Biomedical subjects

L Pereira

Publications and source records attributed to L Pereira.

At least 127 records · Page 7Linked to original sources

PubMed1986

Identification, properties, and gene location of a novel glycoprotein specified by herpes simplex virus 1.

We report the identification of a novel herpes simplex virus 1 (HSV-1) glycoprotein reactive with type specific monoclonal antibody H1379. The monoclonal antibody reacted with two broad bands with apparent mol wt of 60K to 68K and 44K to 48K formed by infected cell lysates subjected to electrophoresis in denaturing polyacrylamide gels and electrically transferred to a nitrocellulose sheet. Early in infection the H1379 reactive protein was found in the faster migrating band. The rate of accumulation was highest late in infection and only the slower migrating form incorporates significant amounts of glucosamine. The epitopic site recognized by H1379 was not uniformly distributed among strains. Analyses of HSV-1 X HSV-2 recombinants with monoclonal antibodies to HSV-1 and HSV-2 glycoproteins mapping in the S component of the HSV genomes and marker transfer experiments indicated that the gene specifying the H1379 reactive protein maps within BamHI fragment J to the left of gD most probably within the open reading frame designated as US4 (D. J. McGeoch, A Dolan, S. Donald, and F. J. Rixon, 1985, J. Mol. Biol. 181, 1-13). The gene specifying a recently discovered HSV-2 glycoprotein designated as gG-2 (B. Roizman, B. Norrild, C. Chan, and L. Pereira, 1984, Virology 133, 242-247) maps in the corresponding domain of the HSV-2 genome and marker transfer experiments suggest that the H1379 reactive protein and gG-2 are collinear. We have therefore designated the novel HSV-1 glycoprotein as gG-1.

Antibodies, Monoclonal↗

PubMed1986

A novel glycoprotein for detection of herpes simplex virus type 1-specific antibodies.

A novel herpes simplex virus type 1 (HSV-1)-specific glycoprotein reactive with monoclonal antibody H1379 was purified by affinity chromatography. This glycoprotein, provisionally designated as gG-1, forms two sets of bands with molecular weights of 40-44,000 and 60-88,000. When used in an immunodot enzymatic assay, gG-1 reacted strongly with rabbit antisera to HSV-1, but not with sera hyperimmune to HSV-2. Specificity of the assay was further established by the lack of reactivity of convalescent sera collected from 20 patients with primary genital HSV-2 infections, and from 100 sero-negative individuals. In contrast, antibodies to gG-1 were detected in 9 of 10 patients with primary HSV-1 infection, and in 63/67 patients with culture-positive, recurrent oral or genital HSV-1 infection. Reproducibility of the gG-1 immunodot assay for HSV-1 antibody detection was 96%. Serological assay with purified gG-1, done in parallel with the assay using purified gG-2 described in an earlier report, provides simple and reliable methods to detect type-specific HSV-1 and HSV-2 antibodies for seroepidemiological studies.

Animals↗

PubMed1986

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.

Animals↗

PubMed1985

Oligosaccharide chains of herpes simplex virus type 2 glycoprotein gG.2.

gG.2 glycoprotein was purified by H966 monoclonal antibodies linked to Sepharose from herpes simplex virus type 2-infected HEp-2 cells labeled with [3H] glucosamine. The glycoprotein was subjected to Pronase digestion and the glycopeptides were fractionated by Con A-Sepharose in a major fraction (88.5% of total radioactivity) unbound to the lectin gel and in a minor species which bound to the lectin as a N-linked diantennary oligosaccharide. Mild and strong acid hydrolysis of Con A-unbound and Con A-bound fractions revealed that (i) both species were highly sialylated; (ii) the Con A-unbound fraction contained mainly labeled N-acetylgalactosamine, as is the case for O-linked oligosaccharides; and (iii) the Con A-bound fraction carried the vast majority of the labeled N-acetylglucosamine present in gG.2. Three size classes of oligosaccharides were separated from mild alkaline borohydride-treated Con A-unbound glycopeptides, which accounted for about 80% of the radioactivity present in the fraction. Galactosaminitol was recovered as the major labeled product in the strong acid hydrolyzates of the oligosaccharides generated by reductive beta-elimination, indicating that they were O-glycosidically linked to the peptide backbone. Thin-layer and DEAE-Sephacel chromatography of the three O-linked oligosaccharide species indicated that disialylated tetrasaccharides and monosialylated trisaccharides were the major components, whereas neutral disaccharide was a minor component. Digestion with neuraminidase and beta-galactosidase of the O-linked oligosaccharides supported the idea that the common disaccharide core was mainly of the structure beta-galactosyl-N-acetylgalactosamine. The large occurrence of O-linked oligosaccharides differentiates this type 2-specific herpes simplex virus glycoprotein from the type-common herpesvirus glycoproteins gB, gC, and gD.

Antibodies, Monoclonal↗

PubMed1985

Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family.

We describe an efficient procedure, which uses monoclonal antibodies directed against specific viral proteins, for the precise mapping of genes on large DNA virus genomes. We have used the technique to locate the gene encoding a family of antigenically related DNA-binding proteins on the 240-kilobase-pair human cytomegalovirus (CMV) genome. A random library of CMV DNA fragments was generated using the prokaryotic vector lambda gt11, which expresses open reading frames as beta-galactosidase fusion proteins in infected Escherichia coli. The library was screened with a mixture of monoclonal antibodies directed against the gene products of interest. The coding sequence for infected cell protein 36 (ICP36) was localized to a 2800-base-pair EcoRI fragment (map coordinates 0.228-0.240) on the CMV(Towne) and CMV(AD169) genomes by using DNA from immunoreactive lambda gt11 as probe. A 5000-nucleotide transcript from this region was detected during the early and late phases of the CMV growth cycle. This transcript directed the synthesis of the predominant member of the ICP36 family when hybrid-selected and translated in vitro. Immunoprecipitation of the in vitro translation product with the same monoclonal antibodies used in the initial mapping confirmed the location of the ICP36 gene. These studies establish the utility of the lambda gt11 expression system for rapid and precise mapping of CMV genes (or other large animal virus genes) that encode proteins for which serological reagents exist.

Antibodies, Monoclonal↗

PubMed1985

Antibody response to cytomegalovirus polypeptides captured by monoclonal antibodies on the solid phase in enzyme immunoassays.

Antibodies to different cytomegalovirus (CMV) polypeptide antigens, captured by monoclonal antibodies coated on the solid phase of an enzyme immunoassay test, were analyzed in 42 serum pairs submitted for serodiagnosis of CMV infection. Three CMV antigens, captured on the solid phase by three monoclonal antibodies of different specificities, designated CH92-1, CH65-1, and CH16-1, were glycoproteins A (gA), gC, and gD, respectively; and one antigen, captured by CH23, was a polypeptide with an apparent molecular weight of 150,000, possibly associated with the nucleocapsid. Of these four CMV antigens, gA captured by CH92-1 was most effective in eliciting an antibody response. Antibody to this antigen was present in serum samples at a higher concentration in primary and reactivated infection and persisted longer than did antibody to the other tested antigens. In contrast, antibody to antigen captured by CH23 was at a lower concentration, rose more slowly in infection, and persisted for a shorter time than did antibody to the other antigens. Antibody response to gC and gD was intermediate in concentration and temporal appearance compared with the antibody response to gA and to the polypeptide bound by CH23. An enzyme immunoassay on paired serum samples with the captured glycoproteins as antigen was equal for the detection of current infection to an enzyme immunoassay with the whole CMV antigen from infected cell lysates. Enzyme immunoassays with either the CMV glycoproteins or the whole CMV antigen from infected cell lysates were superior to a complement fixation test with a glycine extract antigen for serodiagnosis of current infection.

Antibodies, Monoclonal↗

PubMed1985

Detection of herpes simplex virus type 2-specific antibody with glycoprotein G.

A recently described herpes simplex virus (HSV) type 2 (HSV-2)-specific glycoprotein (gG-2) was purified on an immunoaffinity column prepared with monoclonal antibody. This purified antigen was used in an immunodot enzymatic assay on nitrocellulose paper for the detection of HSV-2 antibodies in human serum. The test was very sensitive in that HSV-2 antibodies were detected in the convalescent sera of 132 of 134 patients with recurrent genital infections in which HSV-2 had been isolated earlier. Antibodies to gG-2 were detected in 17% of sera obtained within 10 days after the onset of a primary HSV infection and in 95% of sera obtained more than 10 days after onset. The specificity of the immunodot assay was demonstrated by testing sera from 245 HSV-seronegative adults, 344 children, 29 nuns, and 13 patients with primary genital HSV-1 infections. None of these 631 sera was reactive with the gG-2 antigen. When compared with a microneutralization test, the immunodot assay was found to be more specific in detecting HSV-2 antibodies. Reproducibility of the gG-2 assay, obtained by retesting 391 sera, was 95%. Thus, this assay has the sensitivity, specificity, and reproducibility necessary for the measurement of HSV-2 antibodies in seroepidemiological studies.

Adult↗

PubMed1985

Anatomy of the herpes simplex virus 1 strain F glycoprotein B gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibody-resistant mutants.

In this paper we report the nucleotide sequence and predicted amino acid sequence of glycoprotein B of herpes simplex virus 1 strain F and the amino acid substitutions in the domains of the glycoprotein B gene of three mutants selected for resistance to monoclonal antibody H126-5 or H233 but not to both. Analyses of the amino acid sequence with respect to hydropathicity and secondary structure yielded a two-dimensional model of the protein. The model predicts an N-terminal, 29-amino-acid cleavable signal sequence, a 696-amino-acid hydrophilic surface domain containing six potential sites for N-linked glycosylation, a 69-amino-acid hydrophobic domain containing three segments traversing the membrane, and a charged 109-amino-acid domain projecting into the cytoplasm and previously shown to marker rescue glycoprotein B syn mutations. The nucleotide sequence of the mutant glycoprotein B DNA fragments previously shown to marker transfer or rescue the mutations revealed that the amino acid substitutions cluster in the hydrophilic surface domain between amino acids 273 and 305. Analyses of the secondary structure of these regions, coupled with the experimentally derived observation that the H126-5- and H233-antibody cognitive sites do not overlap, indicate the approximate locations of the epitopes of these neutralizing, surface-reacting, and immune-precipitating monoclonal antibodies. The predicted perturbations in the secondary structure introduced by the amino acid substitutions correlate with the extent of loss of reactivity with monoclonal antibodies in various immunoassays.

Amino Acid Sequence↗

PubMed1984

Mutations affecting conformation or sequence of neutralizing epitopes identified by reactivity of viable plaques segregate from syn and ts domains of HSV-1(F) gB gene.

Three classes of HSV-1(F) mutants expressing a resistance phenotype to two highly potent-type common monoclonal antibodies, H126-5 and H233, to glycoprotein B (gB) were selected. Class 1 mutants, selected for resistance to neutralization from nonmutagenized virus stocks, expressed a gB which reacted in biotin-avidin-enhanced surface immunoassays and in immune precipitation tests with the selecting antibodies. Class 2 and 3 mutants were selected for nonreactivity in the biotin-avidin-enhanced surface immunoassay from BUdR-mutagenized, preneutralized virus stocks, but differ in that the selecting antibodies immune precipitated the gB of Class 2 but not that of Class 3. Mutants expressing a resistance phenotype to one monoclonal antibody (H126-5 or H233) invariably retained reactivity in all tests with the heterologous antibody, and recombinants resistant to both antibodies were produced by cotransfection of intact DNA of one mutant with a cloned DNA fragment from another mutant. Class 1 mutations were mapped by marker transfer to a 1734-bp DNA fragment. Class 2 and 3 mutations were mapped to a region defined by a maximum of 377 bp and a minimum of 46 bp, in a biotin-avidin-enhanced surface immunoassay with a panel of DNA fragments of HSV-1(F) BamHI G carrying staggered deletions across the region encoding gB. This region does not overlap the neutralizing antibody determinant site mapped by T.C. Holland, R.M. Sandri-Goldin, L.E. Holland, S.D. Marlin, M. Levine, and J. Glorioso (1983, J. Virol. 46, 649-652) and is located 3' to the ts lesion of HSV-1(HFEM)tsB5 and 5' to the syn3 locus of that virus. It was concluded that (i) inasmuch as the biotin-avidin-enhanced surface immunoassay does not destroy the virus contained in the plaque, it is a rapid and convenient method for both identification and selection of mutants reactive and nonreactive to specific monoclonal antibodies. (ii) gB may contain multiple domains carrying epitopic sites of neutralizing monoclonal antibodies. (iii) The resistance phenotype may arise from mutations which alter the conformation or the amino acid sequence of the epitope. These mutations might be differentiable on the basis of reactivity of mutated gB with selecting monoclonal antibody in nondenaturing and denaturing environments, respectively.

Amino Acid Sequence↗

PubMed1984

Polymorphism of human cytomegalovirus glycoproteins characterized by monoclonal antibodies.

Monoclonal antibody panels selected in this and preceding studies were employed to begin to characterize the properties of human cytomegalovirus (CMV) glycoproteins. The results were as follows. (i) Four antigenically distinct CMV glycoproteins designated as gA, gB, gC, and gD have been identified. (ii) gA, gC, and gD each form several bands when immune precipitated from infected cell extracts by the corresponding monoclonal antibodies and electrophoretically separated in sodium dodecyl sulfate-polyacrylamide gels. In contrast, gB migrated at one broad band with an apparent molecular weight in the range of 116,000 to 123,000. Bands with different molecular weights were shown to share antigenic determinants by reactivity of monoclonal antibodies with electrophoretically separated polypeptides immobilized on nitrocellulose. (iii) A panel of 16 monoclonal antibodies to gA precipitated a family of glycoproteins 160,000-148,000, 142,000, 138,000, 123,000-107,000, 95,000, and 58,500 in apparent molecular weight designated as gA1 through gA6, respectively. (iv) To identify partially glycosylated precursors of gA, infected cells were treated with tunicamycin or deoxyglucose and reacted with different monoclonal antibodies. Tunicamycin-treated infected cells labeled for a short pulse or longer intervals contained only gA5. Whereas cells treated with deoxyglucose during a pulse contained gA4, those labeled for a longer interval contained gA6 and an additional band approximately 56,500 in apparent molecular weight designated gA7. (v) Precipitates of gA from infected cells labeled for a short pulse contained gA2 and gA3 which appear to be products of rapid glycosylation. After a chase, trace amounts of gA1 and gA6 were also precipitated suggesting that these are products of slow post-translational processing. (vi) Endo-beta-N-acetylglucosaminidase H was used to identify the forms of gA which contain high-mannose oligosaccharide chains. After treatment, the electrophoretic mobility of gA2, gA3, and gA6 increased significantly suggesting that these forms contain high-mannose chains cleaved by the enzyme. A hypothesis for processing gA is presented.

Antibodies, Monoclonal↗

PubMed1984

Identification and preliminary mapping with monoclonal antibodies of a herpes simplex virus 2 glycoprotein lacking a known type 1 counterpart.

The properties of herpes simplex virus 2 (HSV-2)-specific proteins reactive with monoclonal antibody H966 derived from mice immunized with HSV-2 strain G are reported. The reactive proteins contained in infected cell lysates subjected to electrophoresis in denaturing gels and transferred to nitrocellulose sheets form a relatively sharp band characteristic of Mr 124,000 proteins and a diffuse, more slowly migrating band. Antigens reactive with H966 were detected on the surface of viable, unfixed cells. The electrophoretic mobility of the H966-reactive proteins made in the presence of tunicamycin was more rapid than that of the proteins made in the absence of the drug. Direct evidence that the HSV-2-specific antigen was a glycoprotein emerged from purification of [14C]glucosamine-labeled proteins with similar electrophoretic mobilities by immunoabsorption to H966 bound to Sepharose beads. Analyses of the reactivity of HSV-1 X HSV-2 recombinants indicated the gene specifying the glycoprotein maps in the S component of the DNA. The glycoprotein detected by H966 has no known counterpart in HSV-1 and corresponds to the glycoprotein previously designated as gC of HSV-2 and reported to map to the right of gC specified by HSV-1. Inasmuch as an HSV-2 gene colinear with HSV-1 gC has been reported to specify a glycoprotein currently designated as gC of HSV-2 by Para et al. [J. Virol. 45, 1223-1227 (1983)], the glycoprotein identified by H966 should be designated as gG.

Animals↗

PubMed1984

Factors affecting the response to inhibition of drug metabolism by cimetidine--dose response and sensitivity of elderly and induced subjects.

The effect of cimetidine on oxidative drug metabolism was characterised using antipyrine clearance in a group of healthy volunteers. In six subjects cimetidine produced a dose dependent reduction of antipyrine clearance: 400 mg/day (16.8 +/- 2.2%, mean +/- s.e. mean), 800 mg/day (26.3 +/- 1.5%) and 1600 mg/day (33.5 +/- 2.4%). The effect of cimetidine (800 mg/day) was of similar magnitude (approximately 25%) in two groups of six young (21-26 years) and six elderly (65-78 years) subjects. The effect of pretreatment begun just 1 h before administration of antipyrine was similar to that of 24 h pretreatment and that reported for chronic cimetidine pretreatment. The percentage reduction in antipyrine clearance produced by cimetidine 800 mg/day was greater (44 +/- 5 vs 24 +/- 3%; P less than 0.05) in six subjects who had been pretreated with the hepatic enzyme inducer rifampicin (600 mg/day for 21 days) than in the control uninduced state. Although cimetidine was capable of rapidly reversing the effect of rifampicin on antipyrine clearance, following withdrawal of both rifampicin and cimetidine there was still evidence of enzyme induction. These results suggest that the effect of cimetidine on oxidative metabolism is dose dependent, is more marked in enzyme induced subjects, is independent of the duration of pretreatment and is of similar magnitude in young and elderly subjects.

Adult↗

PubMed1984

Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody.

An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.

Animals↗

PubMed1984

Characterization of post-translational products of herpes simplex virus gene 35 proteins binding to the surfaces of full capsids but not empty capsids.

We report on the properties of a genetically and immunologically related family of structural (gamma) polypeptides of herpes simplex virus 1 designated as infected cell polypeptides (ICP) 35. The members of this family were identified and studied with the aid of a panel of monoclonal antibodies exemplified by H745. This monoclonal antibody reacted with six bands (ICP35a to 35f) formed by ICPs contained in either HEp-2 or Vero cell lysates electrophoretically separated in denaturing gels and transferred to nitrocellulose sheets. The six bands had apparent molecular weights in the range 39,000 to 50,000. Traces of ICP35 with apparent molecular weights of 37,000 were also observed in some preparations. On two-dimensional separation ICP35 family members formed at least 20 spots reactive with H745. These differed in both isoelectric properties and electrophoretic mobility in denaturing gels. Pulse-chase experiments, together with results published earlier, indicate that ICP35a to 35d are cytoplasmic precursors to nuclear products. One of these corresponds to virion protein 22a, a component of capsids containing DNA accumulating in the nuclei of infected cells. ICP35 was labeled by 32Pi added to the medium, but the extent of phosphorylation varied and may be a determinant of isoelectric properties. Iodination studies indicate that ICP35e and 35f are the predominant forms of ICP35 present on the surface of full, nuclear capsids containing DNA. None of the members of the ICP35 family were detected in empty capsids. Surface iodination labeled the major capsid protein (ICP5) of empty capsids, but not of full capsids, indicating that ICP35e and 35f coat the surface of the viral capsid and block access to sites for iodination of ICP5, the major capsid protein.

Antibodies, Monoclonal↗

PubMed1984

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).

Animals↗

PubMed1984

Characterization of herpes simplex virus 1 alpha proteins 0, 4, and 27 with monoclonal antibodies.

Analyses of the reactivity and patterns of synthesis of infected cell polypeptides (ICPs) specified by herpes simplex virus (HSV) 1 and 2 and by HSV-1 X HSV-2 recombinants indicated that monoclonal antibody H1183 reacted with HSV-1 alpha ICP0, whereas monoclonal antibody H1113 reacted with both HSV-1 and HSV-2 alpha ICP27. H1083 and H1113 and a monoclonal antibody to ICP4 (H640) similar to one previously described (D. K. Braun et al., J. Virol. 46:103-112.) were then used to study the properties of these alpha proteins. The results were as follows: alpha ICP0, ICP4, and ICP27 accumulated primarily in the nuclei of infected cells. ICP4 and ICP27 were poorly soluble in nondenaturing buffer solutions. ICP0 was considerably more soluble than ICP4 and ICP27. ICP0, ICP4, and ICP27 were readily partially purified by immunoaffinity chromatography from lysates of infected cells solubilized with denaturing agents such as sodium dodecyl sulfate. ICP0 and ICP27 were phosphorylated in cells overlaid with medium containing 32P early (1 to 3 h) or late (18 to 20 h) postinfection. A fraction, but not all, 32P that was incorporated early was chased in the presence of unlabeled phosphate. ICP0, ICP4, and ICP27 labeled with either 32P or [35S]methionine yielded multiple spots upon two-dimensional separations. However, ICP4 quantitatively precipitated at the origin when the migration in the first dimension was from acid to base, and both ICP4 and ICP27 partially precipitated at the origin when the direction of migration was reversed.

Animals↗

PubMed1983

Monoclonal antibodies for rapid diagnosis and typing of genital herpes infections during pregnancy.

Fluorescein-conjugated, type-specific monoclonal antibodies to herpes simplex virus (HSV-1 and HSV-2) and group-specific antibody which recognizes both HSV-1 and HSV-2 were used to detect HSV-infected cells in clinical specimens. Specimens were collected from 66 pregnant women with a past history of herpes genitalis or with suspected lesions. Fourteen of 18 samples from which virus was isolated were positive by immunofluorescence test; four of 18 specimens had an insufficient number of cells (less than 50 per smear) for analysis. In one specimen, a positive reaction by immunofluorescence was not confirmed by virus isolation. HSV was typed directly on smears of clinical specimens in 14 instances: three samples were identified as HSV-1, and 11 as HSV-2. Moreover, the immunofluorescence test was used to type HSV isolates in cell cultures in these cases and 40 additional strains. Examination of viral deoxyribonucleic acid fragments obtained by restriction enzyme digestion confirmed the typings; complete agreement was found among the three methods. Immunofluorescence with monoclonal antibodies is a reliable test for rapid diagnosis and simultaneous typing of genital HSV infections, even in asymptomatic women. With adequate specimens, the specificity and sensitivity of this method approach 100%.

Antibodies, Monoclonal↗

PubMed1983

Requirements for transport of HSV-1 glycoproteins to the cell surface membrane of human fibroblasts and Vero cells.

The intracellular transport of the HSV-1 glycoproteins gA/gB, gC and gD has been followed by the indirect immunofluorescence technique (IIF). Infected tissue culture cells were stained with monoclonal antibodies made to the individual glycoproteins and with fluorochrome-coupled wheat germ agglutinin reacting specifically with Golgi apparatus of the cells. Staining of either infected, human fibroblasts or of VERO cells at 9 hours p.i. with antibodies to gA/gB showed a prominent ring-like nuclear fluorescence and distinct staining of the Golgi apparatus in the cells. Antibodies to gC and gD stained mainly the Golgi apparatus and areas close to or at the surface of the cells. By immunocytolysis of HSV-1-infected VERO cells the viral glycoproteins were demonstrable at the surface of cells but growth of infected cells in the presence of either TM or monensin inhibited the expression of most of the viral glycoproteins at the cell surface. Blocking of the glycosylation of the viral glycoproteins with tunicamycin (TM) was followed by accumulation of the core of the glycoproteins gA/gB and gD in granular structures close to the nucleus as seen by immunofluorescence microscopy. Antibodies to gC did also stain granules close to the nucleus but in addition the periphery of the cells were stained. Inhibition of intracellular transport from the Golgi apparatus by the carboxylic ionophore monensin was followed by accumulation of all the HSV-1 glycoproteins in vesicles derived from the Golgi apparatus in both human fibroblasts and VERO cells. Our data thus support the hypothesis that the HSV-1 glycoproteins are processed in the Golgi apparatus before the transport to and incorporation into the cell surface membrane of infected cells and into virion envelopes.

Animals↗