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PubMed1994

Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor.

Poliovirus initiates infection by binding to its cell receptor and undergoing a receptor-mediated conformational alteration. To identify capsid residues that control these interactions, we have isolated and characterized poliovirus mutants that are resistant to neutralization by a soluble form of the poliovirus receptor. Twenty one soluble receptor-resistant (srr) mutants were identified which still use the poliovirus receptor to infect cells. All but one srr mutant contain a single amino acid change at one of 13 different positions, either on the surface or in the interior of the virion. The results of binding and alteration assays demonstrate that both surface and internal capsid residues regulate attachment to the receptor and conformational change of the virus. Mutations that reduce alteration also affect receptor binding, suggesting a common structural basis for early events in poliovirus infection.

Capsid↗
PubMed2004

Two histidines of the coat protein of turnip yellow mosaic virus at the capsid interior are crucial for viability.

RNA-coat protein interactions in turnip yellow mosaic virus (TYMV) have been shown to involve low pK proton-donating groups. Two different types of interaction have been proposed. In the so-called type I interaction, protonated C-residues interact with acidic amino acids at low pH, thereby providing a rationale for the high C-content (38%) of the genomic RNA. The type II interaction involves charged histidines interacting with phosphates of the RNA backbone. Site-directed mutagenesis of the TYMV coat protein and subsequent in vivo analysis were performed to distinguish between these two types of RNA-protein interaction. The results reveal a prominent role for the histidines H68 and H180, since mutation to an alanine residue inhibits symptom development on secondary leaves, indicating that spreading of the virus in the plant is blocked. Viral RNA and coat protein synthesis are not altered, showing that these two histidines may play a role in the process of RNA encapsidation. Overexpression of the TYMV coat protein in Escherichia coli leads to the formation of bona fide capsids, showing that the two histidines are not critical in capsid assembly. Mutagenesis of the acidic amino acids D11, E135, and D143 to alanine apparently did not interfere with virus viability. The functional role of the histidines during the infection cycle is discussed in terms of the structure of the coat protein, both at the level of amino acid sequence conservation among the members of the Tymoviridae family and as the three-dimensional structure of the coat protein.

Alanine↗
PubMed1995

Docking of a human rhinovirus neutralizing antibody onto the viral capsid.

The structure of the complex between the Fab fragment of a human rhinovirus serotype 2 (HRV2) neutralizing antibody (8F5) and a cross-reactive synthetic peptide derived from the viral capsid protein VP2 has been recently determined by crystallographic methods. The conformation adopted by the peptide was very similar to and could be superimposed onto the corresponding region of the viral protein VP2 of human rhinovirus 1A (HRV1A) whose three-dimensional structure is known. The structure of the Fab fragment determined in the complex was docked onto the viral capsid using the superimposition transformation found for the peptide. In the resulting model the Fab protrudes almost radially to about 60 A from the surface of the virion without any major steric problem. The Fab fragment was then placed on each one of the 60 equivalent epitopes using the T = 1 icosahedral symmetry of the virus. The closest pairs of Fab fragments are related by viral 2-fold axes and run almost parallel to each other without clashing. These axes of symmetry from the viral particle could thus be coincident with the dyad axes of the antibodies. Furthermore, comparison of the three-dimensional structure of the Fab/peptide complex with the structure of the Fab fragment alone indicates that the flexibility of the antibody's elbow would facilitate bivalent attachment to the same viral particle. In accordance with the docking results, experimental determination of the stoichiometry of binding yielded a ratio of 30 IgG molecules per virion also suggesting bivalent attachment of antibody 8F5 onto the viral particle. The neutralization of viral infectivity, being neither aggregation (this paper) nor inhibition of receptor binding, might be mainly achieved by reducing viral spread from cell to cell and/or inhibition of uncoating.

Amino Acid Sequence↗
PubMed1999

Balance of IgG subclasses toward human papillomavirus type 16 (HPV16) L1-capsids is a possible predictor for the regression of HPV16-positive cervical intraepithelial neoplasia.

Human papillomavirus type 16 (HPV16) is known to be a major causative agent of cervical cancer. To test the hypothesis that an enhanced Th1 response favors the natural course of cervical intraepithelial neoplasia (CIN), we measured IgG subclasses toward HPV16 L1-capsids because IgG1/IgG2 balance reflects Th2 and Th1 responses, respectively. We examined IgG2/IgG1 ratios in sera from 67 anti-HPV16 L1-positive women; 18 were cytologically normal women, 29 were CIN patients, and 20 were cervical cancer patients. The IgG2 dominance (IgG2/IgG1 ratio >1) was observed in 94, 48, and 5%, respectively (p < 0.001). The regression rate of CIN lesions was significantly different between patients with and without IgG2 dominance: 83.3% (5/6) versus 16.7% (1/6), respectively (p < 0.05). These findings raise the possibility that IgG2 dominance toward HPV16 L1-capsids, i.e., Th1 dominance, may be a useful marker to predict viral clearance or the regression of HPV16-positive CIN.

Capsid↗
PubMed1997

The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions.

Crystal structures of two complexes between recombinant MS2 capsids and RNA operator fragments have been determined at 2.7 A resolution. The coat protein of the RNA bacteriophage MS2 is bifunctional; it forms the icosahedral virus shell to protect the viral nucleic acid and it acts as a translational repressor by binding with high specificity to a unique site on the RNA, a single stem-loop structure, containing the initiation codon of the gene for the viral replicase. In order to determine the structure of these protein-RNA complexes, we have used chemically synthesized variants of the stem-loop fragment and soaked them into crystals of recombinant capsids. The RNA stem-loop, as bound to the protein, forms a crescent-like structure and interacts with the surface of the beta-sheet of a coat protein dimer. It makes protein contacts with seven phosphate groups on the 5' side of the stem-loop, with a pyrimidine base at position -5, which stacks onto a tyrosine, and with two exposed adenine bases, one in the loop and one at a bulge in the stem. Replacement of the wild-type uridine with a cytosine at position -5 increases the affinity of the RNA to the dimer significantly. The complex with RNA stem-loop having cytosine at this position differs from that of the wild-type complex mainly by having one extra intramolecular RNA interaction and one extra water-mediated hydrogen bond.

Amino Acid Sequence↗
PubMed1998

Structure of the capsid of Pf3 filamentous phage determined from X-ray fibre diffraction data at 3.1 A resolution.

We have recorded X-ray diffraction patterns at 3.1 A resolution from magnetically aligned fibres of the Pf3 strain of filamentous bacteriophage (Inovirus). The patterns are similar to patterns from the higher-temperature form of the Pf1 strain, indicating that the Pf3 and Pf1 virions have the same helix symmetry and similar protein subunit shape. This is of particular interest, given that the primary structures of the two protein subunits are quite different; and the nucleotide/protein subunit ratio in the Pf3 virion is more than twice that in Pf1, indicating important differences in DNA packaging. We have built a molecular model of the Pf3 protein capsid based on the model of Pf1, and refined it against the diffraction data using simulated annealing. The refinement confirms that the two structures are similar, which may reflect a fundamental motif of alpha-helix packing. However, there are some differences between the structures: the Pf3 subunit appears to be completely alpha-helical, beginning at the N terminus, whereas the first few residues of the Pf1 subunit are not helical; and the structure of the C-terminal region of the Pf3 subunit at the inner surface of the tubular capsid indicates that DNA/protein interactions in this virion may involve both aromatic side-chains and positively charged side-chains, whereas those in the Pf1 virion involve predominantly only the latter. In the course of this work, we have developed new approaches to refinement and validation of helical structures with respect to continuous transform fibre diffraction data.

Amino Acid Sequence↗
PubMed1999

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae.

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.

Amino Acids↗
PubMed1993

Analysis of a potential myristoylation site in hepatitis A virus capsid protein VP4.

The VP4 capsid protein of several picornaviruses has been shown to be myristoylated at an N-terminal glycine residue. Myristoylation occurs after removal of an initial methionine residue or a leader peptide, resulting in the exposure of an N-terminal eight amino acid myristoylation signal including a consensus G-x-x-x-T/S motif. Analysis of the amino acid sequence of hepatitis A virus (HAV) capsid protein reveals a potential myristoylation site beginning at position 5 of the VP4 sequence. To assess the significance of this apparent myristoylation signal, mutations were engineered (G to A; T to N) to alter the consensus sequence as well as the potential cleavage site that would be required to remove the short leader. An additional mutant was constructed in which the proposed leader was deleted. Expression of these HAV sequences in BS-C-1 cells showed that leader cleavage did not occur in the wild-type or mutant proteins, although the threonine to asparagine mutation resulted in reduced translation and processing efficiency. Transfection of BS-C-1 or FRhK-4 cells with transcripts derived from the wild-type or mutagenized cDNA clones gave rise to infectious virus, with no detectable incorporation of myristate. The results indicate that HAV does not require leader cleavage and myristoylation of VP4 for growth in cultured cells.

Animals↗
PubMed1993

Identification and expression of the outer capsid protein (VP4) of the IDIR strain of group B rotavirus.

The polypeptide encoded by gene 3 of the IDIR strain of group B rotavirus (GBR) was synthesized by means of a baculovirus expression system. Immunoblot analysis identified the IDIR virus gene 3 product as analogous to the outer capsid protein, VP4, encoded by gene 4 of group A rotaviruses (GAR). This coding assignment had previously been difficult to establish by sequence comparisons, since IDIR virus gene 3 shared < 20% identical amino acid sequences with any GAR protein. Trypsin digestion of the gene 3 protein resulted in the appearance of a product indistinguishable in size from the VP5* outer capsid protein of IDIR agent virions. The recombinant IDIR virus VP4 maintained at least some antigenic epitopes of the native virion protein, as evidenced by reactivity with convalescent antibody obtained following infection of infant rat pups with the IDIR agent. Reactivity was also demonstrated with antisera directed against bovine and porcine isolates of GBR as well as with ADRV, a human strain of GBR.

Amino Acid Sequence↗
PubMed1993

Topology of the major capsid protein P3 of bacteriophage PRD1: analysis using monoclonal antibodies and C-terminally truncated proteins.

Trimeric capsomeres of protein P3 (395 aa) are the main component of the phage PRD1 capsid, which encloses a lipid-protein vesicle containing the viral dsDNA genome. In this study we characterize a panel of monoclonal antibodies (MAb) against P3. The epitopes recognized by the MAbs are analyzed by immunoprecipitation of intact virions or of released P3 trimers, and by Western blotting using a series of C-terminally truncated P3 molecules. Nine of the MAbs recognize epitopes on the virion surface, whereas five require unmasking of epitopes by disruption of the virions. Several of the MAbs are capable of neutralizing the virus; this is most probably due to virus aggregation by the antibodies. Analysis of the C-terminal truncations (the 6 Western blot-positive MAbs were used) delineates three major antigenic regions of the protein. The epitope of MAb 3T74 is included in the 66 N-terminal amino acids, and is not accessible on the virion surface, suggesting that the N-terminus is internally located in the capsid. MAbs 3N81 and 3R2 recognize epitopes in the region of amino acids 159-168, which is part of the first predicted beta-barrel structure of P3. The third antigenic region is in the second predicted beta-barrel, between amino acids 217-242, where the epitopes of 3N180, 3P4, and 3T5 map. The trimerization of P3 was found to be independent of the non-structural assembly factor proteins P10 and P17. Functional studies of the truncated proteins reveal that molecules comprising of 294 or more residues from the P3 N-terminus are capable of trimer formation.

Antibodies, Monoclonal↗
PubMed1994

Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid.

The 25-nm diameter parvovirus capsid is assembled from 60 copies of a sequence common to the overlapping VP1 and VP2 proteins. Here we examine the epitope specificity's of 28 monoclonal antibodies (MAb) prepared against canine parvovirus (CPV), feline panleukopenia virus (FPV), and raccoon-dog parvovirus or blue (Arctic) fox parvovirus. Comparing the reactivity of those MAb with various MAb-selected escape mutants, or with natural variants of CPV or mink enteritis virus (MEV) which differ at known sequences, showed that the binding of 20 of those MAb was strongly affected by variations of two regions on the threefold spike of the CPV capsid. One region was adjacent to the tip of the threefold spike, and the second was around VP2 residue 300, on the shoulder of that structure. MAb recognizing both antigenic sites efficiently neutralized the virus infectivity and inhibited hemagglutination. Mutations leading to natural antigenic variation have also been observed in both those sites in naturally variant strains of CPV or MEV, suggesting that they are important antigenic structures on these parvoviruses. The bindings of several MAb were not affected by the mutations at those antigenic sites, indicating that they recognized other, and perhaps conserved, structures.

Animals↗
PubMed1997

Structure of Broadhaven virus by cryoelectron microscopy: correlation of structural and antigenic properties of Broadhaven virus and bluetongue virus outer capsid proteins.

The three-dimensional structure of Broadhaven virus (BRDV) has been determined to 23 A resolution by cryoelectron microscopy and image processing. As predicted from sequence homology, the BRDV structure resembles that of bluetongue virus (BTV) with the notable exception of one of the outer shell proteins. The cores of BRDV and BTV are identical at medium resolution; they have a diameter of 710 A and the VP7 trimers are arranged on a T = 13 icosahedral lattice. The outer shell proteins, VP5 of BRDV and BTV, have roughly the same molecular weight while VP4 of BRDV is only half the molecular weight of the corresponding VP2 of BTV. This size difference allows unambiguous determination of the identity of the triskelion shape as trimers of VP4 of BRDV (VP2 of BTV). The VP4 of BRDV sits on the VP7 trimers and projects outwards 40 A, giving the capsid an overall diameter of 790 A. This contrasts with VP2 of BTV, which projects outwards 95 A to give the capsid a diameter of 900 A. The difference in accessibility of the outer shell proteins of BRDV and BTV correlates with the difference in antigenic properties of these viral proteins. The shape of the BRDV VP5 indicates that it too is a trimer, thus implying that there are 360 copies of VP5 and 180 copies of VP4 per virion.

Bluetongue virus↗
PubMed1998

One amino acid change on the capsid surface of poliovirus sabin 1 allows the establishment of persistent infections in HEp-2c cell cultures.

Poliovirus mutants (PVpi) selected during the persistent infection of human neuroblastoma cells can establish secondary persistent infections in nonneural HEp-2c cells (I. Pelletier, T. Couderc, S. Borzakian, E. Wyckoff, R. Crainic, E. Ehrenfeld, and F. Colbère-Garapin, 1991, Virology, 180, 729-737). Previous results from our laboratory have also shown that, in the genome of PVpi S11 derived from the Sabin 1 strain, the genomic region involved in this phenotype contains 11 missense mutations which map exclusively to the genes encoding the capsid proteins VP1 and VP2. We report here the identification of precise viral determinants able to confer the capacity to establish persistent infections in HEp-2c cell cultures to the lytic Sabin 1 strain. We used a strategy based on the observation that PVpi, after a few months of persistent infection in HEp-2c cells, tend to regain a more lytic phenotype in uninfected HEp-2c cell cultures. We constructed mutant viruses carrying only a few mutations potentially involved in the phenotype of persistence. Two mutations were identified, one corresponding to the substitution His>Tyr of amino acid 142 of VP2 and another corresponding to the substitution Val>Ile of amino acid 160 of VP1. Mutants carrying one or the other of the two determinants established persistent infections in HEp-2c cell cultures in about 20% of the infections. Higher frequencies were obtained with the mutant carrying both determinants (30%), and with PVpi S11 (63%), indicating that the effects of several determinants can be cumulative. The two determinants are localized on the capsid surface in a region known to be involved in the interactions between poliovirus and its cell receptor and in fact, we demonstrate here that in the case of the two persistent mutants, these interactions are modified.

Amino Acid Substitution↗
PubMed1998

A vibriophage, KVP40, with major capsid protein homologous to gp23* of coliphage T4.

The mcp gene encoding the major capsid protein (Mcp) of vibriophage KVP40, a large-tailed DNA phage, was cloned and sequenced. The nucleotide sequence of the mcp gene was 64.4% similar to that of gene 23 of coliphage T4. Analysis of the N-terminal amino acid sequence of purified native Mcp revealed that the mcp gene actually coded for a precursor, pro-Mcp, whose 62 N-terminal amino acids must be removed upon maturation to Mcp. Thus, mature Mcp would consist of 452 amino acid residues and have a calculated molecular mass of 47,561 Da. Comparison of amino acid sequences of Mcp and gp23*, the major capsid protein of T4, demonstrated 61.8% identity and 89.7% similarity between them. In addition, a sequence, TATAAATA, identical to a typical T4 late promoter sequence was seen in the region upstream of the mcp gene. These findings, together with their morphological similarity, suggest that KVP40 and T4 are phylogenetically related.

Amino Acid Sequence↗
PubMed1998

DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1.

Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells. Plasmids carrying both an SV40 origin of replication (ori) and an E. coli ori were introduced into Cos-1 cells by DNA transfection PV capsid proteins were supplied in trans by recombinant vaccinia viruses. Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E. coli as an indication of packaging efficacy. VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1 + L2 packaged plasmid DNA at least 50 times more effectively. BPV-1 L1 + L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BPV sequences. Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BPV VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences. The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb.

Animals↗
PubMed2000

Interaction of the papillomavirus transcription/replication factor, E2, and the viral capsid protein, L2.

The minor capsid protein L2 of papillomaviruses (PVs) likely plays a role in the selective encapsidation of PV DNA in viral capsids and in the infectivity of PV virions. The L2 protein also can cause the relocalization of the PV early protein, E2TA, to nuclear subdomains known as promyelocytic leukemia oncogenic domains (PODs) in which it is localized. E2TA is a transcriptional transactivator that also plays a critical role in viral DNA replication. In this study, we investigated whether L2, in causing the relocalization of E2TA, alters the activities of E2TA. We provide evidence that L2 inhibits the transcriptional transactivation function of E2, but it does not specifically inhibit the capacity of E2 to support viral DNA replication. We also investigated whether the colocalization of E2 and L2 to PODs and the ability of L2 to inhibit the transcriptional transactivation activity of E2TA might be mediated through a direct interaction between these two proteins. Using an in vitro protein-protein association assay, we found that L2 binds to E2TA. Two regions in E2TA were found to mediate this interaction. One of those domains is present in an alternative E2 gene product, E2TR, which is an antagonist to E2TA. Here we show that the L2 protein also relocalizes the E2 transcriptional repressor, E2TR, to the nuclear subdomains. These data suggest that the ability of L2 to relocalize E2 proteins to PODs is mediated through a direct interaction with L2.

Animals↗
PubMed2003

Analysis of RNA packaging in wild-type and mosaic protein capsids of flock house virus using recombinant baculovirus vectors.

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two positive-sense RNA molecules, RNA1 (replicase gene) and RNA2 (coat protein gene), which are encapsidated into a single virion. Expression of coat protein in Sf21 cells using a baculovirus vector results in formation of virus-like particles (VLPs) whose capsids are structurally indistinguishable from native virions. However, RNA packaging is not specific for RNA2, the coat protein message. Using ribonuclease protection assays, we showed that the fraction of RNA2 in VLPs is 19% relative to the amount present in a population of native virions. To investigate possible reasons for the reduced level of RNA2, we generated two new baculovirus vectors, AcR1delta and AcR2delta, expressing the replicase gene and the coat protein gene, respectively. The inserted genes carried the self-cleaving hepatitis delta ribozyme sequence at the 3' end to allow for synthesis of RNA1 and RNA2 transcripts with authentic 3' ends. Infection of Sf21 cells with AcR2delta yielded VLPs that contained 66% RNA2 relative to native virions. Coinfection of Sf21 cells with AcR1delta and AcR2delta launched self-directed FHV replication and resulted in formation of particles most of which contained RNA1 and RNA2. However, a small fraction of particles containing cellular RNA was detected as well. The latter particles could be eliminated by infecting Sf21 cells with AcR1delta followed by transfection with in vitro synthesized transcripts of RNA2. We have further utilized this system to show that two coat protein deletion mutants with distinct RNA packaging defects form mosaic virus capsids but do not complement each other to rescue specific packaging of FHV RNAs.

Animals↗
PubMed1992

Adenovirus capsid proteins interact with HSP70 proteins after penetration in human or rodent cells.

Soon after penetration of adenovirus serotype 2 in BHK-21 and HeLa cells, HSP70 and HSC70 proteins become associated with the viral capsid. By analysis with a polyclonal antibody derived from a fusion protein containing the C-terminal domain, 290 amino acids of HSP70, and using both immunological methods and infected cells fractionation we observed that a significant amount of HSP70 proteins moved to the nucleus and colocalized with the adenovirus particles. HSP70 proteins of infected cells were isolated as a complex cross-linked with intracytoplasmic adenovirus type 2. By coprecipitation, using a polyclonal-specific antiserum derived from the fusion protein, or two different monoclonal-specific antisera, we showed that HSP70 and HSC70 proteins were associated with hexon, the major adenovirus capsid protein.

Adenoviridae↗