A single amino acid substitution in the capsid protein VP1 of coxsackievirus B3 (CVB3) alters plaque phenotype in Vero cells but not cardiovirulence in a mouse model

We previously described a large plaque attenuant (p14V-1) derived from a cardiovirulent Coxsackievirus B3 (CVB3) and showed that there were no major determinants of either attenuation or plaque phenotype in the 5' nontranslated region (5'NTR). Part of the region encoding the last 124 amino acids of VP3 and the first 106 amino acids of VP1 of the attenuant was then sequenced and compared to the wild-type. Three nucleotide changes were found in the VP1 coding region: a silent single base change at nucleotide position 2467 (C to U) and a double-base change at position 2690-1 (AA to GT), which leads to a change from lysine to serine at amino acid position 80. This mutation maps to the begining of B-C loop of the three-dimensional structure of VP1 of CVB3, where a distinct surface projection is formed. Two infectious chimeric cDNA clones were constructed, based on a cardiovirulent cDNA construct. In one construct, the 5'NTR and the VP3-VP1 region were from p14V-1 and in the other, only the VP3-VP1 region was from this attenuant. Both chimeric viruses produced large plaques on Vero cell monolayers, similar to p14V-1 but larger than the prototypic cardiovirulent virus. In vivo experiments showed that both chimeric viruses induced myocarditis in a murine model, similar to wild-type virus. We conclude that mutation serine-80 in capsid protein VP1 of p14V-1 is a determinant of the large plaque phenotype but is not responsible for attenuation.     

Active cross-protection induced by a recombinant baculovirus expressing chimeric infectious bursal disease virus structural proteins

The VP2 structural gene encoded in the large genomic segment A of the variant GLS strain of infectious bursal disease virus (IBDV) was modified to encode a neutralization epitope (B69), found only on classic strains of IBDV. A chimeric cDNA clone of the large segment A, encoding VP3, VP4, and the modified variant IBDV VP2 structural proteins, was expressed in a recombinant baculovirus. The chimeric protein expressed was assessed with a panel of neutralizing monoclonal antibodies (MAbs), and it contained not only all previously MAb-defined GLS variant strain epitopes but also the B69 neutralization epitope found on classic IBDV strains. Complete active protection was afforded to specific-pathogen-free chickens by a subunit chimeric vaccine against virulent challenge with the classic IM and STC strains, as well as against the variant E/Del and GLS IBDV strains. Compared with a previously tested recombinant subunit vaccine, which incorporated unmodified baculovirus-expressed large-segment A GLS proteins, the recombinant chimeric subunit vaccine resulted in markedly improved active cross-protection against classic IBDV challenge.     

Transgenic expression of replication-restricted enteroviral genomes in heart muscle induces defective excitation-contraction coupling and dilated cardiomyopathy

Numerous studies have implicated Coxsackievirus in acute and chronic heart failure. Although enteroviral nucleic acids have been detected in selected patients with dilated cardiomyopathy, the significance of such persistent nucleic acids is unknown. To investigate the mechanisms by which restricted viral replication with low level expression of Coxsackieviral proteins may be able to induce cardiomyopathy, we generated transgenic mice which express a replication-restricted full-length Coxsackievirus B3 (CVB3) cDNA mutant (CVB3DeltaVP0) in the heart driven by the cardiac myocyte-specific myosin light chain-2v (MLC-2v) promoter. CVB3DeltaVP0 was generated by mutating infectious CVB3 cDNA at the VP4/VP2 autocatalytic cleavage site from Asn-Ser to Lys-Ala. Cardiac-specific expression of this cDNA leads to synthesis of positive- and negative-strand viral RNA in the heart without formation of infectious viral progeny. Histopathologic analysis of transgenic hearts revealed typical morphologic features of myocardial interstitial fibrosis and in some cases degeneration of myocytes, thus resembling dilated cardiomyopathy in humans. There was also an increase in ventricular atrial natriuretic factor mRNA levels, demonstrating activation of the embryonic program of gene expression typical of ventricular hypertrophy and failure. Echocardiographic analysis demonstrated the presence of left ventricular dilation and decreased systolic function in the transgenic mice compared with wild-type littermates, evidenced by increased ventricular end-diastolic and end-systolic dimensions and decreased fractional shortening. Analysis of isolated myocytes from transgenic mice demonstrate that there is defective excitation-contraction coupling and a decrease in the magnitude of isolated cell shortening. These data demonstrate that restricted replication of enteroviral genomes in the heart can induce dilated cardiomyopathy with excitation-contraction coupling abnormalities similar to pressure overload models of dilated cardiomyopathy.     

The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity

The biochemical activities that underlie the genetically defined activator and repressor functions of the VIVIPAROUS1 (VP1) protein have resisted in vitro analysis. Here, we show that a glutathione S-transferase (GST) fusion protein, including only the highly conserved B3 domain of VP1, has a highly cooperative, sequence-specific DNA binding activity. GST fusion proteins that include larger regions of the VP1 protein have very low activity, indicating that removal of the flanking protein sequences is necessary to elicit DNA binding in vitro. DNA competition and DNase I footprinting analyses show that B3 binds specifically to the Sph element involved in VP1 activation of the C1 gene, whereas binding to the G-box-type VP1-responsive element is of low affinity and is nonspecific. Footprint analysis of the C1 promoter revealed that sequences flanking the core TCCATGCAT motif of Sph also contribute to the recognition of the Sph element in its native context. The salient features of the in vitro GST-B3 DNA interaction are in good agreement with the protein and DNA sequence requirements defined by the functional analyses of VP1 and VP1-responsive elements in maize cells.     

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.     

Assaying for structural variation in the parvovirus capsid and its role in infection

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were approximately 31-32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.     

Identification of domains in bluetongue virus VP3 molecules essential for the assembly of virus cores

Bluetongue virus (BTV) cores consist of the viral genome and five proteins, including two major components (VP3 and VP7) and three minor components (VP1, VP4, and VP6). VP3 proteins form an inner scaffold for the deposition on the core of the surface layer of VP7. VP3 also encapsidates and interacts with the three minor proteins. The BTV VP3 protein consists of 901 amino acids and has a sequence that is a highly conserved among BTV serotypes and other orbiviruses (e.g., epizootic hemorrhagic disease virus and African horse sickness virus). To locate sites of interaction between VP3 and the other structural proteins, we have analyzed the effects of a number of VP3 deletion mutants representing conserved regions of the protein, using as an assay the formation of core-like particles (CLPs) expressed by recombinant baculoviruses. Five of the VP3 deletion mutants interacted with the coexpressed VP7 and made CLPs. These CLPs also incorporated the three minor proteins. One mutant, lacking VP3 amino acid residues 499 to 508, failed to make CLPs. Further mutational analyses have demonstrated that a methionine at residue 500 of VP3 and an arginine at residue 502 were both required for CLP formation.     

Epitope mapping of capsid proteins VP2 and VP3 of infectious bursal disease virus

Twenty hybridoma cell lines producing monoclonal antibodies (MAbs) against serotype 1 infectious bursal disease virus (IBDV) of GBF-1 and the attenuated GBF-1E strains were produced. The MAbs recognized major structural proteins VP2 and VP3. MAb recognition sites were mapped using recombinant Escherichia coli clones which expressed N-terminal and (or) C-terminal truncated virus antigens, and competitive-binding assays. At least 3 conformation-dependent serotype 1 specific virus neutralizing antigenic sites and a linear antigenic site were defined on VP2 and VP3, respectively. Two of the conformational virus neutralizing antigenic sites were localized in the central area of VP2 consisting of 156 amino acid residues, and the linear epitope was localized in C-terminal 105 amino acid residues of VP3. Another conformational virus neutralizing antigenic site recognized with the virus neutralizing MAb GK-5 was not defined because GK-5 did not react with virus antigen expressed in recombinant E. coli. The conformational antigenic site was supposed to be composed of tertiary or quaternary protein structure, which may not be constructed in recombinant E. coli.     

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.     

Cerebrosides alter the lyotropic and thermotropic phase transitions of DOPE:DOPC and DOPE:DOPC:sterol mixtures

An infectious cDNA of a highly myocarditic coxsackievirus B3 (CVB3m; Nancy strain) was cloned. Sequence data revealed 43 extra non-viral nucleotides upstream of the initial 5' sequence. However, the authentic 5' end sequence was maintained during replication of viral RNA transfected into HeLa cells, suggesting the RNA synthesizing complex edits the picornaviral 5' terminus sequence. Nucleotide sequences of the 5' nontranslated region and the capsid protein gene sequence of CVB3m were compared with the published sequences of five other CVB3 Nancy strains and two main lineages were found. In comparative assays for cardiovirulence, three of four CVB3 tested were cardiovirulent in adolescent male CD-1 mice. Only one of the three available CVB3 strains was neutralized with several anti-CVB3m monoclonal antibodies, suggesting that mutations in the surface epitopes of the capsid polypeptides contribute to antigenic drift within the serotype, perhaps in part through immunoselective pressures. Thus, phenotypic diversity of CVB3 within the prototype Nancy strain is an example of RNA viruses adapting to changing environments (cells, mice and humans) through mutations and selective pressure.     

The inhibitory effect of astragalus membranaceus on coxsackie B-3 virus RNA replication

Using mice infected with coxsackie B-3 virus (CVB3) as a viral myocarditis model, we observed the inhibitory effect of Astragalus membranaceus (AM) on CVB3-RNA replication in myocardial tissue of mice by RNA-RNA in situ hybridization with negative-strand RNA probes labelled with 35S and quantitative imaging analysis of positive signals. The mechanism of its effect on CVB3-RNA replication has been investigated by detection of beta-interferon (beta-IFN) as well. Results showed that the copy numbers of CVB3-RNA as well as the histologic scores (necrosis) in myocardial tissues of infected-AM treated mice were significantly lower than those in infected and normal saline treated mice, suggesting that AM could inhibit the replication of CVB3-RNA, but its effect on CVB3-RNA replication had no correlation with induction of beta-IFN.     

Effect of substance P, insulin-like growth factor-1 and vasoactive intestinal polypeptide on corneal re-epithelialization in galactosemic rats

Glutathione peroxidase 1 (GPX-1) is a selenium-dependent enzyme with antioxidant properties. Previous investigations determined that mice deficient in selenium developed myocarditis when infected with a benign strain of coxsackievirus B3 (CVB3/0). To determine whether this effect was mediated by GPX-1, mice with a disrupted Gpx1 gene (Gpx1-/-) were infected with CVB3/0. Gpx1-/- mice developed myocarditis after CVB3/0 infection, whereas infected wild-type mice (Gpx1+/+) were resistant. Sequencing of viruses recovered from Gpx1(-/-)-infected mice demonstrated seven nucleotide changes in the viral genome, of which three occurred at the G residue, the most easily oxidized base. No changes were found in virus isolated from Gpx1+/+ mice. These results demonstrate that GPX-1 provides protection against viral-induced damage in vivo due to mutations in the viral genome of a benign virus.     

Structure of a neutralizing antibody bound monovalently to human rhinovirus 2

The structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-A resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45 degrees to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2-Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.     

Increase of heat-shock protein and induction of gamma/delta T cells in peritoneal exudate of mice after injection of live Fusobacterium nucleatum

The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T = 16 icosahedral lattice. The major capsid protein, VP5 MW = 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid promoters, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

Capsid protein VP1 deletions in JC virus from two AIDS patients with progressive multifocal leukoencephalopathy

PCR on 52 cerebrospinal fluid (CSF) specimens and 33 brain biopsies obtained from HIV-1 positive patients utilized pairs of primers from both the early region (JTP) and late region (JLP). In these patients, in whom progressive multifocal leukoencephalopathy (PML) was suspected on the basis of clinical symptoms and magnetic resonance imaging (MRI) studies, eight CSFs (15%) and 14 brain biopsy specimens (42%) contained JCV DNA sequences. In two patients' samples, the CSFs were positive for JCV DNA in the VP1 region using the primer pair for the VP1 region (JLP), but the fragment amplified migrated more rapidly than the 129-bp product obtained from prototype JCV(Mad-1) or the fragment amplified from the antigenic variant of JCV known as Mad-11. These patients died 3-4 months after onset of progressive neurological symptoms. Cycle sequencing of the fragments revealed overlapping deletions of 24 and 27 nucleotides. These strains were of different genotypes, designated strain 107 and strain 206. Computer analysis of the VP1 amino acid sequence predicts that the eight or nine amino acid residue deletions represent a surface loop with a high antigenic index. These naturally occurring deletion mutants are the first examples of a phenomenon observed experimentally in the mouse polyoma virus capsid protein VP2.     

Potentiation of tumour necrosis factor-mediated cell killing by VP16 on human ovarian cancer cell lines. In vitro results and clinical implications

Synergism between recombinant human tumour necrosis factor (rHuTNF) and DNA topoisomerase II inhibitor VP16 during the killing of cells has been studied in six human ovarian cancer cell lines (A2774, A2780, SW626, IGROV-1, SKOV3, Pa1) and a cervical carcinoma cell line (Me180). Studies were performed using an assay of colony formation inhibition (drug treatment for 1 h) and a growth inhibition assay (continuous exposure for 20 h). Concomitant treatment of cells with VP16+rHuTNF enhanced cell killing in all the cell lines tested--an effect observed in both short- and long-term cytotoxicity assays. This study suggests that the activity of VP16 in ovarian cancer cell lines might be enhanced by rHuTNF in in vitro models.