Biomedical subjects

S Person

Publications and source records attributed to S Person.

At least 37 records · Page 2Linked to original sources

PubMed1993

Herpes simplex virus type 1 capsid protein, VP21, originates within the UL26 open reading frame.

The goal of experiments reported here was to identify the genes that encode capsid proteins VP21 and VP24 of herpes simplex virus type 1 (HSV-1). Capsids were isolated from infected cells and the proteins were separated by SDS-PAGE. N-terminal amino acid sequence analysis of partial CNBr digestion products, and of intact VP21, showed that it is encoded within the UL26 open reading frame (ORF) of HSV-1 beginning with codon 248 and probably extending to the end of the ORF (codon 635). Similar analysis of digestion products confirmed that VP24 is specified by codons 1 to 247 at the 5' end of the UL26 ORF. Each of the seven known capsid proteins has now been assigned to an ORF.

Amino Acid Sequence↗

PubMed1993

Mutations in herpes simplex virus type 1 genes encoding VP5 and VP23 abrogate capsid formation and cleavage of replicated DNA.

The herpes simplex virus type 1 capsid is composed of seven capsid proteins which are termed VP5, VP19c, VP21, VP22a, VP23, VP24, and VP26. Major capsid protein VP5 is encoded by the gene UL19. UL18, whose transcript is 3' coterminal with that of VP5, specifies capsid protein VP23. Vero cell lines have been isolated that are transformed with either the BglII N (UL19) or EcoRI G (UL16 to UL21) fragment of KOS. These cell lines, selected for the ability to support the replication of a temperature-sensitive VP5 mutant, were used to isolate VP5 and VP23 null mutants. The mutations in VP5 (K5 delta Z) and VP23 (K23Z) were generated by insertion of the lacZ gene at the beginning of the coding sequences of the genes. Both mutants failed to form plaques on the nonpermissive cell line, and therefore, VP23, like VP5, is an essential gene product for virus replication. Both mutants expressed wild-type levels of infected-cell proteins upon infection of permissive and nonpermissive cell lines. However, the VP5 (150-kDa) and VP23 (33-kDa) polypeptides were absent in lysates prepared from K5 delta Z- and K23Z-infected Vero cells, respectively. No capsid structures were observed by electron microscopic analysis of thin sections of K5 delta Z- and K23Z-infected Vero cells. Following sedimentation of lysates from cells infected by the mutants, capsid proteins were not observed in the fractions where capsids normally sediment. The amounts of DNA replicated in the VP5 and VP23 mutant and in KOS-infected Vero cells were the same as in permissive cells. However, genomic ends were not evident in Vero cells infected with the mutants, suggesting that the DNA remains in concatemers and is not processed into unit length genomes.

Animals↗

PubMed1992

The HLA-E gene encodes two differentially regulated transcripts and a cell surface protein.

An HLA-E-specific oligonucleotide probe was used to study the expression of HLA-E. This probe detects two HLA-E transcripts, 1.8 and 2.7 kb in size, which are present in varying ratios in all tissues and cell lines investigated. We demonstrate that alternative poly(A) site usage accounts for the differential regulation of the two HLA-E mRNA species. Sequence analysis of three cDNA clones, representing the two transcripts of HLA-E, and of an HLA-E gene encoded by cosmid cd3.14, revealed identity of gene and cDNA in the 3' untranslated region. S1 nuclease protection assays confirmed that the two HLA-E transcripts are not alternative splicing products. Introduction of cd3.14, together with human beta 2 m into the murine myeloma cell line P3X63-Ag8.653, resulted in a cell surface expression of an HLA-class I heavy chain detectable by indirect immunofluorescence whereas transfection into the human beta 2m expressing mouse L cell line, J27 was negative with regard to cell surface expression. Cell surface labeling of transfectants and immunoprecipitation with a monomorphic HLA class I-specific antibody or an antibody against human beta 2m confirmed the presence of an HLA-E H chain on the cell surface. These results indicate that the HLA-E gene codes for a class I H chain that can be expressed on the cell surface.

Amino Acid Sequence↗

PubMed1992

Analysis of the gB promoter of herpes simplex virus type 1: high-level expression requires both an 89-base-pair promoter fragment and a nontranslated leader sequence.

To investigate the cis-acting sequences involved in regulation of a herpes simplex virus gamma 1 gene, deletion analyses of the glycoprotein B (gB) gene promoter were performed. In transfection assays with gB-chloramphenicol acetyltransferase plasmids, high-level constitutive expression from the gB promoter was found with an 89-bp sequence (-69 to +20). Additional sequences in the 5'-transcribed noncoding leader region (+20 to +136) were required for full stimulation by herpes simplex virus infection. Plasmids with progressive deletions of the gB leader sequence demonstrated that chloramphenicol acetyltransferase expression in infected cells was proportional to the length of the leader region retained. In recombinant viruses containing a gB-gC gene fusion, a similar 83-bp (-60 to +23) region of the gB gene was found to promote accurately initiated gC mRNA from the viral genome with the same kinetics as the wild-type gB gene. Although the kinetics of expression remained the same, RNA abundance was greater with a 298-bp (-260 to +38) promoter than with the 83-bp promoter.

Animals↗

PubMed1991

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.

Amino Acid Sequence↗

PubMed1989

Identification of mar mutations in herpes simplex virus type 1 glycoprotein B which alter antigenic structure and function in virus penetration.

Analysis of six monoclonal antibody-resistant (mar) mutants in herpes simplex virus type 1 glycoprotein B identified two type-common (II and III) and two type-specific (I and IV) antigenic sites on this molecule. To derive additional information on the location of these sites, mar mutations were mapped and nucleotide alterations were identified by DNA sequencing. Each mutant carried a single amino acid substitution resulting from a G-to-A base transition. Alterations affecting antibody neutralization were identified at residues 473, 594, 305, and 85 for mutants in sites I through IV, respectively. Two clonally distinct site II antibodies each selected mar mutants (Gly to Arg at residue 594) that exhibited a reduction in the rate of entry (roe) into host cells. A site II mar revertant that regained sensitivity to neutralization by site II antibodies also showed normal entry kinetics. DNA sequencing of this virus identified a single base reversion of the site II mar mutation, resulting in restoration of the wild-type sequence (Arg to Gly). This finding demonstrated that the mar and roe phenotypes were the result of a single mutation. To further define structures that contributed to antibody recognition, monoclonal antibodies specific for all four sites were tested for their ability to immune precipitate a panel of linker-insertion mutant glycoprotein B molecules. Individual polypeptides that contained single insertions of 2 to 28 amino acids throughout the external domain were not recognized or were recognized poorly by antibodies specific for sites II and III, whereas no insertion affected antibody recognition of sites I and IV. mar mutations affecting either site II or III were previously shown to cause temperature-sensitive defects in glycoprotein B glycosylation, and variants altered in both these sites were temperature sensitive for virus production. Taken together, the data indicate that antigenic sites II and III are composed of higher-order structures whose integrity is linked with the ability of glycoprotein B to function in virus infectivity.

Amino Acid Sequence↗

PubMed1988

Functional regions and structural features of the gB glycoprotein of herpes simplex virus type 1. An analysis of linker insertion mutants.

Glycoprotein B (gB) of Herpes simplex virus type 1 (HSV-1) plays an essential role in viral entry. A set of more than 100 HpaI (GTTAAC) linker insertion mutations and their derivatives were isolated in plasmids specifying the gB coding and flanking sequences. Mutations including addition, deletion and nonsense mutations at 34 independent sites were identified by DNA sequence analysis of 48 plasmids. A map was constructed for the ability of addition mutants to complement a gB-null virus. The expression of gB activity for some plasmids was temperature-dependent. Many complementation-negative plasmids inhibited the complementation activity of a plasmid specifying wild-type gB, suggesting an interaction between active and inactive molecules to form oligomers. The interaction was localized to 328 of the total of 904 amino acids comprising gB. Partial Endo H digestion of nonsense polypeptides revealed that five of the six potential N-linked oligosaccharide sites are glycosylated; the most C-terminal site appears not to be glycosylated. A number of mutations, including some on the cytoplasmic side, were identified that blocked processing, transport and secretion. Addition mutations that blocked processing of membrane polypeptides also blocked processing and secretion when combined into a nonsense mutant that by itself was processed and secreted. The previously predicted membrane spanning domain and the membrane orientation of the N-terminal portion of gB were confirmed.

Amino Acid Sequence↗

PubMed1988

Monoclonal antibodies define a domain on herpes simplex virus glycoprotein B involved in virus penetration.

In an earlier report (S.D. Marlin, S.L. Highlander, T.C. Holland, M. Levine, and J.C. Glorioso, J. Virol. 59: 142-153), we described the production and use of complement-dependent virus-neutralizing monoclonal antibodies (MAbs) and MAb-resistant (mar) mutants to identify five antigenic sites (I to V) on herpes simplex virus type 1 glycoprotein B (gB). In the present study, the mechanism of virus neutralization was determined for a MAb specific for site III (B4), the only site recognized by MAbs which exhibited complement-independent virus-neutralizing ability. This antibody had no detectable effect on virus attachment but neutralized viruses after adsorption to cell monolayers. These findings implied that the mechanism of B4 neutralization involved blocking of virus penetration. The remaining antibodies, which recognized sites I, II, and IV, required active complement for effective neutralization. These were further studied for their ability to impede virus infectivity in the absence of complement. Antibodies to sites I (B1 and B3) and IV (B6) slowed the rate at which viruses penetrated cell surfaces, supporting the conclusion that antibody binding to gB can inhibit penetration by a virus. The data suggest that MAbs can interfere with penetration by a virus by binding to a domain within gB which is involved in this process. In another assay of virus infection, MAb B6 significantly reduced plaque development, indicating that antibody binding to gB expressed on infected-cell surfaces can also interfere with the ability of a virus to spread from cell to cell. In contrast to these results, antibodies to site II (B2 and B5) had no effect on virus infectivity; this suggests that they recognized structures which do not play a direct role in the infectious process. To localize regions of gB involved in these phenomena, antibody-binding sites were operationally mapped by radioimmunoprecipitation of a panel of truncated gB molecules produced in transient-expression assays. Residues critical to recognition by antibodies which affect penetration by a virus (sites I, III, and IV) mapped to a region of the molecule (amino acid residues 241 to 441) which is centrally located within the external domain. Antibodies which had no effect on penetration (site II) recognized sequences distal to this region (residues 596 to 737) near the transmembrane domain. The data suggest that these gB-specific MAbs recognize two major antigenic sites which reside in physically distinct components of the external domain of gB.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion.

Glycoprotein B (gB) of herpes simplex virus type 1 is an envelope protein that is essential for viral growth. We previously reported the isolation of two gB-null viruses, which form gB-free virions in nonpermissive cells. In the present study, these gB-free virions were shown to bind to the cell surface at the same rate as the wild-type virus. They failed, however, to form plaques and to synthesize virus-specific proteins upon infection. Their plating efficiency was significantly enhanced by treatment with polyethylene glycol, a membrane fusion agent. Therefore, gB is required in a stage after viral attachment but before the expression of the virus-specific proteins. A gB-null syncytial virus was isolated, which contained a gB defect and a syncytial mutation in another genetic locus. It caused complete fusion of gB-transformed cells but no fusion on untransformed cells, indicating the essential role of gB in virus-induced cell fusion. Mutations located at two independent sites in the cytoplasmic domain of gB were transferred to viral DNA and shown to confer a syncytial phenotype to the virus. A transient-expression assay was developed to determine the ability of a set of plasmids containing addition and nonsense mutations in the gB gene to complement the cell-fusion defect in the gB-null syncytial virus. Mutations in plasmids, including those located in the extracytoplasmic domain of gB, were identified that reduced the fusion activity of gB. Therefore, gB contains different functional regions responsible for fusion induction and its inhibition.

Cell Fusion↗

PubMed1987

Silver-coated nylon fiber as an antibacterial agent.

A blend of nylon fiber and silver-coated nylon fiber (the latter known as X-static) was used in these experiments. This fiber was bactericidal when bacteria were exposed to it directly or to an extract derived from its prior incubation in salt solution. At ambient temperatures, a rapid exponential decrease of survival occurred, usually after a delay of approximately 1 h. The rate of killing (decrease of survival) increased with an increase in X-static percentage of the fiber blend, temperature of fiber extraction, concentration of Tris buffer present during extraction, and temperature at which bacteria were exposed to the extract. When bacteria were exposed to the extract at 37 degrees C as opposed to ambient temperature, there was no delay in onset of killing. Escherichia coli was generally the indicator organism tested, but comparable results were also found for Pseudomonas, Klebsiella, Staphylococcus, and Streptococcus species. The rate of killing increased with increasing silver ion concentration of the fiber extract, as determined through atomic absorption spectrophotometry. The rate of killing was greater and the onset was earlier with an extract containing silver ions from fiber than with a salt solution containing the same concentration of silver ions from silver nitrate. Studies of the kinetics of ion release suggested that X-static may be an effective, sustained-release antibacterial agent.

Delayed-Action Preparations↗

PubMed1987

Linker-insertion nonsense and restriction-site deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1.

To study the effects of missense, nonsense, and deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1, a gB-transformed cell line was isolated that, after virus infection, would express sufficient quantities of gB from the cellular chromosome to complement temperature-sensitive gB mutants. The transformed cell line was then used as a permissive cell to transfer two gB mutations from plasmid to viral DNA. One of the mutants, K082, harbored an HpaI linker insertion that introduced one new amino acid and a chain terminator codon within amino acid residue 43. The other mutant contained a 969-base-pair deletion in a part of the gene that includes the membrane-spanning region; a correspondingly shorter gB polypeptide was detected by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation of infected-cell extracts with four pooled monoclonal antibodies. No polypeptide was observed from K082-infected cells. The shortened gB polypeptide was efficiently processed and secreted into the growth medium. Each of the four monoclonal antibodies precipitated full-length gB, and three of the four precipitated the shortened polypeptide. Enveloped virus particles could be purified after infection of nonpermissive cells with either mutant virus. Virus particles appeared to possess normal polypeptide and glycopeptide profiles except for the absence of gB. Therefore, the presence of gB is not essential for viral assembly, including envelopment. Recombinants in virus stocks grown on the gB-transformed cells occurred at frequencies on the order of 10(-7) to 10(-5), compared with a frequency of approximately 10(-2) in mixed infections with the two mutants.

Base Sequence↗

PubMed1986

The nucleotide sequence of the gB glycoprotein gene of HSV-2 and comparison with the corresponding gene of HSV-1.

The nucleotide sequence of the gB glycoprotein gene of HSV-2 has been determined and compared with the homologous gene of HSV-1. The two genes are specified by the same total number of codons (904); eight additional codons of the HSV-1 gene are found within the signal sequence, and eight additional codons of the HSV-2 gene are found at three different sites in the gene. The signal cleavage, membrane-spanning, and eight potential N-linked oligosaccharide sites, as well as 5'- and 3'-regulatory signals are largely conserved. The overall amino acid homology is 85%; least conserved are the N- and C-terminal regions of the protein. Secondary structure plots were determined for the two proteins, and the structures were compared with each other and with alterations in structure due to several mutations in the HSV-1 gB gene for which sequence analysis is available. The high homology in primary and secondary structure suggests a conserved, essential function for the gene.

Amino Acid Sequence↗

PubMed1985

Nucleotide sequence of a herpes simplex virus type 1 gene that causes cell fusion.

The nucleotide sequence (2041 nucleotides) of a genomic region of herpes simplex virus type 1 (KOS strain) associated with virus-induced cell fusion has been determined. The sequence is bounded by a NruI site at 0.732 and a BamHI site at 0.745 prototypic map units. An open reading frame in the left-to-right orientation specifies a protein of 338 amino acids. The protein is positively charged. Since secondary structure analysis predicts four extensive hydrophobic domains the protein is probably a membrane-associated or a transmembrane protein. Transcription of the putative fusion gene is dependent on viral DNA synthesis, characteristic of the late (gamma) viral gene class. Two syncytia-inducing mutations, syn20 and MP, have been previously mapped to a 504-base pair PstI fragment within these genomic coordinates (V. C. Bond and S. Person (1984), Virology 132, 368-376). The nucleotide sequence of the PstI fragment was determined for the two mutants. Both were shown to have an amino acid substitution at residue 40 of the fusion protein. A second change at residue 101 for MP is probably unrelated to the fusion phenotype.

Amino Acid Sequence↗

PubMed1985

Expression in bacteria of gB-glycoprotein-coding sequences of Herpes simplex virus type 2.

A plasmid with an insert that encodes the glycoprotein B(gB) gene of Herpes simplex virus type 2 (HSV-2) has been isolated. DNA sequences coding for a portion of the HSV-2 gB peptide were cloned into a bacterial lacZ alpha expression vector and used to transform Escherichia coli. Upon induction of lacZpo-promoted transcription, some of the bacteria became filamentous and produced inclusion bodies containing a large amount of a 65-kDal peptide that was shown to be precipitated by broad-spectrum antibodies to HSV-2 and HSV-1. The HSV-2 insert of one of these clones specifies amino acid residues corresponding to 135 through 629 of the gB of HSV-1 [Bzik et al., Virology 133 (1984) 301-314].

Antibodies, Viral↗

PubMed1984

Fine structure physical map locations of alterations that affect cell fusion in herpes simplex virus type 1.

Fine structure physical map locations were determined for syncytial mutants (MP, syn-20, syn-102, syn-103, and syn-105) of Herpes Simplex Virus type 1 (HSV-1). All except MP were derived from the KOS strain. MP contains multiple mutations, including one that leads to the loss of accumulation of glycoprotein gC (Ruyechan et al., J. Virol. 29, 677-697, 1979). Overlapping DNA subclones within the prototypic map coordinates 0.707 to 0.810 were constructed from a library of KOS fragments. These were used along with intact mutant DNA to rescue the syn marker. Mutations in all of the mutants were rescued by KOS DNA sequences between 0.732 and 0.745. This cell-dependent syn mutation is the only lesion in the KOS-derived mutants. A second syn mutation in MP was mapped at coordinates 0.745 to 0.753. This lesion produces less fusion and is also cell-type dependent for the fusion phenotype. Cell-type independent fusion requires the presence of both mutations. The locus determining glycoprotein C (gC) production in strain MP was also mapped, using indirect immunofluorescence, to coordinates 0.745 to 0.753. Nucleotide sequences for ICP-27, an immediate early or alpha protein of unknown function, are within these coordinates. Since gC production and the syn phenotype are separable by recombination, they must be caused by independent mutations.

Animals↗

PubMed1984

Nucleotide sequence of a region of the herpes simplex virus type 1 gB glycoprotein gene: mutations affecting rate of virus entry and cell fusion.

The tsB5 isolate of herpes simplex virus type I (HSV-1) enters host cells more rapidly than does KOS, an independent isolate of HSV-1, and this rate-of-entry determinant is located between prototypic map coordinates 0.350 and 0.360 (1). The nucleotide sequence of strain tsB5 has now been determined between prototypic map coordinates 0.347 and 0.360. Comparison of the tsB5 sequence to the homologous KOS sequence revealed that the rate-of-entry difference between these two HSV-1 strains may be due to the single amino acid difference observed within these sequences (0.350 to 0.360). A cell fusion determinant in tsB5 is located between coordinates 0.345 and 0.355 and to the left of the rate-of-entry determinant (1). Nucleotide sequence analysis revealed a second amino acid difference between tsB5 and KOS at coordinate 0.349. The cell fusion determinant was tentatively assigned to this location.

Amino Acid Sequence↗

PubMed1984

Genome locations of temperature-sensitive mutants in glycoprotein gB of herpes simplex virus type 1.

A plasmid containing a herpes simplex virus type 1 (HSV-1) insert from strain KOS, prototypic coordinates 0.345 to 0.368 (3.45 kilobases) was mutagenized in vitro, and potential mutations were introduced into intact viral DNA by cotransfection. Functions normally associated with the glycoprotein gB are in the 1-9 complementation group, and the above coordinates include those that specify the gB glycoprotein gene. Following cotransfection, individual plaques were screened for temperature sensitivity (ts) of viral growth. A total of seven ts mutants was obtained, of which four were spurious mutations due to alterations outside the cloned sequences, presumably mediated by some aspect of the Ca-precipitation-cotransfection method. The remaining three did not complement known mutants of the 1-9 complementation group. These three mutants, along with tsJ12 (P.A. Schaffer, G.M. Aron, N. Biswal, and M. Benyesh-Melnick, 1973, Virology 52, 57-71) and tsJ33 (C.-T. Chu, D.S. Parris, R.A.F. Dixon, F.E. Farber, and P.A. Schaffer, 1979, Virology 98, 168-181), were physically located by marker-rescue experiments to three different restriction fragments between 0.345 to 0.368 map units. Sodium dodecyl sulfate-gel electrophoresis was used to analyze the glycoproteins synthesized during continuous or pulse-chase labeling protocols. All five mutants were found to synthesize a precursor of gB but did not accumulate mature gB during a pulse, a chase, or continuous labeling at the nonpermissive temperature.

Animals↗

PubMed1984

Nucleotide sequence specifying the glycoprotein gene, gB, of herpes simplex virus type 1.

The nucleotide sequence thought to specify the glycoprotein gene, gB, of the KOS strain of herpes simplex virus type 1 (HSV-1) has been determined. A 3.1-kilobase (kb), viral-specified RNA was mapped to the left half of the BamHI-G fragment (0.345 to 0.399 map units). TATA, CAT-box, and possible mRNA start sequences characteristic of HSV-1 genes are found near 0.368 map units. The first available ATG codon is at 0.366 and the first in-phase chain terminator at 0.348 map units. A polyA-addition signal (AATAAA) occurs 17 nucleotides past the chain terminator. Translation of these sequences would yield a 100.3-kilodalton (kDa) polypeptide characterized by a 5' signal sequence, nine N-linked saccharide addition sites, a strongly hydrophobic membrane-spanning sequence, and a highly charged 3' cytoplasmic anchor sequence. Two mutants of KOS, tsJ12 and tsJ20, that are temperature-sensitive for viral growth and for the production of gB, have been physically mapped to 0.357 to 0.360 and 0.360 to 0.364 map units, respectively (DeLuca et al., in preparation). The nucleotide sequence of the mutants was determined in these regions. In both cases a single amino acid replacement within the 100.3-kDa polypeptide is predicted from the sequence analysis.

Amino Acid Sequence↗