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PubMed2006

Deterministic, compensatory mutational events in the capsid of foot-and-mouth disease virus in response to the introduction of mutations found in viruses from persistent infections.

The evolution of foot-and-mouth disease virus (FMDV) (biological clone C-S8c1) in persistently infected cells led to the emergence of a variant (R100) that displayed increased virulence, reduced stability, and other modified phenotypic traits. Some mutations fixed in the R100 genome involved a cluster of highly conserved residues around the capsid pores that participate in interactions with each other and/or between capsid protomers. We have investigated phenotypic and genotypic changes that occurred when these replacements were introduced into the C-S8c1 capsid. The C3007V and M3014L mutations exerted no effect on plaque size or viral yield during lytic infections, or on virion stability, but led to a reduction in biological fitness; the D3009A mutation caused drastic reductions in plaque size and viability. Remarkably, competition of the C3007V mutant with the nonmutated virus invariably resulted in the fixation of the D3009A mutation in the C3007V capsid. In turn, the presence of the D3009A mutation invariably led to the fixation of the M3014L mutation. In both cases, two individually disadvantageous mutations led, together, to an increase in fitness, as the double mutants outcompeted the nonmutated genotype. The higher fitness of C3007V/D3009A was related to a faster multiplication rate. These observations provide evidence for a chain of linked, compensatory mutational events in a defined region of the FMDV capsid. Furthermore, they indicate that the clustering of unique amino acid replacements in viruses from persistent infections may also occur in cytolytic infections in response to changes caused by previous mutations without an involvement of the new mutations in the adaptation to a different environment.

Adaptation, Physiological↗
PubMed1978

Characterization of the mRNA's for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.

Capsid↗
PubMed1981

Molecular genetics of herpes simplex virus. V. Characterization of a mutant defective in ability to form plaques at low temperatures and in a viral fraction which prevents accumulation of coreless capsids at nuclear pores late in infection.

In herpes simplex virus-infected cells, coreless capsids accumulate at the nuclear pores soon after infection, but subsequently disappear, suggesting that, as in adenovirus-infected cells (S. Dales and Y. Chardonnet, Virology 56:465-483, 1973), the release of viral DNA from nucleocapsids takes place at the nuclear pores. A nonlethal mutant, HSV-1(50B), produced by mutagenesis of HSV DNA fragments and selected for delayed production of plaques at 31 degrees C, accumulated coreless capsids at the nuclear pores late in infection in contrast to wild-type viruses. Recombinants selected for ability to produce plaques at 31 degrees C by marker rescue with digests of herpes simplex virus 2 DNA and selected clone fragments of HSV-1 DNA no longer accumulated empty capsids at nuclear pores late in infection. These results suggest that herpes simplex viruses encode a function which prevents accumulation of coreless capsids at nuclear pores, presumably by preventing uptake, unenvelopment, and DNA release from progeny virus, and indicate that the cold sensitivity of plaque formation and accumulation of coreless capsids might be related or comap in the S component of the genome.

Capsid↗
PubMed1984

Bacteriophage P22 capsids with a subgenome length of packaged DNA.

Bacteriophage P22 assembles a DNA-free procapsid that subsequently packages P22 DNA. To study the packaging of bacteriophage P22 DNA, attempts were made to isolate P22 capsids with a subgenome length of packaged DNA. With the use of cesium chloride buoyant density sedimentation and agarose gel electrophoresis, the following capsids with a subgenome length of packaged DNA were isolated and characterized: (i) a capsid with the solid-support-free electrophoretic mobility and radius of the DNA-free P22 procapsid; (ii) a capsid with the solid-support-free electrophoretic mobility and radius of the mature P22 bacteriophage; and (iii) a capsid with a solid-support-free electrophoretic mobility and possibly a radius intermediate to those of the procapsid and bacteriophage.

Bacteriophages↗
PubMed1987

Genetic control of capsid length in bacteriophage T4: phenotypes displayed by ptg mutants.

The phenotypic characteristics of 26 ptg mutations in T4 gene 23 are described. All were located in three tight clusters in that gene and, by definition of ptg mutations, all produced giant phage. Intermediate petite phage, which invariably made up a substantial fraction of the progeny of these mutants, appeared to be a unique product of gene 23 mutations. Isometric petite phage were produced in significant numbers by strains with mutations at only 4 of the 10 sites identified with the PTG phenotype. The data presented indicate that there was little if any variation in the lengths of the normal, the intermediate petite, and the isometric petite classes. The frequencies of those capsid types were fairly specific for the individual mutations. The giant capsids that resulted from ptg mutations also had characteristic length distributions, of which three types were distinguished. These highly specific effects of gene 23 ptg mutations on capsid length regulation of T4 imply that the product of gene 23, gp23, plays a significant role in controlling the length of its capsid. The restrictions these observations place on a model for T4 capsid length regulation are discussed briefly.

Capsid↗
PubMed1989

Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus.

The gene encoding the major capsid protein of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) was identified, sequenced, and transcriptionally mapped. The location of the gene was determined by immunological screening of an expression library of AcMNPV open reading frame-beta-galactosidase fusions with an antibody raised to virus structural proteins. The DNA sequence of the corresponding region, which mapped within 56.6 and 58.0 map units on the AcMNPV genome, revealed a 1,040-base-pair open reading frame capable of encoding a 39-kilodalton polypeptide. The identity of the polypeptide was determined by Western blot (immunoblot) analysis of purified empty capsids with an antibody raised to the capsid-beta-galactosidase fusion protein. The identity of the peptide encoded by the gene was confirmed by immunoprecipitation of an in vitro translation product with RNA selected by hybridization to DNA sequences from the coding region of the gene. Transcripts of the capsid gene were analyzed by Northern (RNA) blots and mapped by nuclease protection and primer extension analysis. The capsid gene is transcribed maximally at 12 and 24 h postinfection but not in the presence of cycloheximide, a protein synthesis inhibitor, or aphidicolin, a viral DNA synthesis inhibitor, and is therefore classified as a late gene. The gene is transcribed in a counterclockwise direction with respect to the circular map. There are three transcriptional start sites, all containing the AGTAAG consensus sequence found at the start site of all late AcMNPV genes.

Amino Acid Sequence↗
PubMed1990

Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane.

The capsids of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus, are preassembled in the infected cell cytoplasm and are then transported to the plasma membrane, where they are enveloped in a virus glycoprotein-containing lipid bilayer. The role of viral glycoprotein in intracellular transport of M-PMV capsids was investigated with a spontaneous mutant (5A) of M-PMV, which we show here to be defective in envelope glycoprotein biosynthesis. DNA sequence analysis of the env gene of mutant 5A reveals a single nucleotide deletion in the middle of the gene, which results in the synthesis of a truncated form of the envelope glycoprotein. Evidence is presented showing that the mutant glycoprotein is not expressed at the cell surface but is retained in the endoplasmic reticulum. Normal levels of gag-pro-pol precursor polyproteins are made and processed in mutant genome-transfected cells, and high levels of noninfectious particles lacking viral glycoprotein are released with normal kinetics into the culture medium. No intracisternal budding of capsids is observed. We conclude that viral glycoprotein is required neither for targeting preassembled capsids of M-PMV to the plasma membrane for final maturation nor for the budding process. Since the presence or absence of M-PMV glycoprotein at the site of budding does not affect the efficiency or kinetics of the targeting process, the preassembled capsid of M-PMV, in contrast to those of intracisternal type A particles, appears to have an intrinsic signal for intracellular transport to the plasma membrane.

Animals↗
PubMed1993

Deletion analysis of the capsid protein of Sindbis virus: identification of the RNA binding region.

The capsid protein of Sindbis virus has multiple functions in the life cycle of the virus. One essential function is to interact with the genomic RNA of the virus to form the nucleocapsid. The experiments described in this article define a region of the protein that is required for binding to Sindbis virus RNA. The assay we used measured the binding of in vitro-translated proteins to RNA on the basis of their migration with the RNA during electrophoresis in an agarose gel. Binding to RNA showed specificity; more protein bound to an RNA containing the previously defined packaging signal in Sindbis virus RNAs than to a similar RNA lacking this sequence. We were able to produce a variety of deleted forms of the capsid protein by constructing cDNAs with in-frame deletions throughout the coding region of the capsid protein gene. These cDNAs were then transcribed into mRNAs and translated in vitro. C-terminal deletions in the capsid protein were obtained by preparing transcripts from cDNAs linearized at sites within the coding region. Our studies identified a 32-amino-acid region that is essential for the specificity in RNA binding, and they defined a 68-amino-acid minimal sequence which displays almost the complete specific RNA binding activity of the intact Sindbis virus capsid protein containing 264 amino acids.

Binding Sites↗
PubMed1994

Mutational analysis of the capsid protein of Leishmania RNA virus LRV1-4.

The virion of Leishmania RNA virus is predicted to be composed of a 742-amino-acid major capsid protein and a small percentage of capsid-polymerase fusion molecules. Recently, the capsid protein alone was expressed and shown to spontaneously assemble into viruslike particles. Since the major structural protein of the virion shell self-assembles into viruslike particles when expressed in the baculovirus expression system, assembly of the virion can be studied by mutational analysis and expression of a single open reading frame. In this study, several deletions and one addition of the capsid protein of Leishmania RNA virus LRV1-4 were generated. These mutants show different degrees of assembly. Assembly domains are being identified such that the capsid protein may be used as a macromolecular packaging and delivery system for Leishmania species.

Animals↗
PubMed1994

Multiple functions of capsid protein phosphorylation in duck hepatitis B virus replication.

We have investigated the role of phosphorylation of the capsid protein of the avian hepadnavirus duck hepatitis B virus in viral replication. We found previously that three serines and one threonine in the C-terminal 24 amino acids of the capsid protein serve as phosphorylation sites and that the pattern of phosphorylation at these sites in intracellular viral capsids is complex. In this study, we present evidence that the phosphorylation state of three of these residues affects distinct steps in viral replication. By substituting these residues with alanine in order to mimic serine, or with aspartic acid in order to mimic phosphoserine, and assaying the effects of these substitutions on various steps in virus replication, we were able to make the following inferences. (i) The presence of phosphoserines at residues 245 and 259 stimulates DNA synthesis within viral nucleocapsids. (ii) The absence of phosphoserine at residue 257 and at residues 257 and 259 stimulates covalently closed circular DNA synthesis and virus production, respectively. (iii) The presence of phosphoserine at position 259 is required for initiation of infection. The results implied that both phosphorylated and nonphosphorylated capsid proteins were necessary for a nucleocapsid particle to carry out all its functions in virus replication, explaining why differential phosphorylation of the capsid protein occurs in hepadnaviruses. Whether these differentially phosphorylated proteins coexist on the same nucleocapsid, or whether the nucleocapsid acquires sequential functions through selective phosphorylation and dephosphorylation, is discussed.

Alanine↗
PubMed1994

Processing of the intracellular form of the west Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study.

According to the existing model of flavivirus polyprotein processing, one of the cleavages in the amino-terminal part of the flavivirus polyprotein by host cell signalases results in formation of prM (precursor to one of the structural proteins, M) and the membrane-bound intracellular form of the viral capsid protein (Cint) retaining the prM signal sequence at its carboxy terminus. This hydrophobic anchor is subsequently removed by the viral protease, resulting in formation of the mature viral capsid protein found in virions (Cvir). We have prepared in vitro expression cassettes coding for both forms of the capsid protein, for the prM protein, for the C-prM precursor, and for the viral protease components of West Nile flavivirus and characterized their translation products. Using Cint and Cvir translation products as molecular markers, we have observed processing of the intracellular form of the West Nile capsid protein by the viral protease in vitro both upon cotranslation of the C-prM precursor and the viral protease-encoding cassette and by incubation of C-prM translation products with a detergent-solubilized extract of cells infected with a recombinant vaccinia virus expressing the active viral protease. The cleavage of Cint by the viral protease at the predicted dibasic site was verified by introduction of point mutations into the cleavage site and an adjacent region. These studies provide the first direct demonstration of processing of the intracellular form of the flavivirus capsid protein by the viral protease.

Amino Acid Sequence↗
PubMed1995

Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria.

The capsid precursor protein (Gag) of Mason-Pfizer monkey virus, the prototype type D retrovirus, has been expressed to high levels in bacteria under the control of the phage T7 promoter. Electron microscopic studies of induced cells revealed the assembly of capsid-like structures within inclusion bodies that formed at the poles of the cells 6 h after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). The inclusion bodies and enclosed capsid-like structures were solubilized completely in 8 M urea, but following renaturation, we observed assembly in vitro of capsid-like structures that demonstrated apparent icosahedral symmetry. These results demonstrate for the first time that retroviral capsid precursors have the propensity to self-assemble in vitro and point to new approaches for the analysis of retroviral assembly and structure.

Capsid↗
PubMed1995

Poliovirus variants selected on mutant receptor-expressing cells identify capsid residues that expand receptor recognition.

Mutations in the predicted C'-C"-D edge of the first immunoglobulin-like domain of the poliovirus receptor were previously shown to eliminate poliovirus binding. To identify capsid residues that expand receptor recognition, 16 poliovirus suppressor mutants were selected that replicate in three different mutant receptor-expressing cell lines as well as in cells expressing the wild-type receptor. Sequence analysis of the mutant viruses revealed three capsid residues that enable poliovirus to utilize defective receptors. Two residues are in regions of the capsid that are known to regulate receptor binding and receptor-mediated conformational transitions. A third residue is located in a highly exposed loop on the virion surface that controls poliovirus host range in mice by influencing receptor recognition. One of the suppressor mutations enables the primate-restricted P1/Mahoney strain to paralyze mice by enabling the virus to recognize a receptor in the mouse central nervous system. Capsid mutations that suppress receptor defects may exert their effect at the binding site or may improve receptor binding by regulating structural transitions of the capsid.

Adaptation, Physiological↗
PubMed1996

Identification of domains in rubella virus genomic RNA and capsid protein necessary for specific interaction.

In rubella virus-infected cells, genomic 40S and subgenomic 24S RNAs are present in the cytoplasm of infected cells. However, encapsidation by rubella virus capsid protein is specific for 40S genomic RNA. As a first step toward understanding the assembly of rubella virus nucleocapsid at the molecular level, the interaction between capsid protein and genomic RNA was studied by Northwestern (RNA-protein) blot analysis. RNA probes prepared by in vitro transcription were used to localize the RNA sequence that participates in binding to the capsid protein. We have identified a 29-nucleotide RNA sequence (nucleotides 347 to 375) that is essential for the binding. By using overlapping synthetic peptides of capsid protein, a peptide domain (residues 28 to 56) that displays specific RNA-binding activity of capsid protein has been located. This result suggests that the specific recognition of viral RNA during rubella virus assembly involves, at least in part, the nucleocapsid protein.

Amino Acid Sequence↗
PubMed1996

Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro.

The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly.

Animals↗
PubMed1996

Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids.

The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.

Animals↗
PubMed1997

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.

Animals↗
PubMed1997

The carboxy-terminal two-thirds of the cowpea chlorotic mottle bromovirus capsid protein is incapable of virion formation yet supports systemic movement.

Previous investigations into recombination in cowpea chlorotic mottle bromovirus (CCMV) resulted in the recovery of an unusual recombinant virus, 3-57, which caused a symptomless infection of cowpeas but formed no detectable virions. Sequence analysis of cDNA clones derived from 3-57 determined that mutations near the 5' terminus of the capsid protein gene introduced an early translational termination codon. Further mutations introduced a new in-frame start codon that allowed translation of the 3' two-thirds of the capsid protein gene. Based on the mutations observed in 3-57, wild-type CCMV clones were modified to determine if the carboxyl two-thirds of the capsid protein functions independently of the complete protein in long-distance movement. Analysis of these mutants determined that while virion formation is not required for systemic infection, the carboxy-terminal two-thirds of the capsid protein is both required and sufficient for systemic movement of viral RNA. This indicates that the CCMV capsid protein is multifunctional, with a distinct long-distance movement function in addition to its role in virion formation.

Bromovirus↗