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Overlapping of the VP2-VP3 gene and the VP1 gene in the SV40 genome.

The nucleotide sequence of the SV40 Hind E fragment has been determined mainly by the partial chemical degradation procedure of Maxam and Gilbert (1977). The sequence of the strand with the same polarity as the late messenger RNA shows only one open reading frame for translation. Considering that VP3 corresponds to the carbosyl terminal part of VP2, and considering various evidence which indicates that the SV40 Hind E segment is part of the amino acid sequence of VP2-VP3. It continues clockwise in Hind K, where it terminates with a UAA signal. The latter is located 110 nucleotides beyond the initiation signal for the major structural protein VP1 (Fiers et al., 1975; Van de Voorde et al., 1976). Hence this small overlapping region of the genome codes for the synthesis of three different proteins in two different reading frames. The deduced amino acid sequence covers a major part of the vp3 poly peptide, and the amino acid composition is in good agreement with published values (Greenaway and Levine, 1973).

Base Sequence↗

[A bivalent VP1 gene vaccine against Coxsackie virus B1/B3].

OBJECTIVE: To construct a bivalent VP1 gene vaccine against Coxsackie virus B1 and B3 (CVB1 and CVB3) and to test its immunogenicity in mice. METHODS: The VP1 gene of CVB1 was amplified by RT-PCR. The VP1 genes of CVB1 and of CVB3 under the control of cytomegalovirus (CMV) promoter and SV40 promoter respectively were inserted oppositely into the pCR3-Uni, a eukaryotic expression vector. The resultant bivalent gene vaccine plasmid was used to inoculate 3-week-old BALB/c mice. Immunofluorescence test and RT-PCR were conducted 24, 48, and 96 hours after the transfection. Sera of the tested animals were sampled at 0, 2, 4, and 6 weeks after the inoculation and detected for CVB-specific antibodies by ELISA. RESULTS: A bivalent gene vaccine plasmid of CVB1 and CVB3 was obtained. Samples of 4 and 6 weeks after the inoculation were shown to be CVB-antibody positive, while those of 0 and 2 weeks after the inoculation shown negative. CONCLUSION: The gene vaccine thus constructed induces CVB-speicific antibodies in mice, and can serve as gene vaccine candidate for human beings.

Animals↗

[Fusion expression of O type foot-and-mouth diseases virus VP1 gene and HSP70 gene and induction of immune responses in mice].

Vp1 gene of O type foot-and-mouth diseases virus and M. tuberculosis HSP70 were expressed in methylotrophic yeast Pichia pastoris expression system. The results of cellular immune responses and humoral immune response were examined after BALB/c mice were immunized with fusion protein expressed in methylotrophic yeast Pichia pastoris. The genes was cloned into the vector pPICZalpha-A by routine molecular technique. The plasmid fusion (pPICZalphaA-vp1-HSP70) was created that HSP70 located downstream of VP1 gene of O type foot-and-mouth disease virus. Vp1 was expressed by fusing to the amino terminus of M. tuberculosis hsp70 in yeast Pichia pastoris. The recombined fusion plasmid was transformed into methylotrophic yeast Pichia pastoris X-33 by electrophoration. The recombinant transformants were selected by Zeocin and induced by the addition of methanol every 24h. The expressived product analyzed by SDS-PAGE and Western blotting. The result indicated that the fusion protein(vp1-HSP70) has specific antigenicity. Mice were inoculated transcutaneous three times at a two-weeks interval with fusion protein, PBS and conventional inactivated vaccines. To evaluate the prophylaxtic efficacy of fusion protein, Titers of antibodies was detected by ELISA and proliferation of lymphocytes were determined by MTT. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses. Compared with conventional inactivated vaccines, fusion protein elicited slightly lower FMDV antibody level but stronger T cell proliferation.

Animals↗

Molecular cloning and expression of the VP1 gene of foot-and-mouth disease virus C1 in E. coli: effect on bacterial cell viability.

The VP1 gene of foot-and-mouth disease virus (serotype C1) has been cloned in Escherichia coli Clts cells, under the control of the bacteriophage lambda pL promoter. The expressed VP1 protein was complete and non-fused, and its molecular weight was indistinguishable from that of the VP1 obtained from virions. Cells harbouring the recombinant vectors exhibited symptoms of plasmid instability and toxicity and died in a few weeks even when never exposed to inducing conditions. A new plasmid clone in which a segment of the VP1 gene was fused with contiguous genes of the viral genome was very stable. The expressed partial VP1 protein contains the two major immunogenic domains of the virion. This system can be used as a tool to design an immunogenic VP1, and to explore possible synthetic vaccines against foot-and-mouth disease.

Animals↗

Differentiation of infectious bursal disease viruses by restriction enzyme analysis of RT-PCR amplified VP1 gene sequence.

In order to differentiate infectious bursal disease virus (IBDV) isolates/strains, a quick method of RT-PCR followed by restriction enzyme analysis of VP1 gene sequence is being reported for the first time. A 480 bp fragment, comprising one of the RNA dependent RNA polymerase motifs of VP1 gene sequence of an Indian classical virus, an attenuated vaccine strain, Georgia and two Indian field isolates, genetically similar to reported very virulent strains of IBDV, was amplified by RT-PCR. Restriction enzyme digestion of PCR products with Taq1 enzyme generated distinct profile for field isolates, different from the classical and attenuated viruses, whereas restriction profile with BstNI restriction enzyme was similar in all the viruses, irrespective of the pathotype. Therefore, the present results suggest that Taq1 digestion can be taken up for the differentiation of field isolates from the classical and vaccine strains. The sequence analysis of VPI gene of reported very virulent IBD viruses from Europe and Japan, using 'MapDraw' programme of Lasergene software, revealed similar restriction enzyme profile as in Indian field isolates.

Animals↗

An approach to a FMD vaccine based on genetic engineered attenuated pseudorabies virus: one experiment using VP1 gene alone generates an antibody responds on FMD and pseudorabies in swine.

Foot-and-mouth disease (FMD) and pseudorabies (PR) are two important infectious diseases in swine. An attenuated pseudorabies virus (PRV) has been successfully used as a gene delivery vector for the development of live-viral vaccines. In this study, a recombinant PRV-VP1 virus was constructed by fusioning the VP1 gene of FMD virus in frame to the N-terminal sequence of the gG gene of PRV. To test the protective immunity, 15 FMDV sero-negative white swine were divided into three groups and immunized with the recombinant PRV-VP1 virus, commercial FMD vaccine and vector virus (TK(-)/gG(-)/LacZ(+)), respectively, and challenged intramuscularly with 20 minimal infecting doses (MID) of virulent type O FMDV 4 weeks after booster immunization. Swine vaccinated with PRV-VP1 acquired antibodies against both FMDV and PRV, however, anti-FMDV antibodies were much lower than those vaccinated with the commercial FMD vaccine. Our results suggested that the recombinant PRV-VP1 virus, which only expressed FMDV VP1 gene controlled by PRV gG promoter, could not protect swine from the challenge of 20 MID type O FMDV, but could delay and reduce the clinical symptoms of FMD.

Animals↗

Fixation of mutations at the VP1 gene of foot-and-mouth disease virus. Can quasispecies define a transient molecular clock?

The number of nucleotide (nt) substitutions found in the VP1 gene (encoding viral capsid protein) between any two of 16 closely related isolates of foot-and-mouth disease virus (FMDV) has been quantified as a function of the time interval between isolations [Villaverde et al., J. Mol. Biol. 204 (1988) 771-776]. One of them (isolate C-S12) includes some replacements found in isolates that preceded it and other replacements found in later isolates. The study has revealed alternating periods of rapid evolution and of relative genetic stability of VP1. During a defined period of acute disease, the rate of fixation of replacements at the VP1 coding segment was 6 x 10(-3) substitutions per nt per year. Only small differences in the rate of evolution were observed between subsegments within the VP1 gene. The observation of a relatively constant rate of evolution during a disease episode was unexpected. We propose that such constancy may be a consequence of random sampling of mutants from the FMDV quasispecies, followed by their amplification in susceptible hosts (to generate a new quasispecies). Successive sampling and amplification events may result in a steady accumulation of mutations.

Amino Acid Sequence↗

Sequence and phylogenetic analysis of the L and VP1 genes of foot-and-mouth disease virus serotype Asia1.

Most of the molecular epidemiological studies of foot-and-mouth disease virus (FMDV) are based on comparison of VP1 gene sequence. In this report, we determine the nucleotide (nt) sequence of the L (603 nt) and VP1 (633 nt) genes of 27 FMDV serotype Asia 1 isolates recovered from different outbreaks in India, and compared with each other and the vaccine strain, IND 63/72, used in the country. Independent phylogenetic analyses on both the aligned gene sequences identified two major lineages (designated A & B) in the Asia 1 isolates. Both L- and VP1-based trees were congruent with respect to the major branching pattern of the isolates. The lineage A is represented by the isolates of 1986-2000 including the vaccine strain IND 63/72, whereas, lineage B appeared to be dominant and responsible for most of the recent outbreaks. A correlation was observed between the clustering of the isolates in the phylogenetic tree and the amino acid changes at many of the positions in VP1 as well as in L protein. The annual rate of evolution in L and VP1 genes was found similar and estimated to be 4.0 x 10(-3) and 3.8 x 10(-3) substitutions per nucleotide, respectively. Our result, largely from the congruence in phylogenetic trees and the rate of evolution in both the genes, suggests the possibility for the use of L gene sequence in phylogenetic comparison of FMDV.

Amino Acid Sequence↗

Genetic mapping of the Sorghum bicolor vp1 gene and its relationship with preharvest sprouting resistance.

The plant vp1 gene encodes a transcription factor originally identified in maize that participates in the control of the transition from embryogenesis to seed germination. Different lines of evidence suggest that vp1 participates in preharvest sprouting (PHS) resistance in cereals, but the genetic relationship is not yet established. Thus, the aim of this study was to investigate if there is a connection between vp1 and the formerly documented quantitative trait loci for PHS in Sorghum bicolor. This gene was mapped in sorghum using two well-studied inbred lines displaying contrasting PHS phenotypes. Linkage analysis revealed that the S. bicolor vp1 (Sbvp1) locus is linked to markers located on chromosomes 3 and 8 in maize. Analysis indicated that this gene is not correlated with PHS at a statistically significant level. Structural analysis of the Sbvp1 gene from both inbred lines showed a high degree of overall conservation (low polymorphism), even in the promoter region. These findings, taken together with the previously observed correlation between the pattern of expression of this gene and PHS performance, suggest that Sbvp1 might act downstream in the signalling pathway that leads to seed germination, or even be differentially regulated as a consequence rather than as a cause of the phenotypic behaviour.

Chromosome Mapping↗

Structure and function of the vp1 gene homologue from the resurrection plant Craterostigma plantagineum Hochst.

A gene that is homologous to the vp1 genes from maize and rice, the Arabidopsis abi3 gene and PvAlf from Phaseolus vulgaris was isolated from the resurrection plant, Craterostigma plantagineum Hochst, The C. plantagineum gene (epvp1) encodes a protein of 688 amino acids and contains five introns at positions identical to those in the Arabidopsis, maize and rice homologues. The cpvp1 transcript is present in mature seeds and in young seedlings up to 8 days following germination. The ability of the cpvp1 gene product to activate target genes was demonstrated by transient expression experiments in tobacco protoplasts using reporter gene constructs containing the beta-glucuronidase (GUS) gene fused to the promoter of two late embryogenesis abundant (LEA)-like genes, CDeT27-45 and CDeT6-19 from C. plantagineum, or the promoter of a maize gene encoding a 22-kDa zein seed storage protein.

Abscisic Acid↗

Intrinsic curvature in the VP1 gene of SV40: comparison of solution and gel results.

DNA restriction fragments that are stably curved are usually identified by polyacrylamide gel electrophoresis because curved fragments migrate more slowly than normal fragments containing the same number of basepairs. In free solution, curved DNA molecules can be identified by transient electric birefringence (TEB) because they exhibit rotational relaxation times that are faster than those of normal fragments of the same size. In this article, the results observed in free solution and in polyacrylamide gels are compared for a highly curved 199-basepair (bp) restriction fragment taken from the VP1 gene in Simian Virus 40 (SV40) and various sequence mutants and insertion derivatives. The TEB method of overlapping fragments was used to show that the 199-bp fragment has an apparent bend angle of 46 +/- 2 degrees centered at sequence position 1922 +/- 2 bp. Four unphased A- and T-tracts and a mixed A3T4-tract occur within a span of approximately 60 bp surrounding the apparent bend center; for brevity, this 60-bp sequence element is called a curvature module. Modifying any of the A- or T-tracts in the curvature module by site-directed mutagenesis decreases the curvature of the fragment; replacing all five A- and T-tracts by random-sequence DNA causes the 199-bp mutant to adopt a normal conformation, with normal electrophoretic mobilities and birefringence relaxation times. Hence, stable curvature in this region of the VP1 gene is due to the five unphased A- and T- tracts surrounding the apparent bend center. Discordant solution and gel results are observed when long inverted repeats are inserted within the curvature module. These insertion derivatives migrate anomalously slowly in polyacrylamide gels but have normal, highly flexible conformations in free solution. Discordant solution and gel results are not observed if the insert does not contain a long inverted repeat or if the long inverted repeat is added to the 199-bp fragment outside the curvature module. The results suggest that long inverted repeats can form hairpins or cruciforms when they are located within a region of the helix backbone that is intrinsically curved, leading to large mobility anomalies in polyacrylamide gels. Hairpin/cruciform formation is not observed in free solution, presumably because of rapid conformational exchange. Hence, DNA restriction fragments that migrate anomalously slowly in polyacrylamide gels are not necessarily stably curved in free solution.

Birefringence↗

[Expression of antigenic region of VP1 gene of swine vesicular disease in Escherichia coli].

The antigenic region of VP1 gene of swine vesicular disease virus was amplified by reverse transcription polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (nPCR). After the amplified fragment was cloned into the expression vector pProEX-HTb. The insert position,the size and the reading frame of the insertion were identified by PCR, restriction digestion and sequence analysis of the recombinant plasmids. SDS-PAGE and Western blot indicated that the transformed BL21(DE3) by the recombinant plasmids and induced by IPTG could express the antigen region of VP1 of swine vesicular disease virus, the expressed antigen protein could be recognized by the positive serum of SVDV.

Animals↗

Does the VP1 gene of foot-and-mouth disease virus behave as a molecular clock?

We have carried out a phylogenetic study of the evolution of the VP1 gene sequence from different serological types and subtypes of foot-and-mouth disease virus (FMDV). The maximum-likelihood method developed by Hasegawa and co-workers (Hasegawa et al. 1985) for the estimation of evolutionary parameters and branching dates has been used to decide between alternative models of evolution: constant versus variable rates. The results obtained indicate that a constant rate model, i.e., a molecular clock, seems to be the most plausible one. However, additional information suggests the possibility that the appearance of serotype CS has been accompanied by an episode of rapid evolution (Villaverde et al. 1991). We discuss the possibility that this evolution of RNA viruses was due to episodic positive Darwinian selection, which would have helped the new variant to escape the immunogenic pressure from the hosts.

Aphthovirus↗

Missense mutations in the VP1 gene of simian virus 40 that compensate for defects caused by deletions in the viral agnogene.

Simian virus 40 mutants lacking sequences in the late leader region are viable but produce smaller plaques than does wild-type virus. Within three passages at low multiplicities of infection, virus stocks of several such mutants accumulated variants that synthesized an altered form of the major virion protein, VP1, having a slightly faster mobility in sodium dodecyl sulfate-polyacrylamide gels than did the wild-type protein. Because these variants overgrew the original virus stocks, we consider them to be second-site revertants. By construction and characterization of a series of recombinants, the second-site mutations were shown to map to at least two different regions of the VP1 gene. Nucleotide sequence analysis indicated that single-amino-acid changes were responsible for the rapid mobility of VP1. When combined in cis with either a wild-type or mutant leader region, these VP1 mutations sped up by 10 to 20 h the time course of accumulation of infectious progeny but not of viral DNA or VP1. LP1, the protein encoded by the agnogene, was shown previously to be necessary for the efficient transport of the virion proteins to the nucleus or for their efficient assembly with viral minichromosomes. The VP1 missense mutations reported here compensate for the lack of LP1 by facilitating this process. On the basis of these findings and findings reported previously by us and others, we hypothesize that LP1 facilitates the formation of infectious particles by inhibiting the polymerization of VP1 molecules until the time they interact with viral minichromosomes; the VP1 mutations reported here compensate for the loss of LP1 by lessening the potential of VP1 for self-polymerization.

Animals↗

Nucleotide sequence of the restriction fragment Hind-F-EcoRI1 of simian-virus-40 DNA (part of the VP1 gene).

The nucleotide sequence of the simian virus 40 (SV40) genome region between the cleavage sites for restriction endonucleases EcoRI (map position 0) and HindII (map position 0.05) has been determined mainly by the partial chemical DNA degradation procedure of Maxam and Gilbert. This fragment represents 5.3% of the genome of SV40 and is located in the late region, internally in the VP1 gene. The message strand shows only one open reading frame for translation into protein, which connects to the one for the preceding fragment. On this basis part of the amino acid sequence of the VP1 protein is presented.

Base Sequence↗

Identification of optimal regions for phylogenetic studies on VP1 gene of foot-and-mouth disease virus: analysis of types A and O Argentinean viruses.

An analysis of the informative content of sequence stretches on the foot-and-mouth disease virus (FMDV) VPI gene was applied to two important viral serotypes: A and O. Several sequence regions were identified to allow the reconstruction of phylogenetic trees equivalent to those derived from the whole VPI gene. The optimal informative regions for sequence windows of 150 to 250 nt were predicted between positions 250 and 550 of the gene. The sequences spanning the 250 nt of the 3' end (positions 400 to 650), extensively used for FMDV phylogenetic analyses, showed a lower informative content. In spite of this, the use of sequences from this region allowed the derivation of phylogenetic trees for type A and type O FMDVs which showed topologies similar to those previously reported for the whole VP1 gene. When the sequences determined for viruses isolated in Argentina, between 1990 and 1993, were included in these analyses, the results obtained revealed features of the circulation of type A and type O viruses in the field, in the months that preceded the eradication of the disease in this country. Type A viruses were closely related to an Argentinean vaccine strain, and defined an independent cluster within this serotype. Among the type O viruses analysed, two groups were distinguished; one was closely related to the South American vaccine strains, while the other was grouped with viruses of the O3 subtype. In addition, a detailed phylogeny for type A FMDV is presented.

Amino Acid Sequence↗

A granule cell neuron-associated JC virus variant has a unique deletion in the VP1 gene.

The human polyomavirus JC (JCV) typically infects glial cells and is the aetiological agent of progressive multifocal leukoencephalopathy (PML), which occurs in immunosuppressed individuals. The full-length sequence of a granule cell neuron-tropic JCV variant, JCV(GCN1), associated with lytic infection of granule cell neurons and cerebellar atrophy in a human immunodeficiency virus-infected patient with PML was determined and compared with the sequence of the JCV isolate from the classic PML lesions present in the hemispheric white matter of the same individual (JCV(HWM)). A unique deletion was found in the C terminus of the VP1 gene of JCV(GCN1), which encodes the major capsid protein, resulting in a frame shift and a total change of the C-terminal amino acid sequence of this protein. This deletion was not present in JCV(HWM), suggesting that this mutation may be instrumental in facilitating entry or replication of JCV into granule cell neurons.

AIDS-Related Opportunistic Infections↗

Genetic diversity in the VP1 gene of foot-and-mouth disease virus serotype Asia 1.

Complete nucleotide sequence of the 1D (VP1-encoding) gene of 61 foot-and-mouth disease (FMD) serotype Asia I virus isolates recovered from different outbreaks in India between 1985 and 1999 including two vaccine strains currently used were determined. The sequences were compared with each other and those from other Asian countries. On the basis of phylogenetic analysis the viruses could be grouped into four genotypes (genotypes I-IV). All the 61 isolates from India belong to a single genotype (genotype-II) which is further subdivided into three lineages (B1, B2 and B3) under the same genotype. The viruses of the lineage B1 and B3 were found to be more prevalent before 1996 while the viruses of lineage B2 appeared to be new variants responsible for most of the recent outbreaks. Most of the isolates of lineage B1 lack one amino acid in the VP1 protein (position 44) whereas most of the isolates of lineage B2 and B3 contain it which indicates the possibility of these lineages having evolved independently. The rate of evolution of FMDV Asia 1 virus was also estimated and found to be 2.7 x 10(-2) synonymous substitutions per nucleotide per year.

Amino Acid Sequence↗