Outer Capsid Protein VP2 Research Articles

High level expression of the two outer capsid proteins of bluetongue virus serotype 10: their relationship with the neutralization of virus infection

DNA representing RNA segments 2 and 5 of bluetongue virus (BTV) serotype 10, corresponding to the genes that code for the outer capsid proteins VP2 and VP5, have been inserted into a baculovirus transfer vector in lieu of the coding region of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). After co-transfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the recombinant transfer vector DNAs polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with these recombinant viruses proteins of similar size and antigenic properties to BTV VP2 and VP5 were synthesised. The recombinant VP2, but not the recombinant VP5, was shown to be capable of inducing antibodies that neutralized the infectivity of BTV-10 in vitro.

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.

Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype

Analyses of reassortant and parental strains of BTV serotypes 3 and 10, in serum neutralization tests, confirmed the major role of outer capsid protein VP2 in determination of virus serotype and its involvement in serum neutralization. However, a reassortant BTV strain (R70), containing protein VP5 derived from BTV 3 and VP2 derived from BTV 10, cross-neutralized with both parental virus strains (BTV 3 and BTV 10). It is concluded that VP5 also plays some part in serotype determination of these virus isolates, as analyzed by serum-neutralization, but its role may be less significant than that of VP2.

The rhesus rotavirus outer capsid protein VP4 functions as a hemagglutinin and is antigenically conserved when expressed by a baculovirus recombinant

Rhesus rotavirus (RRV) gene 4 was cloned into lambda bacteriophage, inserted into a polyhedrin promoter shuttle plasmid, and expressed in Sf9 cells by a recombinant baculovirus. The baculovirus-expressed VP4 protein made up approximately 5% of the Spodoptera frugiperda-infected cell protein. Monoclonal antibodies that neutralize the virus bound to the expressed VP4 polypeptide, indicating that the expressed VP4 protein was antigenically indistinguishable from viral VP4. In addition, we have determined that the baculovirus-expressed VP4 protein bound to erythrocytes and functions as the RRV hemagglutinin. The endogenous hemagglutinating activity of the VP4 protein, like the virus, was inhibited by guinea pig antirotavirus hyperimmune serum and by VP4-specific neutralizing monoclonal antibodies. The human erythrocyte protein, glycophorin, also inhibited hemagglutination by RRV or the expressed VP4 protein and appears to be the rotavirus erythrocyte receptor. The baculovirus-expressed VP4 protein was conserved functionally and antigenically in the absence of other outer or inner capsid rotavirus components and represents a logical candidate for future immunological studies.

A comparison of six different bluetongue virus isolates by cross-hybridization of the dsRNA genome segments

The relationship between six different isolates of BTV was analyzed by cross-hybridization of genomic dsRNA using blotting and probe techniques (using an alakali fragmented probe made from BTV dsRNA). The viruses compared in this way included BTV serotype 1 from South Africa, serotypes 3 and 4 from Cyprus, serotype 10 from North America, and serotypes 1 and 20 from Australia. Under the hybridization and washing conditions used, which were calculated to allow stable duplex formation between RNA molecules containing greater than 90% sequence homology, two of the genome segments (segments 2 and either 5 or 6, which encode the two major outer capsid proteins VP2 and VP5) appeared to contain serotype-specific RNA sequences. Significant cross-hybridization between these segments from different serotypes was detected only with serotypes 4 and 20, which are known to have a particularly close antigenic relationship. The amounts of homologous sequence that were detected in segments other than 2 and 5 between different viruses indicated some correlation between their geographical origins and a degree of relatedness, which is independent of the virus serotype. High levels of sequence homology were detected between the isolates from Cyprus and Africa and to a slightly lesser extent from North America, suggesting a common ancestry. These results also indicated that within the limited number of viruses studied, the Australian isolates form a separate interrelated group of bluetongue viruses

Genetic relatedness among human rotavirus genes coding for VP7, a major neutralization protein, and its application to serotype identification.

Antigenic characterization of human rotaviruses by plaque reduction neutralization assay has revealed four distinct serotypes. The outer capsid protein VP7, coded for by gene 8 or 9, is a major neutralization protein; however, studies of rotaviruses derived from genetic reassortment between two strains have confirmed that another outer capsid protein, VP3, is in some cases equally important in neutralization. In this study, the genetic relatedness of the genes coding for VP7 of human rotaviruses belonging to serotypes 1 through 4 was examined by hybridization of their denatured double-stranded genomic RNAs to labeled single-stranded mRNA probes derived from human-animal rotavirus reassortants containing only the VP7 gene of their human rotavirus parent. A high degree of homology was demonstrated between the VP7 genes of strain D and other serotype 1 human rotaviruses, strain DS-1 and other serotype 2 human rotaviruses, strain P and other serotype 3 human rotaviruses, and strain ST3 and other serotype 4 human rotaviruses. Hybrid bands could not be demonstrated between the VP7 gene of D, DS-1, P, or ST3 and the corresponding gene of human rotaviruses belonging to a different serotype. RNA specimens extracted from the stools of 15 Venezuelan children hospitalized with rotavirus diarrhea were hybridized to each of the reassortant probes representing the four human serotypes. All five viruses with short RNA patterns showed homology with the DS-1 strain VP7 gene; two of these were previously adapted to tissue culture and shown to be serotype 2 strains by tissue culture neutralization. Of the remaining 10 viruses with long RNA patterns, 2 hybridized only to the D strain VP7 gene, 6 hybridized only to the P strain VP7 gene, and 2 hybridized only to the ST3 strain VP7 gene. Hybridization using single human rotavirus gene substitution reassortants as probes may provide an alternative method for identifying the VP7 serotype of field isolates that would circumvent the need for tissue culture adaptation.

Further analysis of the role of calcium in rotavirus morphogenesis

Previously we reported that calcium plays an important role in the maturation of bovine rotavirus ( M. S. Shahrabadi and P. W. K. Lee, 1986. Virology 152, 298–307). We now demonstrate that the formation of mature double-shelled (L) particles was strictly dependent on the concentration of calcium present in the growth medium. The formation of single-shelled (D) particles did not appear to be a calcium-mediated process. Subsequent labeling studies using 45Ca revealed that calcium was incorporated into the L particles but not the D particles. The previously noted decreased level of the outer capsid protein VP7 (42K) in calcium-deprived cultures was now found to be due to the preferential degradation, and not to the impaired synthesis, of this protein in the absence of calcium. It was further demonstrated that calcium had a stabilizing effect on VP7 and that VP7 synthesized in the presence of calcium was not degraded upon subsequent calcium deprivation. Protein degradation during calcium deprivation was apparently limited to the mature form of VP7 since the unglycosylated precursor (pVP7), formed in the presence of tunicamycin, was found to be stable under this condition. Electron microscopic examination of infected cells revealed that in the presence of calcium, virus maturation took place by the budding of viral cores through the endoplasmic reticulum (ER). No such budding was observed in calcium-deprived cells. In these cells mature virions were absent and membrane fragments could be found associated with viral cores or single-shelled particles.

Identification of the two rotavirus genes determining neutralization specificities.

Bovine rotavirus NCDV and simian rotavirus SA-11 represent two distinct rotavirus serotypes. A genetic approach was used to determine which viral gene segments segregated with serotype-specific viral neutralization. There were 16 reassortant rotaviruses derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant rotavirus double-stranded RNA segments was determined by gene segment mobility in polyacrylamide gels and by hybridization with radioactively labeled parental viral transcripts. We found that two rotavirus gene segments found previously to code for outer capsid proteins vp3 and vp7 cosegregated with virus neutralization specificities.