected homologous and heterologous rotavirus strains when tested by immunofluorescence, immunoprecipitation, and enzyme-linked immunosorbent assays.     

Antibodies specific for the carboxy-terminal region of the major surface glycoprotein of simian rotavirus (SA11) and human rotavirus (Wa)

Antibodies specific for the major outer capsid protein (VP7) of the simian rotavirus SA11 were obtained by immunization of rabbits with a synthetic peptide, Ser-Ala-Ala-Phe-Tyr-Tyr-Arg-Val, corresponding to the eight carboxy-terminal amino acids of the viral protein predicted from the nucleotide sequence of the gene segment 9 of the SA11 genome. As the carboxy-terminal region of the VP7 of human rotavirus Wa has an identical sequence, cross-reactivity of the raised antibodies was observed with this strain.     

Vaccinia virus recombinants expressing the SA11 rotavirus VP7 glycoprotein gene induce serotype-specific neutralizing antibodies.

A cDNA copy of the gene coding for the major outer neutralizing protein (VP7) of simian 11 rotavirus was incorporated into the vaccinia virus genome under the control of the vaccinia promoter (molecular weight, 7,500). A deletion mutant of this gene which codes for a secreted form of VP7 when expressed under the control of the simian virus 40 late promoter (M. S. Poruschynsky, C. Tyndall, G. W. Both, F. Sato, A. R. Bellamy, and P. H. Atkinson, J. Cell Biol. 101:2199-2209, 1985) was also inserted. Each recombinant vaccinia virus directed the synthesis of a rotavirus protein in infected cells, and the product encoded by the mutated gene was secreted. Rabbits immunized with the two types of recombinant vaccinia virus generated antibodies that were able both to recognize simian 11 rotavirus in an enzyme-linked immunosorbent assay and to neutralize the virus in a plaque-reduction test. Antibodies induced by the recombinant vaccinia viruses expressing either form of VP7 were serotype specific.     

Gene protein products of SA11 simian rotavirus genome

When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns equal to those of viral proteins observed in tunicamycin-treated infected cells. Nine of the 11 double-stranded RNA genome segments were resolved by polyacrylamide gel electrophoresis and were translated individually. Two were not resolved from each other and therefore were translated together. Correlation of each synthesized polypeptide with an individual RNA segment allowed us to make a probable gene-coding assignment for the different SA11 genome segments.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.