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PubMed1977

Nucleotide sequence of the restriction fragment Hind F-Eco RI2 of SV40 DNA.

The nucleotide sequence of the SV40 genome region between the Hind K fragment and the Eco RI cleavage site has been determined by a combination of three different approaches : analysis of RNA products obtained by transcription with Escherichia coli DNA dependent RNA polymerase, partial degradations with snake venom exonuclease and base-specific chemical degradation of 5'-terminal labeled restriction fragments. This nucleotide sequence shows only one open reading frame and allows the deduction of a small segment of the amino acid sequence of VP1, the major structural protein.

Alkaline Phosphatase↗

PubMed1990

Delineation of canine parvovirus T cell epitopes with peripheral blood mononuclear cells and T cell clones from immunized dogs.

Three synthetic peptides derived from the amino acid sequence of VP2 of canine parvovirus (CPV) which were recently shown to represent three distinct T cell epitopes for BALB/c mice could prime BALB/c mice for a CPV-specific proliferative T cell response upon immunization. Proliferative responses of peripheral blood mononuclear cells (PBMC) from CPV-immunized dogs upon stimulation with these and other peptides, covering the major part of the sequence of VP2', identified the presence of T cell epitopes for this species. Most of these epitopes were recognized by PBMC from only a minority of the dogs tested. With three newly generated canine Thyl+ T cell clones, which recognized CPV antigen in association with major histocompatibility complex class II molecules, two distinct T cell epitopes were identified within the unique sequence of VP1.

Amino Acid Sequence↗

PubMed1999

A peptide construct containing B-cell and T-cell epitopes from the foot-and-mouth disease viral VP1 protein induces efficient antiviral protection.

A new peptide construct Palm135-158-GGA-170-188(Acm) has been synthesized and investigated in a number of in vitro and in vivo test systems. The construct contains a virus specific T-helper epitope within the 170-188 sequence of VP1, in addition to the main antigenic 135-158 region of the foot-and-mouth disease viral VP1 protein (strain A22). The construct has higher protective, antigenic, immunogenic and T-cell proliferative activity then the previously described shorter peptide Palm(2)135-159. The 170-188 part of the construct serves as a virus specific T-epitope, responsible for the enhanced immunogenic and protective activity of the construct.

Amino Acid Sequence↗

PubMed1990

Modelling of poliovirus. HIV-1 antigen chimaeras.

We have used laboratory-based molecular modelling to identify structural features of antigen chimaeras of poliovirus expressing epitopes from human immunodeficiency virus (HIV-1) that may affect virus viability. Chimaeras were constructed by replacement of antigenic site 1 of VP1 by sequences corresponding to epitopes from HIV-1. Loop volume, estimated by approximating the loop to an ellipsoid was significantly (P less than 0.001) lower in viable (2062.1 A3 +/- 400.2) than in non-viable (3617 A3 +/- 650.7) constructs. Our results suggest that viable virus will only be formed when antigen chimeras modified at antigenic site of VP1 have a loop occupying a similar volume in space to that occupied by the antigenic site 1 loop. In addition, the modified loop must fit with the peptide bond angles and distances at the top of the beta-barrel of VP1.

Amino Acid Sequence↗

PubMed1983

Expression of poliovirus capsid polypeptide VP1 in Escherichia coli.

A recombinant plasmid (pPV1-958) carrying a poliovirus cDNA insert corresponding to nucleotides 1-5750 of the poliovirus RNA genome was constructed through in vitro recombination of two plasmids carrying overlapping poliovirus cDNA inserts (Van der Werf et al., Proc. Natl. Acad. Sci. USA 78 (1981) 5983-5987). pPV1-958 was then cleaved with PstI and the fragment carrying the sequence encoding capsid polypeptide VP1 was isolated and subcloned at the PstI site of pBR322. The VP1 sequence was successfully fused with the 5' sequence of the beta-lactamase gene by deleting 300-350 bp from each side of the PvuI site of the plasmid with nuclease BAL31. One of the resulting plasmids, pSW119, expressed an Mr 49 000 VP1-beta-lactamase-fusion protein, which was specifically immunoprecipitated with anti-VP1 immune serum but not by hyperimmune sera raised against native or heat-inactivated virus.

Capsid↗

PubMed1995

Overlap of Viviparous1 (VP1) and abscisic acid response elements in the Em promoter: G-box elements are sufficient but not necessary for VP1 transactivation.

The relationship between promoter sequences that mediate Viviparous1 (VP1) transactivation and regulation by abscisic acid (ABA) in the wheat Em promoter was investigated using deletion analysis and directed mutagenesis. The Em1a G-box is strongly coupled to VP1 transactivation as well as to ABA regulation; however, the Em promoter includes additional components that can support VP1 transactivation without ABA responsiveness or synergism. Oligonucleotide tetramers of several G-box sequences, including Em1a, Em1b, and the dyad G-box element from the UV light-regulated parsley chalcone synthase gene, were sufficient to confer VP1 transactivation and the synergistic interaction with ABA to the -45 cauliflower mosaic virus 35S core promoter. These data suggest that VP1 can activate transcription through at least two classes of cis-acting sequences, including the G-box elements and the Sph regulatory motif found in the C1 promoter. The contrasting roles of these motifs in the Em and C1 promoters suggest a basis for the differential regulation of the corresponding genes by VP1.

Abscisic Acid↗

PubMed1997

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.

Aleutian Mink Disease Virus↗

PubMed2002

Phylogenetic analysis of Ljungan virus and A-2 plaque virus, new members of the Picornaviridae.

In addition to the viruses belonging to the nine proposed genera of the Picornaviridae, Enterovirus, Rhinovirus, Cardiovirus, Aphtovirus, Hepatovirus, Parechovirus, Kobuvirus, Erbovirus and Teschovirus, two new members of this family have recently been discovered. Three strains of Ljungan virus (LV) were isolated from bank voles (Clethrionomys glareolus) and A-2 plaque virus (A-2) was isolated from human sera. To study the genetic relationship between these recently discovered viruses and the members of the family Picornaviridae, an evolutionary analysis has been carried out using the amino acid sequences of the two nonstructural proteins 2C and 3D. Phylogenetic analysis using prime members of the nine genera support the division of picornaviruses into the proposed genera. The study also supports a previous suggestion based on analysis of partial sequences of the structural proteins that LV is more related to the genus of Parechovirus than to other picornaviruses, but also shows that the three LV strains used in the comparison constitute a distinct monophyletic group, clearly separated from the parechoviruses. The analyses using the 2C and 3D sequences clearly showed that A-2 was related to the genera of Rhinovirus and Enterovirus, but it was not possible to group the A-2 with high confidence into one of the genera. Comparison using the VP1 protein sequences of Enterovirus and Rhinovirus showed that although the A-2 virus is positioned between the two genera, the virus is more related to the genus of Enterovirus than to Rhinovirus. Our analysis of the three LV strains based on the phylogenetic analysis of the 2C and 3D proteins suggests that the strains used in this study constitute a monophyletic group clearly related to Parechovirus of Picornaviridae. The taxonomic position of the A-2 virus is presently uncertain but available data indicate that this virus may be classified as a member of the genus of Enterovirus.

Animals↗

PubMed1991

Identification of two determinants that attenuate vaccine-related type 2 poliovirus.

The poliovirus P2/P712 strain is an attenuated virus that is closely related to the type 2 Sabin vaccine strain. By using a mouse model for poliomyelitis, sequences responsible for attenuation of the P2/P712 strain were previously mapped to the 5' noncoding region of the genome and a central region encoding VP1, 2Apro, 2B, and part of 2C. To identify specific determinants that attenuate the P2/P712 strain, recombinants between this virus and the mouse-adapted P2/Lansing were constructed and their neurovirulence in mice was determined. By using this approach, the attenuation determinant in the central region was mapped to capsid protein VP1. Candidate attenuating sequences in VP1 and the 5' noncoding region were identified by comparing the P2/P712 sequence with that of vaccine-associated isolate P2/P117, and the P2/117 sequences were introduced into the P2/Lansing-P2/P712 recombinants by site-directed mutagenesis. Results of neurovirulence assays in mice indicate that an A at nucleotide 481 in the 5' noncoding region and isoleucine (Ile) at position 143 of capsid protein VP1 are the major determinants of attenuation of P2/P712. These determinants also attenuated neurovirulence in transgenic mice expressing human poliovirus receptors, a new model for poliomyelitis in which virulent viruses are not host restricted. These results demonstrate that A-481 and Ile-143 are general determinants of attenuation.

Animals↗

PubMed1988

Antigenic and immunogenic properties of a hepatitis A virus capsid protein expressed in Escherichia coli.

We have constructed a recombinant plasmid producing, in bacteria, a hepatitis A virus (HAV) capsid protein that may be useful as a subunit vaccine. HAV VP1 coding sequences were fused in-frame to NH2-terminal Escherichia coli TrpE coding sequences under the control of the tryptophan promoter. In the absence of exogenous tryptophan, E. coli containing this recombinant plasmid produced high levels of an 88-kilodalton fusion protein that was recognized in immunoblots by antibodies to TrpE and HAV. The TrpE/HAV VP1 protein was gel-purified and used to immunize rabbits. The resulting antiserum reacted with denatured HAV VP1 in immunoblots but did not react with intact virus. However, subsequent inoculation of an immunized animal with a subimmunogenic dose of inactivated, whole HAV resulted in the rapid appearance of a stable, virus-neutralizing antibody response.

Animals↗

PubMed2002

Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14.

Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.

Base Sequence↗

PubMed1995

Complete sequence of the RNA genome of human rhinovirus 16, a clinically useful common cold virus belonging to the ICAM-1 receptor group.

We report here the complete nucleotide sequence and predicted polyprotein sequence of HeLa cell-adapted human rhinovirus 16 (HRV16). This virus is more suitable than human rhinovirus 14 (HRV14) for clinical studies, and its growth and physical properties are favorable for biochemical and crystallographic analysis. The complete message-sense RNA genome of HRV16 is composed of 7124 bases, not including the poly(A) tail. An open reading frame, extending from base 626 to 7084 predicts a polyprotein containing 2152 amino acid residues. Comparison with other rhinovirus sequences shows HRV16 is much more representative of human rhinoviruses than HRV14. No apparent relationship was found between receptor group and amino acid sequence in VP1, the capsid protein bearing the binding site for the intercellular adhesion molecule-1 (ICAM-1) in both HRV14 and HRV16.

Base Sequence↗

PubMed2004

Complete nucleotide sequence analysis of a vaccine strain and a field isolate of foot-and-mouth disease virus serotype Asia1 with an insertion in VP1 genomic region.

Complete nucleotide sequences except the poly (C) tract and poly (A) tail of a vaccine strain (IND 491/97) and an atypical field isolate (IND 321/01) of Foot-and-mouth disease virus (FMDV) serotype Asia1 are described. Amino acid (aa) sequence analysis of the VP1 protein of the field isolate revealed that the latter has 212 instead of 210 or 211 aa found in the so far available sequences of other FMDV isolates of Asia1 serotype. The insertion was localized in the hypervariable region of aa 130-160 of VP1 protein. Nucleotide sequencing of the entire genome was therefore carried out to detect changes in other parts of the genome, if any, besides VP1, which could contribute to its fitness. An 8.16 kb sequence of IND 491/97 and an 8.162 kb sequence of IND 321/01 were compared with each other and also with the known sequence of IND 63/72, another vaccine strain of serotype Asia1. Comparison of the entire polyprotein coding (L to 3D) region of IND 321/01 with those of the two Asia1 vaccine strains (IND 63/72 and IND 491/97) revealed no significant differences. A similar comparison of IND 491/97 with IND 63/72 revealed variability across the entire length of the genome. In addition to the capsid-coding region, sequence variability was also observed in non-structural proteins albeit to different extent. This study shows that in the gene pool of serotype Asia1 at least three groups of isolates/strains are present with respect to the length of VP1 protein.

Amino Acid Sequence↗

PubMed1999

Controlled conformational transitions in the MVM virion expose the VP1 N-terminus and viral genome without particle disassembly.

Antisera were raised against peptides corresponding to the N-termini of capsid proteins VP1 and VP2 from the parvovirus minute virus of mice. Epitopes in the 142-amino-acid VP1-specific region were not accessible in the great majority of newly released viral particles, and sera directed against them failed to neutralize virus directly or deplete stocks of infectious virions. However, brief exposure to temperatures of 45 degreesC or more induced a conformational transition in a population of full virions, but not in empty viral particles, in which VP1-specific sequences became externally accessible. In contrast, the VP2 N-terminus was antibody-accessible in all full, but not empty, particles without prior treatment. An electrophoretic mobility shift assay, in which particles were heat-treated and/or preincubated with antibodies prior to electrophoresis, confirmed this pattern of epitope accessibility, showing that the heat-induced conformational transition produces a retarded form of virion that can be supershifted by incubation with VP1-specific sera. The proportion of virions undergoing transition increased with temperature, but at all temperatures up to 70 degreesC viral particles retained structure-specific antigenic determinants and remained essentially intact, without shedding individual polypeptide species or subunits. However, despite the apparent integrity of its protective coat, the genome became accessible to externally applied enzymes in an increasing proportion of virions through this temperature range, suggesting that the conformational transitions that expose VP1 likely also allow access to the genome. Heating particles to 80 degreesC or above finally induced disassembly to polypeptide monomers.

Amino Acid Sequence↗

PubMed1989

[Antigenic structure of foot-and-mouth virus. IV. Synthesis and immunogenic properties of new fragments of the VP1 protein of food-and-mouth virus strain A22].

A synthesis of new fragments of VP1 protein with the specificity of A22 strain of foot-and-mouth disease virus is described. Immunization with the free 136-152 peptide and KLH-conjugates of the peptides 136-152 and 197-213 induced 60-80% protection of guinea pigs against challenge with the A22 virus. Synthetic peptides corresponding to the 10-24, 50-69 and 175-189 sequences of VP1 did not show any protective activity. We have found that uncoupled peptides 175-189 and 197-213 are able to induce antipeptide antibodies. However, these antibodies did not possess any neutralizing activity. Immunization of animals with the mixture of (136-152)O1K and (175-189)A22 has led to inhibition of the immune response to the (136-152)O1K fragment.

Amino Acid Sequence↗

PubMed2000

A molecular genetic analysis of Eragrostis tef (Zucc.) Trotter: non-coding regions of chloroplast DNA, 18S rDNA and the transcription factor VP1.

The non-coding chloroplast DNA sequences of the trnL (UAA) intron and the trnL-trnF (GAA) intergeneric spacer (IGS), the coding sequences of nuclear 18S rDNA, and the transcription factor Vp1 of the cereal tef (Eragrostis tef (Zucc.) Trotter) were studied. No intraspecific variation was found among the 6 studied tef varieties. However, the study displayed that Eragrostis tef has a number of unique traits compared to other grasses. Phylogenetic analysis of the chloroplast DNA gave three grass clades, joining Eragrostis with sorghum and maize in one. In the analysis of the 18S rDNA sequences, the three grass species were joined in a monophyletic trichotomy in the cladogram, in which maize is the most divergent, rice the least and tef intermediate. The Vp1 is highly conserved. The Vp1 phylogeny showed that the tef Vp1-sequence is the hitherto most divergent Vp1-sequence reported from a grass.

Amino Acid Sequence↗

PubMed2000

Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998.

A large scale outbreak of hand-foot-and-mouth disease (HFMD) occurred in Taiwan in 1998, in which more than 80 children died of shock syndrome with pulmonary edema/hemorrhage. Enterovirus 71 was implicated as the cause of this outbreak. In order to understand the virological basis responsible for mortality on this scale, nucleotide sequences of VP1 that is important for serotypic specificity, and the 5'-non-coding region (5'-NCR) that is important for replication efficiency, were analyzed comparatively. Phylogenetic analysis of both VP1 and 5'-NCR of nine EV71 isolates derived from specimens of fatal patients and seven isolates derived from uncomplicated HFMD patients showed that all but one isolate fell into genotype B. The one distinct isolate from a case of uncomplicated HFMD belonged to genotype C that was clustered along with one isolate from Taiwan in 1986. Complete sequence analysis of two selected isolates, one from the spinal cord of a fatal case and one from the vesicle fluid of a patient with mild HFMD, confirmed a high degree (97-100%) of identity in nucleotide sequence throughout the entire genome, except focal regions of 3C and 3'-NCR where the nucleotide homology was 90-91%. The identity of the deduced amino acid sequence in the 3C region that encodes viral proteinase dropped further to 86%, a result of missense mutations at the first nucleotide position of many codons.

5' Untranslated Regions↗

PubMed1998

Enhancement of the immunogenicity of a synthetic peptide bearing a VP3 epitope of hepatitis A virus.

The immune responses elicited in mice by different forms of the VP3(110-121) B-epitope of the hepatitis A virus (HAV) were studied. Different forms of incorporation in liposomes were tested, encapsulation, rather than surface exposure, being the best antigenic preparation. Three larger peptides of the VP3 epitope, two of them containing a hepatitis B virus T-epitope, and a third containing a putative T-epitope of HAV (VP3(102-121)) were assayed. While this latter T-epitope induced an enhancement of the response against the VP3 B-epitope, the artificially coupled T-epitopes failed to induce a significant increase. The administration of two multiple antigenic peptide (MAP) constructs, the first containing the VP3(110-121) and VP1(11-25) HAV sequences and the second only the VP1(11-25) sequence, also suggested the presence of a T-epitope, since the response against the VP1 peptide was increased in the first construct.

Animals↗