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PubMed1996

An enhancer of recombination in polyomavirus DNA.

Previous work from this laboratory has indicated that intramolecular homologous recombination of polyomavirus (Py) DNA is dependent upon promoter structure or function. In this report, we demonstrate that Py DNA contains not two but three binding sites for transcription factor YY1, all located on the late side of viral origin of replication (ori) and the third well within the VP1 coding sequence. This third site (Y3), which may or may not play a role in transcription regulation, is immediately adjacent to a previously described recombination hot spot (S1/S2). We found that Py replicons carrying an altered Y3 site recombined in a manner suggesting partial inactivation of the S1/S hot spot. Point mutations precluding the binding of YY1 to Y3 in vitro depressed hot spot activity in vivo; however, of the two reciprocal products reflecting recombination at this spot, only that carrying the mutated Y3 site arose at a reduced rate. These results are interpreted in light of a model assuming that recombination occurs within a transcriptionally active viral chromatin tethered to the nuclear matrix by YY1.

Animals↗
PubMed2006

VP2 cleavage and the leucine ring at the base of the fivefold cylinder control pH-dependent externalization of both the VP1 N terminus and the genome of minute virus of mice.

Cylindrical projections surrounding the fivefold-symmetry axes in minute virus of mice (MVM) harbor central pores that penetrate through the virion shell. In newly released DNA-containing particles, these pores contain residues 28 to 38 belonging to a single copy of VP2, disposed so that its extreme N-terminal domain projects outside the particle. Virions are metastable, initially sequestering internally the N termini of all copies of the minor capsid protein, VP1, that is essential for entry. This VP1 domain can be externalized in vitro in response to limited heating, and we show here that the efficiency of this transition is greatly enhanced by proteolysis of VP2 N termini to yield VP3. This step also renders the VP1 rearrangement pH dependent, indicating that VP2 cleavage is a maturation step required to prime subsequent emergence of the VP1 "entry" domain. The tightest constriction within the cylinder is created by VP2 leucine 172, the five symmetry-related copies of which form a portal that resembles an iris diaphragm across the base of the pore. In MVMp, threonine substitution at this position, L172T, yields infectious particles following transfection at 37 degrees C, but these can initiate infection only at 32 degrees C, and this process can be blocked by exposing virions to a cellular factor(s) at 37 degrees C during the first 8 h after entry. At 32 degrees C, the mutant particle is highly infectious, and it remains stable prior to VP2 cleavage or following cleavage at pH 5.5 or below. However, upon exposure to neutral pH following VP2 cleavage, its VP1-specific sequences and genome are extruded even at room temperature, underscoring the significance of the VP2 cleavage step for MVM particle dynamics.

Animals↗
PubMed2002

Molecular characterization of hepatitis-A-virus infections, in the context of two outbreaks in southern Thailand.

As hepatitis A virus (HAV) is usually transmitted through the faecal-oral route, hepatitis A is a communicable disease. In countries of intermediate to low endemicity, sudden outbreaks of human infection with the virus may occur. Between September 2001 and April 2002, there were two outbreaks of HAV infection in the Ruso and Yeengor districts of Narathiwas province, in southern Thailand. Isolates of HAV were recovered during these outbreaks, from 14 in-patients with acute hepatitis in Ruso (12 positive for anti-HAV IgM and all positive for HAV RNA), 16 children with asymptomatic infection in Yeengor (14 positive for anti-HAV IgM and nine for HAV RNA), and four isolated cases in Bangkok (all positive for anti-HAV IgM). Molecular characterization of the VP1-P2A region of each isolate was followed by phylogenetic analysis. All of the isolates from Narathiwas province were found to be of genotype 1a, to have the same VP1 nucleotide sequence, and to show a high level of sequence homology (>/= 99.5%) with the isolates from Bangkok and with previous Thai isolates. These results should facilitate further research into HAV transmission and genotype identification in community outbreaks.

Adolescent↗
PubMed2005

[Cytoplasmic expression of VP1 gene of coxsackievirus B3].

OBJECTIVE: To increase the immune effect of gene vaccine, T7 RNA polymerase was used to establish a system of cytoplasmic expression. METHODS: (1) The plasmid pT7 EMCVP1, including T7 promoter sequence, 5'-untranslated sequence of encephalomyocarditis (EMC) virus, VP1 sequence of coxsackievirus B3 (CVB3), was cotransfected with the plasmid pAR 3132, which codes for the T7 RNA polymerase, into HeLa cells and murine peritoneal macrophages. (2) The plasmid pT7 EMCVP1 and pAR 3132 were respectively transformed into the attenuated Salmonella typhimurium SL 7207. The two kinds of transformed bacteria were coinfected into murine peritoneal macrophages. RESULTS: (1) The target antigen VP1 in the cytoplasm was about 2-4-fold higher than that of pcDNA3 VP1 singly transfected. (2) After the murine peritoneal macrophages were coinfected by two kinds of transformed bacteria, the target antigen VP1 could also be detected. CONCLUSION: The pT7 EMCVP1 and pAR 3132 could be expressed in the cytoplasm of HeLa cells and murine peritoneal macrophages and the amount of the antigen VP1 increased remarkably as compared with that of pcDNA3 VP1 singly transfected.

Animals↗
PubMed1976

The initiation region of the SV40 VP1 gene.

The sequence of 15 nucleotides located at the 5' terminus of the plus strand of the SV40 Hind K fragment has been determined as (5') A-G-C-T-T-A-T-G-A-A-G-A-T-G-G (3'). The 3' on OH terminal G of this segment is part of the G-C-C codeword for the N terminal alanine of the VP1 protein. This region therefore presumably corresponds to a ribosome binding site on the 16S late mRNA. Complementarily to the 3' OH of eucaryotic 18S ribosomal RNA and homology with the BMV coat ribosome binding site are discussed.

Bacterial Proteins↗
PubMed2005

New sequence polymorphisms in the outer loops of the JC polyomavirus major capsid protein (VP1) possibly associated with progressive multifocal leukoencephalopathy.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML) in patients with decreased immune competence. To elucidate genetic changes in JCPyV associated with the pathogenesis of PML, multiple complete JCPyV DNA clones originating from the brains of three PML cases were established and sequenced. Although unique rearranged control regions occurred in all clones, a low level of nucleotide variation was also found in the coding region. In each case, a parental coding sequence was identified, from which variant coding sequences with nucleotide substitutions would have been generated. A comparison between the parental and variant coding sequences demonstrated that all 12 detected nucleotide substitutions gave rise to amino acid changes. Interestingly, seven of these changes were located in the surface loops of the major capsid protein (VP1). Finally, 16 reported VP1 sequences of PML-type JCPyV (i.e. derived from the brain or cerebrospinal fluid of PML patients) were compared with their genotypic prototypes, generated as consensus sequences of representative archetypal isolates belonging to the same genotypes; 13 VP1 proteins had amino acid changes in the surface loops. In contrast, VP1 proteins from isolates from the urine of immunocompetent and immunosuppressed patients rarely underwent mutations in the VP1 loops. The present findings suggest that PML-type JCPyV frequently undergoes amino acid substitutions in the VP1 loops. These polymorphisms should serve as a new marker for the identification of JCPyV isolates associated with PML. The biological significance of these mutations, however, remains unclear.

Adult↗
PubMed2001

The VP1-unique region of parvovirus B19: amino acid variability and antigenic stability.

The unique region of structural protein VP1 of parvovirus B19 (erythrovirus B19) is important for eliciting neutralizing antibodies that are responsible for eliminating the virus from the peripheral blood and for inducing lifelong immunity. Neutralizing human MAbs bind a conformationally defined epitope spanning VP1 residues 30-42. The DNA sequence encoding the VP1-unique region was determined in parvovirus B19 isolated from peripheral blood and amniotic fluid of nine acutely infected pregnant women, five arthritis patients and two chronically infected children. The amino acid sequences of the VP1-unique region exhibited higher variability in comparison with other B19-specific proteins. To analyse the influence of amino acid variations on antibody binding and protein conformation, two variants of the VP1-unique region were selected and expressed in E. coli as intein-fusion proteins. The selected variants displayed a number of amino acid exchanges in the VP1-unique region and had mutations in the determined epitope and adjacent regions. After purification via affinity chromatography, the dissociation constants K(D) of VP1-specific human MAbs interacting with the variant antigens and a viral prototype of the VP1-unique region were determined with a quartz crystal microbalance biosensor. A value of 5.4 x 10(-8) M was determined for the prototype isolate pJB; the affinity constants for the variant VP1-unique regions were similar. Comparable values were obtained for interaction of antibodies with non-infectious VP1/VP2 capsids produced by recombinant baculovirus and with B19 virions from amniotic fluid. It is concluded that the conformation of the epitope is unaffected by mutations or the environment of the VP1-unique region in virus capsids.

Amino Acid Sequence↗
PubMed1988

Neutralization sites of type O1 foot-and-mouth disease virus defined by monoclonal antibodies and neutralization-escape virus variants.

Monoclonal antibodies (MAbs) were derived from mice infected with foot-and-mouth disease virus type O1 Brugge (FMDV 01B) or immunized with inactivated virions (140 S) or viral subunits (12 S). A total of 19 neutralizing MAbs were characterized of which 17 recognized conformationally determined epitopes and two recognized amino acid sequences on isolated VP1. Neutralizing MAbs were used to select antigenic variants of FMDV O1B. Based on cross-neutralization and binding assays with MAbs the variants were divided into discrete groups demonstrating the presence of three unique neutralization sites on FMDV O1B. One site was present only on intact 140 S virions, a second was present on both 140 S virions and 12 S subunits, and the third was present on 140 S virions, 12 S subunits, and isolated VP1. Comparison of the deduced nucleic acid sequence of parental FMDV O1B with those of the O1B variants demonstrated that the epitope recognized by the VP1-reactive, neutralizing MAbs included amino acid residues 138, 144, and 148. Cross-neutralization assays demonstrated that these neutralization sites of FMDV O1B function on other type O1 strains of FMDV.

Animals↗
PubMed2003

Serial recombination during circulation of type 1 wild-vaccine recombinant polioviruses in China.

Type 1 wild-vaccine recombinant polioviruses sharing a 367-nucleotide (nt) block of Sabin 1-derived sequence spanning the VP1 and 2A genes circulated widely in China from 1991 to 1993. We surveyed the sequence relationships among 34 wild-vaccine recombinants by comparing six genomic intervals: the conserved 5'-untranslated region (5'-UTR) (nt 186 to 639), the hypervariable portion of the 5'-UTR (nt 640 to 742), the VP4 and partial VP2 genes (nt 743 to 1176), the VP1 gene (nt 2480 to 3385), the 2A gene (nt 3386 to 3832), and the partial 3D gene (nt 6011 to 6544). The 5'-UTR, capsid (VP4-VP2 and VP1), and 2A sequence intervals had similar phylogenies. By contrast, the partial 3D sequences could be distributed into five divergent genetic classes. Most (25 of 34) of the wild-vaccine recombinant isolates showed no evidence of additional recombination beyond the initial wild-Sabin recombination event. Eight isolates from 1992 to 1993, however, appear to be derived from three independent additional recombination events, and one 1993 isolate was derived from two consecutive events. Complete genomic sequences of a representative isolate for each 3D sequence class demonstrated that these exchanges had occurred in the 2B, 2C, and 3D genes. The 3D gene sequences were not closely related to those of the Sabin strains or 53 diverse contemporary wild poliovirus isolates from China, but all were related to the 3D genes of species C enteroviruses. The appearance within approximately 2.5 years of five recombinant classes derived from a single ancestral infection illustrates the rapid emergence of new recombinants among circulating wild polioviruses.

Base Sequence↗
PubMed1999

Capsid protein-encoding genes of hamster polyomavirus and properties of the viral capsid.

On the basis of its genome organization the hamster polyomavirus (HaPV) is closely related to the murine polyomavirus Py. But HaPV infection, in contrast to Py infection, gives rise to two different tumor types; depending on the hamster strain used for infection, HaPV induces either epitheliomas or lymphomas. Although the HaPV virions were shown to be similar to those of Py and SV40, more precise information about the structure and protein composition of the HaPV capsid was still missing. Here we describe the primary structure of the capsid protein-encoding HaPV genes and the structure and protein composition of the HaPV capsid. Virions isolated from epitheliomas in HaPV-infected hamsters were shown by electron microscopy to be spherical particles with the typical icosahedral structure of polyomaviruses. However, in contrast to the capsids of SV40 and Py, a T = 7 laevo symmetry of HaPV capsids was observed. Separation of HaPV virions in SDS polyacrylamide gels and Western blotting with VP1-specific antisera identified VP1 as the major capsid protein species corresponding in its molecular weight to the predicted value of 41.8 kDa. Because of the presence of two potential translational initiation sites in the VP1 gene, the N-terminal amino acid sequence of virion VP1 was determined and found to start at the second initiation site. The amino acid homologies of HaPV capsid proteins shared with Py varied between 65.5% (VP1), 45.4% (VP3) and 44.6% (VP2), whereas the homologies to the relevant proteins of other polyomaviruses were found to range between 49.6-57.9% for VP1 and 28.9-41% for VP2/VP3.

Amino Acid Sequence↗
PubMed1991

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.

Animals↗
PubMed1990

The effect of peptides containing the arginine-glycine-aspartic acid sequence on the adsorption of foot-and-mouth disease virus to tissue culture cells.

Sequencing of the VP1 of a large number of subtypes of foot-and-mouth disease virus (FMDV) has revealed the presence of a conserved arginine-glycine-aspartic acid (RGD) sequence located in a highly exposed region. This sequence has been shown to be essential for the interaction of certain extracellular matrix and adhesion proteins with a superfamily of cell-surface receptors called integrins. We have examined the effects of synthetic peptides containing the RGD sequence on the binding of eight different subtypes of FMDV to tissue culture cells. The results showed that such peptides inhibited viral adsorption by 50-80%. The inhibition was dose dependent but not as great as that achieved by using a saturating amount of virus as an inhibitor. Substitution of other amino acids for any of the three main residues lowered the inhibitory properties of the peptides. These results suggest that the RGD sequence in FMDV VP1 appears to be important for the interaction of virus with cellular receptor sites.

Adsorption↗
PubMed2006

Unique BK virus non-coding control region (NCCR) variants in hematopoietic stem cell transplant recipients with and without hemorrhagic cystitis.

Hematopoietic stem cell transplant recipients frequently develop BK virus (BKV)-associated hemorrhagic cystitis, which coincides with BK viruria. However, the precise role of BKV in the etiology of hemorrhagic cystitis in hematopoietic stem cell transplant recipients remains unclear, since approximately 50% of all such adult transplant recipients excrete BKV, yet do not develop this clinical condition. In the present study, BKV were analyzed to determine if mutations in the non-coding control region (NCCR), and specific BKV sub-types defined by sequence analysis of major capsid protein VP1, were associated with development of hemorrhagic cystitis in hematopoietic stem cell transplant recipients. The regions encoding VP1 and NCCRs of BKV in urine samples collected from 15 hematopoietic stem cell transplant recipients with hemorrhagic cystitis and 20 without this illness were amplified and sequenced. Sequence variations in the NCCRs of BKV were identified in urine samples from those with and without hemorrhagic cystitis. Furthermore, five unique sequence variations within transcription factor binding sites in the canonical NCCR, O-P-Q-R-S, were identified, representing new BKV variants from a population of cloned quasi-species obtained from patients with and without hemorrhagic cystitis. Thirty-five BKV VP1 sequences were analyzed by phylogenetic analysis but no specific BKV sub-type was associated with hemorrhagic cystitis. Five previously unrecognized naturally occurring variants of the BKV are described which involve amplifications, deletions, and rearrangements of the archetypal BKV NCCRs in individuals with and without hemorrhagic cystitis. Architectural rearrangements in the NCCRs of BKV did not appear to be a prerequisite for development of hemorrhagic cystitis in hematopoietic stem cell transplant recipients.

Adolescent↗
PubMed2007

Analysis of the full-length genome of a subgenotype IIIB hepatitis A virus isolate: primers for broadly reactive PCR and genotypic analysis.

Among six known subgenotypes (IA, IB, IIA, IIB, IIIA, and IIIB) of human hepatitis A virus (HAV), the complete genomic sequence has not been determined for IIIB. In this study, the full-length genomic sequence of a IIIB HAV isolate (HA-JNG06-90F) recovered from a Japanese patient who contracted sporadic hepatitis A in 1990, was determined. The HA-JNG06-90F genome, which comprised 7462 nt excluding the poly(A) tail, was related most closely to NOR-21 of subgenotype IIIA with an identity of 89.1%, and was only 82.6-83.4% similar to human HAV isolates of genotypes I and II over the entire genome. Comparison of full-length genomic sequences of 20 reported isolates and HA-JNG06-90F generated optimal results for separation of different levels: the nucleotide identities were 80.7-86.6% at the genotype level, 89.1-91.9% at the subgenotype level, and 94.6-99.7% at the isolate level. Similar ranges of nucleotide identity were observed when comparing partial nucleotide sequences of the VP1-2B (481 nt; primer sequences at both ends excluded) and 3C/3D (590 nt) regions, which were amplifiable by PCR with primers designed from well-conserved areas of the HAV genome. All 66 samples with IgM-class HAV antibodies tested positive for HAV RNA by both VP1-2B (481 nt)-PCR and 3C/3D (590 nt)-PCR: subgenotype assignment was concordant in all samples tested (IA [n = 61], IB [n = 1], IIIA [n = 2] and IIIB [n = 2]). These results suggest that two broadly reactive PCRs using primers derived from the VP1-2B and 3C/3D regions, respectively, may be applicable to universal detection and phylogenetic analysis of various HAV strains.

DNA Primers↗
PubMed2002

Laboratory diagnosis and genetic analysis of an echovirus 30-associated outbreak of aseptic meningitis in Taiwan in 2001.

A large outbreak of aseptic meningitis occurred from April to November 2001 in Taiwan. Of the 1,130 enterovirus-infected patients, echovirus 30 (E30) infection was diagnosed in 188 (16.6%), with the patients having various clinical manifestations including aseptic meningitis (73.9%), young infant fever (6.9%), respiratory symptoms or herpangina (13.3%), or others (5.9%). The majority of the E30-infected patients were between 3 and 10 years old. Of the 264 E30 strains identified, 94.3, 71, and 67.4% were isolated from RD, MRC-5, and A549 cells, respectively. Primary isolation of E30 required mean times of 3.7 days for RD cells and 4.1 days for MRC-5 and A549 cells. Among all E30-positive patients, virus was most frequently isolated from throat swab specimens (85.2%) and, to a lesser extent, stool (76.4%) or cerebrospinal fluid (70.1%) specimens. The virus isolates were initially identified as echovirus 4 (E4) on the basis of immunofluorescence staining with anti-E4 and anti-E30 (Bastianni prototype) monoclonal antibodies. However, upon performance of the neutralization test, E30-specific reverse transcription-PCR, and sequencing of the VP1 gene, the results identified these isolates as E30, not E4, indicating that the reagent used to type E30, which is produced with the Bastianni strain as the immunogen, is inadequate for the identification of recent E30 isolates in Taiwan. Phylogenetic analyses of the VP1 genes of these isolates showed that their sequences differed from those of E30 isolates from the GenBank database by 9.1 to 25.2%, suggesting that this outbreak was caused by a new variant strain of E30 introduced into Taiwan in 2000 that resulted in the widespread aseptic meningitis epidemic in 2001.

Adolescent↗
PubMed1983

Chemical cleavage of polyomavirus major structural protein VP1: identification of cleavage products and evidence that the receptor moiety resides in the carboxy-terminal region.

As a first step toward identifying the various functional regions of the polyomavirus major capsid protein VP1, we used recently developed methods for the chemical cleavage of proteins and the available polyomavirus sequence data to devise a scheme to produce large, identifiable peptides and generate a cleavage map of VP1. Formic acid (75%) was found to cleave VP1 at only two sites, producing three peptides of apparent molecular weights of 29,000, 16,000, and 2,000. The order of peptides in intact VP1 was determined by recleavage of partial products and was found to be 29,000, 16,000, and 2,000. Two-dimensional peptide mapping studies of 125I-labeled VP1 formic acid peptides established that the limit products of formic acid digestion contained mutually exclusive sets of labeled peptides when either trypsin or chymotrypsin was used and that together the formic acid peptides contained all of the 125I-labeled tryptic and chymotryptic peptides found in VP1. Iodosobenzoic acid (IBA) digestion produced four peptides separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 12,000, 8,000, 7,000, and 5,000. The approximate positions of the IBA peptides in the VP1 sequence were determined by cleavage of formic acid fragments with IBA. The number of peptides produced, their respective sizes, and their order in the intact VP1 molecule agree with predictions made from available sequence data, both for formic acid cleavage and IBA cleavage. In addition, the numbers of 125I-labeled tryptic peptides produced from digestion of VP1 formic acid peptides also agree with predictions made from the sequence information. These data establish with reasonable certainty that the peptides produced by formic acid cleavage and IBA cleavage of VP1 are indeed those predicted. Antibodies raised against spontaneously produced, previously undefined polypeptides resulting from degradation of VP1 reacted exclusively with the formic acid peptides derived from the C-terminal portion of VP1. These antibodies inhibited hemagglutination and neutralized polyomavirus virions. We interpret this to mean that at least some of the antigenic determinants of the receptor moiety reside in this portion of the VP1 sequence.

Amino Acid Sequence↗
PubMed2004

Amplification of Echoviruses genomic regions by different RT-PCR protocols--a comparative study.

In the present report, the results of a comparative study in the detection of all Echoviruses reference strains as well as of 38 clinical isolates are presented. Using RT-PCR with already published primer pairs (UG(52)-UC(53), 292-222, 012-011 and EUG2a, 2b, 2c-EUC2) from the 5'UTR, the VP1 region as well as a long genomic fragment including the VP1 3' end, the entire coding sequence of 2A, 2B, and the 5' moiety of the 2C-coding region amplification was effective with all reference and clinical Echovirus isolates with primer pair UG(52)-UC(53) while with 292-222 and 012-011 were amplified 27/28 reference Echovirus strains and all clinical isolates. As far as EUG2a,2b,2c-EUC2 is concerned, the RT-PCR gave a positive result for 26/28 reference Echovirus strains and 34/38 clinical isolates. The sequence analysis of a large part of the 5'UTR has revealed that there is no correlation between 5'UTR identity and the currently recognized human enterovirus species. It has been suggested that part of VP1 coding sequence would correlate well with serotype since a number of important neutralization epitopes, as well as receptor recognition sequences, lie within the VP1 coding sequence. Therefore, UG(52)-UC(53) and 292-222 primer pairs seem to be the most appropriate for Echovirus detection and, moreover, UG(52)-UC(53) is useful for the classification of enteroviruses into genetic clusters (sub-groups) while 292-222 for the identification of enteroviruses by amplicon sequencing.

DNA Primers↗