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PubMed1984

Identification and genetic mapping of a herpes simplex virus capsid protein that binds DNA.

Herpes simplex virus virion protein 19C (VP19C) is a constituent of both unenveloped (nuclear) and enveloped (cytoplasmic) capsids. In this paper we report that 32P-labeled DNA, either supercoiled or linear double stranded, efficiently bound to VP19C electrically transferred from denaturing polyacrylamide gels containing electrophoretically separated proteins from purified capsids. Analyses of the polypeptides specified by herpes simplex virus type 1 X herpes simplex type 2 recombinants with respect to electrophoretic mobility and binding of 32P-labeled DNA indicate that VP19C maps at the same location as infected cell polypeptide 32 and is derived from it.

Animals↗
PubMed1984

Adeno-associated virus proteins: origin of the capsid components.

The three primary capsid proteins (A, B, and C) of adeno-associated viruses have been shown previously to contain overlapping amino acid sequences (R. McPherson and J. Rose, J. Virol. 46:523-529, 1983). In the present study we demonstrate definitively that these proteins are encoded in the right half of the adeno-associated virus 2 genome, and one or both of the smallest adeno-associated RNA species (2.3- or 2.6-kilobase RNA) account for their synthesis. Protein A (90 kilodaltons) apparently initiates from a site within the intervening sequence, which is intact in the larger (unspliced) 2.6-kilobase mRNA, and may read through one or more termination codons, including a strong stop signal (UAA) that lies 31 bases downstream from the end of the intervening sequence. Proteins B (72 kilodaltons) and C (60 kilodaltons) are not derived from protein A but apparently originate from independent, in-frame initiations that lie downstream from the splice junction. It thus seems likely that production of the three adeno-associated virus capsid proteins involves at least two mRNA species. The B and C proteins presumably arise from the spliced 2.3-kilobase RNA, whereas protein A should be generated by the 2.6-kilobase RNA or a hitherto unidentified spliced RNA species.

Base Sequence↗
PubMed1985

Characterization of a major protein with a molecular weight of 160,000 associated with the viral capsid of Epstein-Barr virus.

A monoclonal antibody designated V3 was produced against a late protein associated with the Epstein-Barr virus-induced viral capsid antigen complex. The antibody reacted with discrete patches in the nuclei of infected cells as well as with virus particles, as shown by immunofluorescence and ultrastructural immunoperoxidase staining. The molecular weight of the protein precipitated by this monoclonal antibody was ca. 160,000. All anti-viral capsid antigen antibody-positive sera tested in an enzyme-linked immunosorbent assay reacted with this purified protein. The synthesis of the antigen was inhibited by phosphonoacetic acid but was not affected by tunicamycin, indicating that this was a late nonglycosylated viral protein. No differences were noted between the protein isolated from the P3HR-1 and B-95-8 cell lines as determined by immunoprecipitation and peptide mapping. By isoelectric focusing, this protein had a pI on the basic side ranging from 7.5 to 9.0.

Antibodies, Monoclonal↗
PubMed1986

Structure of the hepatitis A virion: peptide mapping of the capsid region.

Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-MW HAV polypeptide. These reactions enabled the identification of the gene order of the larger HAV P1 region gene products. Our data indicate the following molecular weights: HAV VP2 or 1B, 27,000; HAV VP3 or 1C, 29,000; and HAV VP1 or 1D, 33,000.

Amino Acid Sequence↗
PubMed1986

Formation of deletions after initiation of simian virus 40 replication: influence of packaging limit of the capsid.

Transfected DNA is frequently broken and rejoined in mammalian cells by recombination processes that depend on minimal nucleotide sequence homology. Although measurements of breakage and joining account reasonably well for the frequent formation of deletions during transfection, they are inadequate to explain the high frequency of deletion formation by simian virus 40 (SV40) genomes that are slightly larger than the packaging limit of the capsid. To investigate this anomaly, we constructed and transfected into CV-1 cells a series of modified SV40 genomes containing 136, 284, 460, and 656 extra base pairs in the intron of the gene encoding T antigen. These experiments indicate that the effective packaging limit of an SV40 capsid lies between 284 and 460 extra base pairs. Further analysis of these transfections suggests that molecules just above the effective packaging limit may be encapsidated and transmitted between cells at low efficiency, thereby allowing multiple rounds of replication and multiple opportunities to generate and package genomes that contain deletions. The junctional sequences in several such deletions were determined; they were similar to the junctions in deletions that were formed before replication began, suggesting that the enzymatic machinery responsible for both types of deletion may be similar.

Animals↗
PubMed1986

Quantitation, with a new assay, of Theiler's virus capsid protein in the central nervous system of mice.

We developed a quantitative assay for antigens at the single-cell level. Tissue sections were reacted with a primary antibody, a biotinylated secondary antibody, or 35S-streptavidin. Binding of streptavidin to cells was quantitated by microscopic autoradiography. We showed that the number of autoradiographic grains was proportional to the amount of antigen per cell. With this assay, we studied the synthesis of Theiler's virus capsid proteins VP1, VP2, and VP3 in permissive BHK cells grown in vitro and in mouse central nervous system (CNS) cells during a persistent infection. We found that synthesis of the three capsid proteins was restricted in mouse CNS cells. Restricted virus replication could play a major role in the persistence of Theiler's virus in mouse CNS cells.

Animals↗
PubMed1987

Effects of S-adenosylhomocysteine and analogs on Epstein-Barr virus-induced transformation, expression of the Epstein-Barr virus capsid antigen, and methylation of Epstein-Barr virus DNA.

S-Adenosylhomocysteine was found to have no effect on Epstein-Barr virus-induced transformation of B-lymphocytes and to stimulate viral capsid antigen expression only slightly in the FF41-1 cell line. In contrast, the S-adenosylhomocysteine analogs sinefungin and S-isobutyladenosine inhibited Epstein-Barr virus transformation and induced a significant increase in the numbers of cells expressing the viral capsid antigen. An inverse relationship between levels of viral DNA methylation and gene expression was demonstrated.

Capsid↗
PubMed1987

Genetic control of capsid length in bacteriophage T4: clustering of ptg mutations in gene 23.

Fifty-two new bacteriophage T4 ptg mutations have been isolated by selecting for the giant-capsid phenotype they display. Genetic mapping placed all of them at eight sites, all located in gene 23. These sites were clustered in three locations, one near amber B17 (gene 23 nucleotide [NT] 268), another centrally placed between amE506 (NT 706) and amE1270 (NT 925), and the third between amC208 (NT 1297) and amE1236 (NT 1489). The lack of a selective system for identifying recombinant genotypes when dealing with the very close linkages found within these clusters opens the possibility that more than eight sites are represented in this set of mutations. Since one site was represented by only one mutation, it seems likely that further searching might uncover additional sites. It is suggested that the clustering of mutations observed here identifies regions of the gene 23 product that play a role in regulating the capsid length of T4.

Base Sequence↗
PubMed1987

Genetic control of capsid length in bacteriophage T4: DNA sequence analysis of petite and petite/giant mutants.

The T4 gene 23 product (gp23) encodes the major structural protein of the mature capsid. Mutations in this gene have been described which disrupt the normal length-determining mechanism (A.H. Doermann, F.A. Eiserling, and L. Boehner, J. Virol. 12:374-385, 1973). Mutants which produce high levels of petite and giant phage (ptg) are restricted to three tight clusters in gene 23 (A.H. Doermann, A. Pao, and P. Jackson, J. Virol. 61:2823-2827, 1987). Twenty-six of these ptg mutations were cloned, and their DNA sequence alterations were determined. Each member of this set of ptg mutants arose from a single mutation, and the set defined 10 different sites at which ptg mutations can occur in gene 23. Two petite (pt) mutations in gene 23 (pt21-34 and ptE920g), which produce high frequencies of petite particles but no giants, were also sequenced. Both pt21-34 and ptE920g were shown to include multiple mutations. The phenotypes attributed to both pt and ptg mutations are discussed relative to the mechanism of capsid morphogenesis. A site-directed mutation (SD-1E) was created at the ptgNg191 site, and its phenotypic consequences were examined. Plaque morphology revertants arising from a gene 23 mutant derivative of pt21-34 and from SD-1E were isolated. A preliminary mapping of the mutation(s) responsible for their revertant phenotypes suggested that both intra- and extragenic suppressors of the petite phenotype can be isolated by this method.

Amino Acid Sequence↗
PubMed1989

A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self-assembles into capsid particles but fails to bind nucleic acids.

We have cloned in Escherichia coli both the complete core gene of hepatitis B virus and a truncated version of it, leading to the synthesis of high levels of a core-antigen-equivalent polypeptide (r-p22) and of an e-antigen-equivalent polypeptide (r-p16), respectively. We then compared the structural and antigenic properties of the two polypeptides, as well as their ability to bind viral nucleic acids. r-p16 was found to self-assemble into capsid-like particles that appeared similar, when observed under the electron microscope, to those formed by r-p22. In r-p16 particles, disulfide bonds linked the truncated polypeptides in dimers, assembled in the particle by noncovalent interactions. In r-p22 capsids, further disulfide bonds, conceivably involving the carboxy-terminal cysteines of r-p22 polypeptides, joined the dimers together, converting the structure into a covalently closed lattice. The protamine-like domain was at least partly exposed on the surface of r-p22 particles, since it was accessible to selective proteolysis. Finally, r-p22, but not r-p16, was shown to bind native and denatured DNA as well as RNA. Taken together, these results suggest that the protamine-like domain in core polypeptides is a nucleic acid-binding domain and is dispensable for the correct folding and assembly of amino-terminal and central regions.

Capsid↗
PubMed1989

Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide.

We established a human cell line which was persistently infected (PI) by the normally cytolytic echovirus 6. All of the cultured PI cells contained genome-size viral RNA which was synthesized continuously and incorporated into virus particles. This steady-state infection has been maintained for more than 6 years. In contrast to RNA of wild-type echovirus 6, the viral RNA from PI cells was not lytic when transfected into uninfected, susceptible cells. The capsid polypeptides of the virus particles produced during lytic infections were compared with those of virus particles from PI cells. Wild-type virions contained five polypeptides with molecular masses of 31.5, 27, 25.8, 21.2, and 9.5 kilodaltons. Comparison of polypeptide profiles of virions and empty immature capsids along with peptide analyses by immunoblotting and partial proteolysis of isolated viral proteins identified the cleavage products of the 31.5-kilodalton polypeptide (VP0) as the two smaller polypeptides (VP2 and VP4). The virus particles produced by PI cells as well as cellular extracts of PI cells contained only the three largest proteins (VP0, VP1, and VP3), indicating that VP0 was not processed during persistent infection. The lack of VP2 and VP4 in the defective virus particles coincided with their inability to attach to uninfected, susceptible cells. The maintenance of the steady-state infection of echovirus 6 was not dependent upon the release of virus particles from PI cells.

Capsid↗
PubMed1989

Proteolytic cleavage of encephalomyocarditis virus capsid region substrates by precursors to the 3C enzyme.

Picornavirus protease 3C is normally released from its P3 precursor by two successive self-cleavage reactions. The free enzyme can then catalyze most of the remaining processing events within the viral polyprotein. To investigate the role of the 3C precursors in the processing cascade, we constructed cDNA clones which expressed genetically altered forms of the encephalomyocarditis P3 region in vitro. Site-specific substitutions were introduced into the Gln-Gly residues at the 3B-3C and 3C-3D junctions, and the resulting proteins were tested for their ability to self-process and to catalyze cleavage of viral capsid precursors in cell-free protease assays. We determined that three P3 region precursor proteins (3ABC, 3CD, and P3), harboring inactive cleavage sites, were as active as the free enzyme (3C) in processing assays with capsid substrates. Further, we found that in addition to the naturally occurring Gln-Gly and Gln-Ser amino acid pairs, the encephalomyocarditis 3C enzyme was able to process Gln-Cys but not Gln-Thr, Gln-Ile, Gln-Tyr, Arg-Gly, or Leu-Gly combinations when these residues were substituted into normal cleavage site contexts.

Capsid↗
PubMed1989

Physical mapping and nucleotide sequence of a herpes simplex virus type 1 gene required for capsid assembly.

In this report, we describe some phenotypic properties of a temperature-sensitive mutant of herpes simplex type 1 (HSV-1) and present data concerning the physical location and nucleotide sequence of the genomic region harboring the mutation. The effect of shifts from the permissive to the nonpermissive temperature on infectious virus production by the mutant A44ts2 indicated that the mutated function is necessary throughout, or late in, the growth cycle. At the nonpermissive temperature, no major differences were detected in viral DNA or protein synthesis with respect to the parent A44ts+. On the other hand, electron microscopy of mutant-infected cells revealed that neither viral capsids nor capsid-related structures were assembled at the nonpermissive temperature. Additional analyses employing the Hirt extraction procedure showed that A44ts2 is also unable to mature replicated viral DNA into unit-length molecules under nonpermissive conditions. The results of marker rescue experiments with intact A44ts2 DNA and cloned restriction fragments of A44ts+ placed the lesion in the coordinate interval 0.553 to 0.565 (1,837 base pairs in region UL) of the HSV-1 physical map. No function has previously been assigned to this region, although it is known to be transcribed into two 5' coterminal mRNAs which code in vitro for a 54,000-molecular-weight polypeptide (K. P. Anderson, R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner, J. Virol. 37:1011-1027, 1981). We sequenced the interval 0.551 to 0.565 and found an open reading frame (ORF) for a 50,175-molecular-weight polypeptide. The predicted product of this ORF exhibits strong homology with the product of varicella-zoster virus ORF20 and lower, but significant, homology with the product of Epstein-Barr virus BORF1. For the three viruses, the corresponding ORFs lie just upstream of the gene coding for the large subunit of viral ribonucleotide reductase. The ORF described here corresponds to the ORF designated UL38 in the recently published nucleotide sequence of the HSV-1 UL region (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988).

Amino Acid Sequence↗
PubMed1990

Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture.

Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability. Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100). Only 5 of 15 mutations in P1 of R100 were silent. Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date. Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing. The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1. Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies. In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.

Amino Acid Sequence↗
PubMed1990

Identification of precursor to cytomegalovirus capsid assembly protein and evidence that processing results in loss of its carboxy-terminal end.

The 37-kilodalton (kDa) assembly protein of cytomegalovirus (strain Colburn) B capsids is shown to have a 40-kDa precursor. Pulse-chase radiolabeling experiments revealed that conversion of the precursor to the product was slow, requiring over 6 h for completion, and correlated with movement from the cytoplasmic to the nuclear fraction of Nonidet P-40-disrupted cells. Of these two proteins, only the 40-kDa precursor was synthesized in vitro from infected-cell RNA, consistent with its being the primary translation product. Amino acid sequence data obtained from CNBr-treated, high-performance liquid chromatography-purified assembly protein indicated that precursor translation begins at the first of two closely spaced potential initiation sites and that precursor maturation involves the loss of at least 32 amino acids from its carboxy-terminal end. It is also shown by immunological cross-reactivity and peptide similarity that three low-abundance B-capsid proteins (i.e., the 45-kilodalton [45K], 39K, and 38K proteins) are closely related to the assembly protein; the nature of this relatedness is discussed.

Amino Acid Sequence↗
PubMed1990

Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease.

The structural proteins of Sindbis virus are translated as a polyprotein precursor that is cleaved upon translation. The capsid protein is postulated to be a serine protease that releases itself from the N terminus of the nascent polyprotein by autoproteolysis. We have tested the importance in autoproteolysis of His-141, Asp-147, and Ser-215, previously postulated to form the catalytic triad of the protease, and of Asp-163. Several site-specific mutations were constructed at each of these positions, and the release of the capsid protein during translation in a cell-free system was examined. Because proteolysis occurs in cis during translation, the kinetics of release cannot be determined in this system, but the extent of proteolysis can be ascertained. Ser-215 appears to be the catalytic serine of the proteinase. Cys or Thr could substitute inefficiently for Ser-215, but substitution with Ala or Ile led to complete loss of activity. His-141 was also important for proteolysis. Substitution with Ala or Pro led to total loss of activity. Surprisingly, substitution with Arg resulted in complete proteolysis in vitro. Changes at the two Asp residues resulted in complete proteolysis of the substrate in vitro. All mutations that resulted in at least partial cleavage in vitro were incorporated into a full-length clone of Sindbis virus and an attempt was made to recover mutant virus. All changes tested were lethal for the virus except Asp-163 to Asn. Thus, production of infectious virus is either a more sensitive measure of the catalytic rate than the extent of in vitro cleavage, or these residues have necessary functions in addition to their possible role in proteolysis.

Amino Acid Sequence↗
PubMed1990

Lack of myristoylation of poliovirus capsid polypeptide VP0 prevents the formation of virions or results in the assembly of noninfectious virus particles.

We previously described the generation of a set of mutations into a cDNA of poliovirus type 1 in the myristoylation signal of the capsid polypeptide VP4 (D. Marc, G. Drugeon, A.-L. Haenni, M. Girard, and S. van der Werf, EMBO J. 8:2661, 1989). Genomic transcripts synthesized in vitro from the mutated cDNAs were found to be noninfectious upon transfection of permissive cells, and this property correlated with the lack of VP0 myristoylation in vivo. In the study presented here, we analyzed the assembly intermediates that could be recovered from cells transfected with the mutated transcripts. We found that 14S pentamers could still assemble to a certain extent with an unmyristoylated VP0. Furthermore, viral particles sedimenting at 150S and containing capsid polypeptides VP1 to VP4 and virus-specific RNA were detected in the transfected cells. However, these mature virions were less abundant than those recovered after transfection with an infectious transcript, and they were devoid of infectivity. The results suggest that VP0 myristoylation plays a role in the late steps of poliovirus assembly and that the myristate moiety of VP4 may be required in the early steps of poliovirus infection.

Amino Acid Sequence↗
PubMed1991

The 45-kilodalton protein of cytomegalovirus (Colburn) B-capsids is an amino-terminal extension form of the assembly protein.

Intranuclear B-capsids from cytomegalovirus (strain Colburn)-infected cells contain an abundant 37-kDa assembly protein, thought to be involved in capsid formation, and three minor protein constituents (i.e., 45, 39, and 38 kDa) that are immunologically and structurally related to the assembly protein. In the experiments reported here, antisera produced against synthetic peptides were used in conjunction with chemical protein cleavage to examine the structural relationship of these proteins in more detail. Results of these experiments verify that the carboxyl end of the 39-kDa assembly protein precursor is lost during maturation and suggest that the 38-kDa protein may be a processing intermediate. It is shown that the 45-kDa protein is coterminal with the mature assembly protein at its carboxyl end but differs by a predicted 115-amino-acid extension at its amino terminus. In addition, evidence is presented that the 45-kDa protein has a 48-kDa precursor and a 47-kDa putative processing intermediate which have the same carboxy-terminal sequences and undergo the same maturational events as those of the assembly protein. A working model considering the structural relationship of these proteins is presented.

Amino Acid Sequence↗