Serotypic characterization of outer capsid spike protein VP4 of vervet monkey rotavirus SA11 strain

Summary.  The vervet monkey rotavirus SA11, a prototype strain of group A rotaviruses, has been shown to possess VP7 serotype 3 specificity but its neutralization specificity with regard to the other outer capsid protein VP4 has not been elucidated. We thus determined its VP4 specificity by two-way cross-neutralization with guinea pig antiserum prepared with a single gene substitution reassortant that had only the VP4-encoding gene from the simian rotavirus SA11 strain and remaining ten genes from human rotavirus DS-1 strain (G serotype 2). The SA11 VP4 was related antigenically in a one-way fashion to rhesus monkey rotavirus MMU18006 VP4 (a P5B strain) and marginally to human and canine rotavirus VP4s with P serotype 5A specificity. In addition, the SA11 VP4 was shown to be distinct antigenically from those of other known P serotypes (1–4, and 6–11) as well as those of uncharacterized equine, lapine, and avian rotavirus strains. The SA11 VP4 is thus proposed for classification as a P5B serotype. Received September 2, 1997 Accepted January 8, 1998     

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2.

A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.     

Comparative studies of the antigenic polypeptide species VP4, VP6, and VP7 of three strains of bovine rotavirus.

Three bovine rotavirus strains belonging to two distinct serotype groups, serotype 6 (NCDV and B641) and B223, distinct from the other six mammalian rotavirus serotypes but not yet assigned to a serotype group, were compared with each other and with canine rotavirus (K9, serotype 3) by studying the properties of their cognate polypeptide species VP4, VP6, and VP7. The three viruses showed distinct differences in the polyacrylamide gel electrophoretic migration rates of protein species VP4 and VP7, with minor differences in VP6. Differences were also observed among the migration patterns of genome segments 4, 6, and the 7-8-9 triplet, which encode VP4, VP6, and VP7, respectively. Monoclonal antibodies (MAbs) to B223, which were directed against VP4 or VP7, showed homologous specificity for neutralization and immunofluorescence (IF), although one MAb reactive with VP4 also reacted by IF and by immunoprecipitation (IP) with all four viruses and weakly neutralized B641 and K9. This MAb may react with the epitope responsible for the B223-induced one-way neutralizing and protection response of calves against B641 observed in earlier studies. MAbs reactive with VP6 by IP showed enzyme-linked immunosorbent assay and IF reactivity with all three bovine viruses and the canine virus. The two serotype 6 viruses could be distinguished by the two B641 MAbs, B641-N2b reacting by neutralization and IF with both viruses and B641-N1 reacting with B641 and the serotype 3 canine rotavirus but not with NCDV. One nonneutralizing B641 MAb reacted by IP and IF with VP7 of all four rotaviruses examined, and one B223 MAb neutralized B223 and, to low titer, B641 and K9 although reacting by IP and IF with all four viruses. Three MAb-resistant mutants were selected by passage of B223 in the presence of one of three selected B223 MAbs at concentrations which only neutralized approximately 90% of the infectious virions. The resulting mutants were 100% resistant to neutralization with their respective MAb but remained neutralizable by the same selection of MAbs as the parent B223 virus.     

Cloning of bovine rotavirus (RF strain): nucleotide sequence of the gene coding for the major capsid protein

The genes of the RF strain of bovine rotavirus have been cloned into pBR 322 following the synthesis and hybridization of cDNA transcribed from both strands of in vitro polyadenylated genomic RNA. Cloned rotavirus DNAs were assigned to most of the 11 genomic RNA segments by Northern blot hybridization. The complete sequence of gene 6 that codes for the major inner capsid protein has been determined. The gene is 1356 nucleotides long and possesses an unique long open reading frame that could encode a protein (397 amino acids) of similar size to the known gene 6 product. Comparison of the RF bovine rotavirus gene 6 sequence with the sequence of the simian rotavirus gene 6, showed these genes to be very similar in nucleotide sequence (87% homology). Most of the base changes are silent and the predicted amino acid sequences are almost identical (97% homology).     

Amino acid sequence analysis of bovine rotavirus B223 reveals a unique outer capsid protein VP4 and confirms a third bovine VP4 type.

The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and aa 257 to 271) which show high homologies between strains, demonstrate that the region 5' to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3' to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5' flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of B223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613-615 in the VP5* subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.     

Nucleotide sequence of gene 5 encoding the inner capsid protein (VP6) of bovine group C rotavirus: comparison with corresponding genes of group C, A, and B rotaviruses.

To further study the molecular characteristics of group (gp) C rotaviruses, we produced, cloned, and sequenced cDNA to gene 5 of the Shintoku strain of bovine gp C rotavirus. The resulting clone was specific for gene 5 and was genetically related to the human and porcine gp C rotaviruses, as demonstrated by Northern blot hybridization analysis. The Shintoku gene 5 is 1352 nucleotides in length and has one open reading frame encoding a polypeptide of 395 amino acids with a predicted molecular mass of 44.5 kDa. Comparative sequence analysis indicated that: (i) the Shintoku gene 5 protein shared 88.4 to 90.6% homology with the VP6 of the human (Bristol and 88-220) and porcine (Cowden) strains of gp C rotaviruses, but only low homology with the VP6 of bovine gp A (RF) and human gp B (ADRV) rotaviruses (41.3 and 16.3%, respectively); (ii) the predicted secondary structure was highly conserved among the gene 5 proteins of the bovine, porcine, and human gp C rotaviruses; and (iii) seven highly conserved regions were identified for the first time in the deduced primary amino acid sequences of gene 5 of gp C and gene 6 of gp A rotaviruses. However, only three of these highly conserved areas were present in the regions of VP6, where the secondary structure was predicted to be similar for the rotavirus strains examined. These three regions may contribute to common epitopes between the two groups of rotaviruses. Our results, in comparison with data for other rotaviruses, indicate that gene 5 of the bovine gp C rotavirus codes for the major inner capsid protein (VP6).     

Characterization of a nuclear localization sequence in the polyomavirus capsid protein VP1.

Expression of VP1-beta-galactosidase fusion proteins in the yeast Saccharomyces cerevisiae was used to identify a domain of the polyomavirus VP1 capsid protein which targets this protein to the nucleus. Fusion of the first 17 amino acids of VP1 to beta-galactosidase was sufficient for nuclear localization, whereas fusion of the first 12 amino acids gave a "mixed" cytoplasmic-nuclear phenotype. Mutation of a putative targeting sequence MAPKR(5)K from R to S changed the localization of a 21 amino acid fusion protein from the nucleus to cytoplasm. These results define a nuclear location signal in the amino terminus of polyomavirus VP1 and separate this function from the high-affinity DNA binding function previously defined for this region.     

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.     

Reactivity patterns to human rotavirus strains of a monoclonal antibody against VP2, a component of the inner capsid of rotavirus

Summary A non-neutralizing monoclonal antibody (YO-60) against human rotavirus was found to be directed to VP2 (90,000-dalton protein), one of the two major components of the inner capsid. The reactivity patterns of the YO-60 antibody were very similar, though not identical, to those of subgroup II-specific YO-5 monoclonal antibody directed to VP6 (42,000-dalton protein), the other major component of the inner capsid.These results indicated the possible presence of a subgroup-specific antigen on VP2 in addition to the one on VP6.With 1 FigureThis study was supported in part by a grant no. 58570213 from the Ministry of Education, Science and Culture of Japan.     

Nucleotide sequence of the cDNA for porcine rotavirus VP7 gene (strain K)

The nucleotide sequence of the cDNA encoding one of the neutralizing proteins VP7 of the new porcine strain K is determined. The deduced VP7 amino acid sequence of the K strain showed a high homology (93%) and a lower homology (75%) to those of the Gottfried and OSU strains, respectively. This finding suggests that strain K is more closely related to the Gottfried strain serotype 4.     

Comparative sequence analyses of the cognate structural protein VP6 genes of five US bluetongue viruses.

The S3 segment (the small segment 3), encoding the structural protein, VP6, from the five United States (US) prototype bluetongue virus (BTV) serotypes were amplified by the Clamp-R method and cloned as full-length entities. The complete nucleotide sequence of each cognate gene segment was determined. Each cognate S3 segment of BTV-10, 11, 13 and 17 was 1049 nucleotides long and contained an open reading frame (ORF) capable of encoding a 325-amino acid protein. However, the S3 segment of BTV-2, which also contained 1049 nucleotides, had a longer 5'-non-coding region of 99-nucleotide and contained an ORF capable only of encoding a 301-amino acid protein. Comparative analyses of the predicted amino acid sequences of S3 segments of BTV-2, 10, 11, 13 and 17 revealed that VP6 was unusually high in glycine and contained few aromatic amino acids, but a high concentration of charged amino acids, which is a characteristic of a hydrophilic protein. Phylogenetic analyses indicated that BTV-11, 13 and 17 were more closely related than the other two US BTV serotypes. BTV-2 was the most distantly related.     

Molecular characterization of VP4, VP6, VP7, NSP4, and NSP5/6 genes identifies an unusual G3P[10] human rotavirus strain

An unusual strain of human rotavirus G3P[10] (CMH079/05) was detected in a stool sample of a 2‐year‐old child admitted to the hospital with severe diarrhea in Chiang Mai, Thailand. Analysis of the VP7 gene sequence revealed highest identities with unusual human rotavirus G3 strain CMH222 at 98.7% on the nucleotide and 99.6% on the amino acid levels. Phylogenetic analysis of the VP7 sequence confirmed that the CMH079/05 strain formed a cluster with G3 rotavirus reference strains and showed the closest lineage with the CMH222 strain. Analysis of partial VP4 gene of CMH079/05 revealed highest degree of sequence identities with P[10] rotavirus prototype strain 69M at nucleotide and amino acid levels of 92.9% and 94.6%, respectively. Phylogenetic analysis of the VP4 sequence revealed that CMH079/05 and 69M clustered closely together in a monophyletic branch separated from other rotavirus genotypes. To our knowledge, this is a novel G–P combination of G3 and P[10] genotypes. In addition, analyses of VP6, NSP4, and NSP5/6 genes revealed these uncommon genetic characteristics: (i) the VP6 gene differed from the four other known subgroups; (ii) the NSP4 gene was identified as NSP4 genetic group C, an uncommon group in humans; and (iii) the NSP5/6 gene was most closely related with T152, a G12P[9] rotavirus previously isolated in Thailand. The finding of uncommon G3P[10] rotavirus in this pediatric patient provided additional evidence of the genetic diversity of human group A rotaviruses in Chiang Mai, Thailand. J. Med. Virol. 81:176–182, 2009. © 2008 Wiley‐Liss, Inc.     

B组轮状病毒WH-1株 NSP2基因序列和蛋白质结构分析

目的 克隆成人腹泻标本WH-1中B组轮状病毒组(group Brotavirus，GBRV)非结构蛋白NSP2基因，分析其核苷酸序列，比较与其它GBRV基因同源性，预测其mRNA二级结构、蛋白质二级结构。方法利用逆转录-聚合酶链反应(RT-PCR)技术，从成人腹泻标本WH-1中扩增GBRV NSP2基因，克隆载体pUCmT，对NSP2基因进行核苷酸序列分析。利用GeneBee软件比较与其它GBRV毒株NSP2基因同源性，Rnaviz2．0软件绘制NSP2基因的二级结构，PredictProtein软件分析NSP2蛋白结构。结果成人腹泻标本WH-1中GBRV非结构蛋白NSP2基因全长1007bD，与ADRV核苷酸序列的同源性达98％，与印度加尔各达分离株CAI-1达82％，与IDIR(鼠)同源性仅为79％。氨基酸序列与ADRV的同源性达98．4％，与CAL-1达97．7％，而与IDIR仅为89．0％。其mRNA折叠形成多达26个发卡环状结构。NSP2蛋白是由301个氨基酸残基组成的多肽，含有2个潜在的N-糖基化位点和多个磷酰化位点。结论成人腹泻标本WH-1中GBRV非结构蛋白NSP2基因和氨基酸序列与人GBRV有较高的同源性，而与动物中GBRV同源性相对较低，其中与ADRV的同源性最高，推测GBRV WH-1株与ADRV具有相同起源。     

Characterization of novel VP7, VP4, and VP6 genotypes of a previously untypeable group A rotavirus

Rotavirus is the most common cause of acute gastroenteritis among infants and young children throughout the world, but rotavirus cases in developing countries account for nearly all of the ∼ 600,000 annual deaths. We studied the epidemiology of rotavirus in 22 rural communities in northern coastal Ecuador over a five-year period. From 250 rotavirus positive stool specimens, the percentage that could not be RT-PCR genotyped for VP4 and VP7 was 77% and 63%, respectively. The possibility of sample degradation was considered but discounted after an experimental examination of rotavirus stability and EM visualization of rotavirus-like particles in several untypeable samples. Finally, alternate primers were used to amplify Ecu534, a sample that was untypeable using most published VP4 and VP7 primers. Characterization of the VP7, VP4, and VP6 full gene segments revealed novel genotypes and nucleotide mismatches with most published primer sequences. When considered with other findings, our results suggest that primer mismatch may be a widespread cause of genotyping failure, and might be particularly problematic in countries with greater rotavirus diversity. The novel sequences described in this study have been given GenBank accession numbers EU805775 (VP7), EU805773 (VP4), EU805774 (VP6) and the RCWG has assigned them novel genotypes G20P[28]I13, respectively.     

腺病毒载体表达轮状病毒G1型外壳蛋白VP7及其诱导的免疫应答研究

构建表达地方流行株轮状病毒G1型外壳蛋白VP7的复制缺陷型重组腺病毒,免疫小鼠评价其体液及细胞免疫反应效果,探讨基因工程轮状病毒疫苗的实验基础及可行性。RT-PCR扩增轮状病毒VP7基因并克隆至pshuttle-CMV穿梭质粒,pshuttle-VP7与腺病毒骨架质粒同源重组后转染293细胞包装重组腺病毒rAd-VP7。RT-PCR、western blot检测rAdVP7在体外细胞中的转录及表达。rAd-VP7经肌注或滴鼻免疫小鼠后检测其血清IgG、肠道IgA及中和抗体效价;流式、ELISPOT检测淋巴细胞亚群变化及IFN-γ分泌情况。结果显示ELISA可检测到免疫小鼠血清IgG和滴鼻组肠道IgA抗体的产生;肌注和滴鼻免疫组中和抗体平均滴度分别为228和322.5;免疫小鼠脾细胞IFN-γ的分泌增加。表达轮状病毒VP7的重组腺病毒不但能够激发体液及细胞免疫反应,滴鼻免疫途径还可诱导粘膜免疫应答。     

Molecular analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 genes of a buffalo rotavirus strain: identification of the rare P[3] rhesus rotavirus-like VP4 gene allele

We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P[3] rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B[3]), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P[3] genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.     

Mapping of antigenic sites on the major inner capsid protein of avian rotavirus using anEscherichia coli expression system

Summary The cDNA encoding the VP6 gene of avian rotavirus PO-13 strain was inserted into the bacterial expression vector pET-3a. Upon isopropyl-l-thio--D-galactoside induction, theE. coli BL21 (DE3) harboring the vector containing cDNA of the VP6 gene produced an approximately 45-kDa polypeptide, which reacted with rabbit serum against PO-13 strain in Western blotting. To study the antigenic sites on VP6, various deletion mutants were constructed, expressed inE. coli and the reactivity with antigenic site I- and II-specific MAbs analyzed by Western blotting. Site I, which is shared with all group A mammalian and avian rotaviruses except for chicken rotavirus, was found to be located at amino acid positions 45 to 65, and site II, which probably contributes to an authentic group A antigen common to both mammalian and avian rotaviruses, at amino acid positions 134 to 142.     

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS–PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.