Effect of trypsin and chymotrypsin on polypeptides of human rotavirus KUN strain

In view of the fact that trypsin enhances the infectivity of human rotavirus and decreases its hemagglutination, the trypsin-mediated structural modification of the viral polypeptides was analysed, using the KUN strain, a cultivable human rotavirus isolate, grown in the absence of trypsin. A major polypeptide sensitive to trypsin treatment was Vp4 with a molecular weight of 80,000, being cleaved into three polypeptides with molecular weights of 54,000 (P54), 30,000 (P30), and 24, 000 (P24). Vp4 was also sensitive to chymotrypsin treatment, generating cleavage products different from those obtained with trypsin.     

Polypeptides of bovine rotavirus.

Polyacrylamide gel electrophoresis of bovine rotavirus or neonatal calf diarrhoea virus (NCDV) grown in cell culture resolved eight species of polypeptide. The inner shell particles contained five polypeptides and the outer shell three polypeptides. A major polypeptide of the outer shell was glycosylated. The infectivity of NCDV was enhanced by treatment with trypsin in vitro. All eight polypeptides were affected by trypsin treatment as judged by diminished intensity of polypeptide bands by radiography and several new bands appeared. The intracellular synthesis of NCDV polypeptides was studied by pulse and pulse-chase experiments. Infected cells contained all eight virus capsid proteins and, in addition, three presumably virus-specific polypeptides which were non-capsid polypeptides (NCVP). There was no evidence that any of these polypeptides was processed after synthesis. It is suggested, therefore, that all these polypeptides are primary gene products.     

The structural polypeptides of avian paramyxoviruses

Summary The structural polypeptides of twenty-three avian paramyxovriuses from five serotypes were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate under reducing and non-reducing conditions. All virus polypeptide profiles consisted of 7–10 polypeptides of which two were glycosylated. Some variation was seen in the profiles of viruses from the same serotype, but groups formed on the basis of their serological relationships in haemagglutination inhibition tests were identical to those formed on the basis of similarities in their polypeptide profiles.With 6 Figures     

Biosynthesis and morphogenesis of group C rotavirus in swine testicular cells

Summary Polypeptide synthesis and morphogenesis of a group C rotavirus (AmC-1) adapted to a continuous swine testicular cell line was examined. SDS-PAGE analysis of³⁵S methionine labeled infected cell lysates revealed 9 viral polypeptides (122, 98, 79, 78, 43, 41, 35, 24, and 20 kD). Viral polypeptide synthesis appeared to be maximal at 7–10 h post infection. Purified group C virus grown in the presence of trypsin was found to contain seven structural polypeptides (122, 98, 79, 53, 43, 41, and 30 kD) by protein blotting and five polypeptides (98, 79, 78, 43, and 41 kD) by immunoprecipitation with a hyperimmune rabbit antisera. Tunicamycin treatment, Concanavalin A binding, protein blotting, endo-H treatment and^2,6H-mannose labeling suggested that group C rotavirus contains one structural glycoprotein (41 kD) with a corresponding precursor mol. wt. of 37 kD and one not previously identified non-structural glycoprotein (24 kD) with a corresponding precursor mol. wt. of 20 kD. Electron microscopy of infected swine testicular cells revealed an assembly process for group C rotavirus similar to group A, with single-shelled particles budding through the rough endoplasmic reticulum with concomitant acquisition of a transient membrane.     

Characterization of neutralization specificities of outer capsid spike protein VP4 of selected murine,lapine, and human rotavirus strains

Neutralization specificities of outer capsid spike protein VP4 of murine rotavirus strains EW (P?[16],G3) and EHP (P?[20],G3) and lapine rotavirus strains Ala (P?[14],G3), C11 (P?[14],G3), and R2 (P?[14],G3) as well as human rotavirus strains PA169 (P?[14],G6) and HAL1166 (P?[14],G8) were determined by two-way cross-neutralization. This was done by generating and characterizing (i) three murine x human, three lapine x human, and two human x human single gene substitution reassortant rotaviruses, each of which bore identical human rotavirus DS-1 strain VP7 (G2), and (ii) guinea pig hyperimmune antiserum raised against each reassortant. Reference rotavirus strains employed in the study represented 10 established VP4 (P) serotypes, including 1A[8], 1B[4], 2A[6], 3[9], 4[10], 5A[2], 5B[2], 5B[3], 6[1], 7[5], 8[11], 9[7], and 10[16] as well as a P serotype unknown P[18]. Murine rotavirus strains EW and EB were demonstrated to share the same P serotype (P10[16]) distinct from (i) 9 established P serotypes, (ii) lapine and human rotavirus strains bearing the P[14] genotype, and (iii) an equine rotavirus strain bearing the P[18] genotype. Both lapine (Ala, C11, and R2) and human (PA169 and HAL1166) rotaviruses were shown to belong to the same VP4 serotype, which represented a distinct new P serotype (P11[14]). P serotype 13[20] was assigned to murine rotavirus EHP strain VP4, which was shown to be distinct from all the P serotypes/genotypes examined in the present study.     

Structural polypeptides of canine distemper virus

Summary The structural polypeptides of two strains of canine distemper virus and the Lec strain of measles virus were analysed by SDS-polyacrylamide-slab-gel electrophoresis. One strain of canine distemper virus derived from a live vaccine (Convac, Dumex), contained six major structural polypeptides with mol. wt. of 85, 78, 59, 43, 41 and 34×10³. The 85K polypeptide was glycosylated. It was interpreted to be equivalent to the 79K glycoprotein of the measles hemagglutinin.The second strain, a rapidly growing variant of the Onderstepoort strain of canine distemper virus characterized by extensive syncytium forming cytopathic effects in tissue culture, contained the 59, 43, 41 and 34K polypeptides, but the 85 and 78K polypeptides were not present in detectable amounts. The 43K polypeptide was identified as cellular actin by limited proteolysis. By use of monospecific rabbit hyperimmune sera against each of the major structural polypeptides of measles virus, the 59, 41 and 34K structural polypeptides could be identified as nucleocapsid protein (NP), fusion (F) polypeptide, and the membrane (M) polypeptide, respectively. In neutralization tests with rabbit hyperimmune sera against each of the two strains, this Onderstepoort strain, which contained reduced amounts of the hemagglutinin glycoprotein, gave higher neutralization titers than the vaccine strain.With 7 Figures     

Machupo virus polypeptides: Identification by immunoprecipitation

Summary The most abundant protein in purified Machupo virions (Corvallo strain) labelled with¹⁴C-Protein hydrolysate is a 64K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78K polypeptide and a minor 50K polypeptide have been detected.In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64K, NP), and the others were glycosylated polypeptides (78K and 37K). The synthesis of NP and 78K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78K is a precursor for structural glycoproteins of Machupo virions.With 4 Figures     

Characterization of a second bovine rotavirus serotype

Summary Bovine rotavirus (BRV) V 1005 was characterized by two-way cross-neutralization tests as a second serotype of BRV. Virions and inner shell particles of 65 nm and 55 nm diameter respectively, and empty capsids of 65 nm and 55 nm diameter were separated by density gradient centrifugation. Three polypeptides of molecular weight 60,000, 36,000 and 28,000 (minor protein) could be identified in the outer shell of virions and in the larger empty capsids. Inner shell particles contained three polypeptides of molecular weight 105,000, 83,000 and 43,000. Both sizes of empty capsids showed two polypeptides of molecular weight 75,000 and 55,000 not found in virions. Pulse-labelling of infected cells revealed eight major and three minor intracellular viral polypeptides. Viral polypeptide synthesis started at about 6 hours p.i. and correlated in time with double-stranded RNA synthesis. As soon as viral polypeptide synthesis was detectable, newly synthesized viral polypeptides were incorporated into intracellular viral particles. Radioactive viral polypeptides appeared without a longer lag period in extracellular viruses from 6 hours p.i. onwards.With 6 Figures     

Analysis of early and late Epstein-Barr virus associated polypeptides by immunoprecipitation.

The Epstein-Barr virus-producing cell lines P3HR-1 and B95-8 and the nonproducer cell lines Raji clone No. 7 and NC37 were induced to viral antigen synthesis by the tumor promoter TPA and then analyzed by immunoprecipitation with human sera for early and late virus-associated polypeptides. After labeling of producer cells for a 4-day period with [³⁵S]methionine, two polypeptides with molecular weights of 140,000 and 150,000 were identified reacting predominately with virus capsid antigen (VCA⁺) sera. Analysis of purified Epstein-Barr virus demonstrated that the 140,000 polypeptide presumably represents an envelope protein while the 150,000 polypeptide is a nucleocapsid protein. In 4-hr radioactively labeled producer cells an additional polypeptide with a molecular weight of 130,000 was found to be immunoreactive with VCA⁺ sera. Immunoprecipitation of [³⁵S]methionine-labeled cell extracts from nonproducer cells resulted in the specific precipitation of two polypeptides with molecular weights of 85,000 and 35,000 which most likely represent early EBV-associated proteins. Producer cells exhibit three additional apparently early EBV-associated polypeptides with molecular weights of 120,000, 18,000, and 16,000. None of these polypeptides could be detected in EBV genome-negative Ramos cells after TPA treatment.     

Two types of glycoprotein precursors are produced by the simian rotavirus SA11

The rotavirus genome codes for two glycoproteins: an outer capsid structural glycoprotein (VP7, apparent molecular weight 38,000 (38K)) and a nonstructural glycoprotein (NS28K). The synthesis of these glycoproteins was analyzed in infected cells and in a cell-free system derived from rabbit reticulocyte lysates supplemented with dog pancreatic microsomes. The data showed a 37K product synthesized in the cell-free system is the precursor to the 38K glycoprotein and that the 37K polypeptide contains a cleavable signal sequence (apparent molecular weight 1.5K). The 37K polypeptide was glycosylated in vitro in the presence of microsomal membranes to a polypeptide of 38K that was immunoprecipitated by monospecific antiserum to VP7. Endo H digestion of the 38K polypeptides from either infected cells or the cell-free system produced polypeptides of identical molecular weight, 35.5K (the glycoprotein precursor lacking the signal sequence). These results were confirmed by comparative studies with a variant of SA11 that is defective in glycosylation of VP7. Similar experiments with the 20K precursor to the 29K nonstructural glycoprotein showed the 20K polypeptide contains a noncleavable signal sequence. Both glycoproteins were inserted into microsomal membranes and were processed via oligosaccharide trimming.     

Effect of hypertonic conditions on protein synthesis in MA104 cells infected with human rotavirus

When a high NaCl concentration was used to decrease selectively the synthesis of cell proteins, the synthesis of most cellular polypeptides was greatly diminished relative to human rotavirus proteins. Thus, in the presence of 150 mM excess NaCl, 11 viral polypeptides were clearly identified. However, hypertonic conditions also reduced viral protein synthesis to a different extent with individual proteins. No significant changes in viral protein synthesis occurred during incubation under the hypertonic condition for up to 6 h, and infectious virus yields of MA104 cells incubated in the hypertonic medium did not differ from the yields of untreated MA 104 cells. These results indicate that hypertonic conditions provide a useful tool for qualitative studies of viral protein synthesis in human rotavirus infected cells.     

Genomic characterization of human rotavirus G8 strains from the African rotavirus network: Relationship to animal rotaviruses

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. During 1996–2004 surveillance within the African Rotavirus Network (ARN), six P[8],G8 and two P[6],G8 human rotavirus strains were identified. Gene fragments (RT‐PCR amplicons) of all 11‐gene segments of these G8 strains were sequenced in order to elucidate their genetic and evolutionary relationships. Phylogenetic and sequence analyses of each gene segment revealed high similarities (88–100% nt and 91–100% aa) for all segments except for gene 4 encoding VP4 proteins P[8] and P[6]. For most strains, almost all of the genes of the ARN strains other than neutralizing antigens are related to typical human strains of Wa genogroup. The VP7, NSP2, and NSP5 genes were closely related to cognate genes of animal strains (83–99% and 97–99% aa identity). This study suggests that the ARN G8 strains might have arisen through VP7 or VP4 gene reassortment events since most of the other gene segments resemble those of common human rotaviruses. However, VP7, NSP2 (likely), and NSP5 (likely) genes are derived potentially from animals consistent with a zoonotic introduction. Although these findings help elucidate rotavirus evolution, sequence studies of cognate animal rotavirus genes are needed to conclusively determine the specific origin of those genes relative to both human and animal rotavirus strains. J. Med. Virol. 81:937–951, 2009. © 2009 Wiley‐Liss, Inc.     

Detection of anti-rotavirus IgG, IgM, and IgA antibodies in healthy subjects, rotavirus infections, and immunodeficiencies by immunoblotting

Immune responses to individual simian rotavirus (SA 11) structural polypeptides were studied with emphasis on specific IgG, IgM, and IgA in paired sera from four children with rotavirus infections. Responses to simian rotavirus (SA 11) were also studied in 103 healthy children, 10 patients with primary immunodeficiency who received intravenous immunoglobulin, and 11 human immunodeficiency virus antibody-positive patients. All samples were immunoblotted for two major polypeptides--VP2 and VP6--of IgG. Immunoblotted IgA and IgM were elevated 2-4 weeks after the onset of the disease (stage II) in patients with rotavirus infection. Immunoblotted IgG remained almost the same at the onset of the disease and stage II. The scores for primary and secondary immunodeficiency patients were lower than for healthy children. The score obtained by totaling all polypeptides found in each individual was compared with the optical density (OD) value obtained by enzyme immunoassay.     

Measles virus polypeptide synthesis in infected cells

The synthesis of measles virus polypeptides has been studied using an inhibitor of virus-induced cell fusion, carbobenzoxy-d-phenylalanyl-l-phenylalanyl-nitro-l-arginine (SV4814). Cells infected at a multiplicity of 10 fuse extensively by 17 hr and die shortly thereafter, making it difficult to detect viral polypeptides. Cells protected from fusion by SV4814 survive and continue to produce virus, and the synthesis of viral polypeptides can be followed for 4 days. The previously described measles virion polypeptides G, 2, NP, 5, and M have been identified in infected cells, and, in addition, a polypeptide (L) with a molecular weight of ～200,000 has been found in infected cells and in small amounts in virions. (New designations suggested for polypeptides G, 2, and 5 are H, P, and F₁, respectively.) A glycosylated polypeptide (F₀, MW ～62,000) has also been found in infected cells, but not in virions. This polypeptide is thought to be the precursor of two polypeptides which appear under pulse-chase conditions: F₁ (MW ～40,000), which is not glycosylated, and F₂, a small glycosylated polypeptide detected with [³H]glucosamine labeling. In addition to facilitating studies of measles virus polypeptide synthesis, the use of SV4814 has shown that cell fusion is the major factor in early cell death caused by measles virus, but that cell death ultimately ensues in the absence of cell fusion, indicating another mechanism of measles virus-induced cell damage.     

A comparison of the polypeptides of human and bovine respiratory syncytial viruses and murine pneumonia virus

Human and bovine respiratory syncytial (RS) viruses and pneumonia virus of mice (PVM) are morphologically similar viruses and are the only known members of the metamyxovirus (or pneumovirus) group. Comparison of the polypeptides of these viruses by polyacrylamide-gel electrophoresis (PAGE) has shown that the serologically related human and. bovine RS viruses have similar polypeptide profiles. However, PVM does not resemble the other two viruses closely. The molecular weights of RS virus polypeptides were established by discontinuous SDS-PAGE in gradient slab gels and were comparable to previous determinations by continuous SDS-PAGE (Wunner and Pringle, 1976), apart from the detection of an additional 10,000-MW polypeptide. Comparison of 11 strains of human RS virus isolated from different localities, or from the same locality at different dates, showed that the relative mobility of VP32 (a nonglycosylated polypeptide of unknown function) was a stable characteristic of each strain. The mobilities of the other viral polypeptides showed little variation. The 11 strains fell into three groups in which the molecular weights of VP32 were estimated to be 31,000, 32,000, and 35,000, but there was no clear correlation with date or place of origin.     

cDNA probes of individual genes of human rotavirus distinguish viral subgroups and serotypes

The use of cDNA probes for detection of rotaviruses has been investigated using plasmids containing inserts specific for each of the eleven genes of human rotavirus strain Wa. In a dot-blot detection system in which radioactive DNA probes were hybridized to viral RNA extracted from cultivatable rotavirus strains, cDNAs of genes 7, 8, 10 and 11, were found to be the most reliable probes for detecting a range of rotavirus strains. Unexpectedly, rotaviruses could be distinguished with respect to subgroup and subtype specificities when cDNAs of genes 6 and 9, which encode the immunologically relevant proteins VP6 (group-specific antigen) and VP7 (type-specific antigen), were used as probe, even though the nucleic acid sequences of these genes are known to have a high degree of sequence homology.     

Structural homologies between RNA gene segments 10 and 11 from UK bovine, simian SA11, and human Wa rotaviruses

The nucleotide sequences of gene segments 10 and 11 from UK bovine rotavirus have been determined. Gene 10 is 751 nucleotides long and contains a single long open reading frame capable of coding for a protein of 175 amino acids. When compared with the published data for gene 10 of the simian rotavirus SA11 and human Wa strains it was found to be more closely related to the SA11 structure (92% nucleotide sequence homology; 97% amino acid sequence homology) than to the human Wa structure (84% nucleotide, 86% amino acid sequence homology). All three strains have two potential N-glycosylation sites in the hydrophobic N terminus of the gene 10 protein. Gene 11 from UK bovine rotavirus is 667 nucleotides long with a single long open reading frame capable of coding for a protein of 198 amino acids. When compared with the published sequence of gene 11 from the human rotavirus Wa, the UK bovine rotavirus gene 11 was found to be one nucleotide longer in the 5'-noncoding region and three nucleotides longer in the coding region. The nucleotide sequence homology was 86%. The predicted proteins coded by segment 11 in UK and Wa rotaviruses are both rich in serine and threonine (23%) and very hydrophilic, but differ appreciably in amino acid sequence (83% homology).     

Characterization of monoclonal antibodies against human rotavirus hemagglutinin

Three monoclonal antibodies capable of specifically inhibiting hemagglutination of human rotavirus were produced. Their hemagglutination inhibition (HI) activity was specific to the homologous strain (KUN) used for immunization. The monoclonal antibodies with HI activity were highly effective in neutralizing the infectivity of the KUN strain. These antibodies reacted with Vp80, and 80,000 molecular weight (MW) protein present in the viral outer shell. It was confirmed by immunoblotting assay with the monoclonal antibodies that the antigenic site of human rotavirus hemagglutinin (HA) resides on Vp80 and on its smaller trypsin cleavage products Vp30 (MW 30,000) and Vp24 (MW 24,000). Immunofluorescence studies using the antibodies revealed that the HA antigen of the KUN strain developed at the final stage of virus maturation.     

Nucleotide Sequence Analysis of Rotavirus Gene 11 from Two Tissue Culture-adapted ATCC Strains,RRV and Wa

We report here nucleotide sequence and characterization of gene 11 from two tissue culture-adapted ATCC¹ rhesus (RRV) and human (Wa) strains of rotavirus. Gene 11 sequence encodes a nonstructural protein, NSP5 and also encodes NSP6, from an out of phase open reading frame. Sequence of RRV^ATCC gene 11 represents the first report from a rhesus rotavirus which has more than 90% homology at the nucleotide and deduced amino acid sequence level with that of its closely related simian SA11 strain. The Wa^ATCC gene sequence differed from that of published Wa (Wa^Pub) at three nucleotide positions, one at 264 (G^Wa-Pub to A^ATCC-Wa), another a nucleotide insertion (A) at position 388 and the third, a deletion (A) at 416. The latter two changes in Wa^ATCC NSP5 resulted in drastic amino acid changes within a 10-residue region (123–132) from VHVYQFQLTN in Wa^Pub to DSCVSISTNH in Wa^ATCC NSP5 protein. In this region, Wa^ATCC NSP5 is closer to published sequences from other strains, suggesting the authenticity of the present sequence. The nucleotide difference between Wa^Pub and Wa^ATCC NSP5 sequences, however, did not affect the NSP6 deduced amino acid sequence, which is overall highly conserved among all the strains compared. Sequence-based phylogenetic analysis of gene 11 identified a high degree of conservation within the Group A rotaviruses. In addition, it also separated RRV^ATCC and Wa^ATCC, suggesting rotavirus segregation by genogroup. An anti-NSP5 monoclonal antibody of SA11 recognized RRV NSP5 protein but not Wa^ATCC NSP5 from the infected cells, further supporting the phylogenetic segregation of RRV^ATCC and Wa^ATCC strains based on their NSP5 coding sequence.     

Synthesis of human rotavirus polypeptides in cell culture

The replication strategy of a human serotype 1 rotavirus, adapted to rapid growth in CV-1 cells, was investigated. A single cycle growth curve revealed eclipse and latent periods of 3 and 4 hours, respectively. Although the extent of reduction of host cell protein synthesis was directly related to the multiplicity of infection of the virus, incorporation of actinomycin D and excess NaCl into the medium resulted in significant reduction in host cell background and enabled observation of viral polypeptides as early as 2 hours post infection. Five polypeptides were found to be structural components of the virion, and a further eight appeared to be nonstructural proteins or intermediates. Five polypeptides were glycosylated during virus replication, but only one of these, VP7, was a definite structural glycoprotein. Pulse-chase experiments revealed that four low molecular weight polypeptides underwent post-translational modifications.