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W Gibson

Publications and source records attributed to W Gibson.

At least 109 records · Page 6Linked to original sources
PubMed1990

Identification of the lytic origin of DNA replication in human cytomegalovirus by a novel approach utilizing ganciclovir-induced chain termination.

Infection with human cytomegalovirus in the presence of the antiviral nucleotide analog ganciclovir results in continuing low-level viral DNA synthesis and the accumulation of relatively small fragments of double-stranded progency DNA. These fragments consistently proved to represent amplification of sequences from only one small section of the viral genome (EcoRI-V) lying near the center of the unique L segment. Further mapping revealed that the viral sequences represented in these fragments occurred in gradients of abundance that decreased in both directions from a point near 0.35 to 0.4 map unit. The proportion of amplified sequences increased with both time after infection and dosage of ganciclovir used. We conclude that the primary lytic cycle replication origin of human cytomegalovirus lies within a 3- to 4-kb region immediately upstream and to the right of the promoter for the single-stranded DNA-binding protein (DB140). The amplified origin-containing DNA molecules appeared to arise by continuing rounds of bidirectional initiation on truncated fragments of the genome that were generated as a result of chain termination effects induced by the incorporation of ganciclovir into the viral DNA. Inspection of the DNA sequence in the vicinity of ori-Lyt revealed a large complex upstream region that may be a noncoding intergenic domain and that bears no homology to any previously described herpesvirus origin. This 2.5-kb region includes many duplicated and inverted sequences, together with consensus CRE/ATF and other transcription factor-binding sites, and an interesting set of 23 copies of an interspersed decamer consensus element AAAACACCGT that is also conserved at the equivalent locus in simian cytomegalovirus. This work represents the first identification of an origin domain in a cytomegalovirus genome and is the first demonstration of a bidirectional mechanism for any herpesvirus lytic cycle origin.

Base 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

Sequence requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne.

Truncated versions of the human cytomegalovirus (CMV) strain Towne glycoprotein B (gB) gene were stably expressed in CHO cell lines. The calcium-specific ionophore A23187 inhibited proteolytic cleavage of C-terminal-truncated gB expressed by cell line 67.77. These inhibition studies also showed that the 93-kilodalton cleavage product most likely represents the N-terminal cleavage fragment of gB. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that proteolytic cleavage of gB did not occur in the endoplasmic reticulum. Two-dimensional polyacrylamide gel electrophoresis demonstrated that the N- and C-terminal cleavage products of gB remained associated by disulfide linkages after cleavage. Expression studies using constructs in which 80% or all of the N terminus was deleted demonstrated that the N terminus was required for secretion of the gB molecule. The amino acid sequence at the site of cleavage was shown to be critical for cleavage by a cellular protease. Our results indicate that an arginine-to-threonine change at either amino acid 457 or 460, a lysine-to-glutamine change at amino acid 459, or all three substitutions together block gB cleavage. The effect on proteolysis of the arginine-to-threonine amino acid change at residue 457 (position -4 relative to the cleavage site) demonstrated that a basic pair of amino acids at the endoproteolytic processing site is not the only requirement in cis for gB cleavage.

Amino Acid Sequence↗
PubMed1990

Hearing loss in Stickler's syndrome: a family case study.

Stickler's syndrome is an autosomal dominant hereditary condition that presents with pathognomonic facial features, dysplasia, myopia and auditory deficits. This paper describes the auditory history of a family with Stickler's syndrome. Given the expressive variability, a complete audiologic and otologic evaluation is encouraged when Stickler's syndrome is suspected.

Abnormalities, Multiple↗
PubMed1989

Primate cytomegalovirus assembly protein: genome location and nucleotide sequence.

A cDNA encoding the 37-kilodalton (kDa) capsid assembly protein of cytomegalovirus (CMV) strain Colburn was isolated from a lambda gt11 library constructed from CMV Colburn-infected human fibroblast RNA. RNA transcribed in vitro from this cDNA was translated in vitro to give a 40-kDa protein whose electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fragmentation pattern following partial proteolysis were indistinguishable from those of authentic assembly protein precursor. The position of the assembly protein gene was mapped to the EcoRI F, XbaI R, and SalI U restriction fragments, near the middle of the CMV Colburn genome, by Southern hybridizations using the cloned assembly protein cDNA as a probe. Similar sequences were identified by cross-hybridizations in colinear regions of the genomes of human CMV strains Towne and AD169: specifically, in the HindIII H, BamHI V, and EcoRI A fragments of Towne and in the HindIII L and BglII S fragments of AD169. The predominant transcript of the assembly protein gene was determined to be approximately 1 kilobase in size; however, a larger transcript (1.8 kilobases) was also identified. The nucleotide sequence of the assembly protein cDNA was determined and found to contain a single long open reading frame predicted to encode a polypeptide of 36.6 to 37 kDa, close to the 40-kDa size determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the assembly protein precursor. The presence of a single cysteine residue at the carboxy-terminal end of this open reading frame is consistent with data from biochemical studies and indicates that processing of the assembly protein precursor includes a proteolytic cleavage that removes its carboxyl end.

Amino Acid Sequence↗
PubMed1988

Primate cytomegalovirus assembly: evidence that DNA packaging occurs subsequent to B capsid assembly.

Results presented here show that when cytomegalovirus (strain Colburn)-infected cells are treated with the DNA synthesis inhibitor hydroxyurea or phosphonoformate, one type of intranuclear capsid accumulates. These particles appeared to contain symmetrically organized internal material, and had a protein composition and sedimentation rate characteristic of B capsids. Radiolabeling experiments provided evidence that a population of B capsids lacking DNA is present during the course of a normal infection. These capsids sedimented slightly slower than the peak of viral DNA in the same region of the gradient, and had a ratio of DNA/protein that was estimated to be sevenfold lower than that of the faster sedimenting C capsids. DNA in both the B and C capsid regions of such gradients was found to be relatively resistant to digestion with DNase. The possibility is considered that herpesvirus B capsids lacking DNA may be counterparts of unexpanded proheads in the bacteriophage assembly pathway.

Capsid↗
PubMed1988

Virion basic phosphoprotein from human cytomegalovirus contains O-linked N-acetylglucosamine.

A 149-kDa virion protein of human strains of cytomegalovirus is the principal acceptor for galactose added in vitro by bovine milk galactosyltransferase. Peptide comparisons with other biochemical characteristics of the galactosylated protein identified it as the virus-encoded basic phosphoprotein. This protein is an abundant constituent of the virion and is located in the tegument region, between the capsid and the envelope, rather than in the envelope layer with the recognized virion glycoproteins. The galactosylated carbohydrate was resistant to a commercial preparation of endoglycosidase F but was sensitive to removal by alkali-induced beta-elimination, indicating an O-linkage to the protein. Chromatographic and electrophoretic determinations identified the beta-eliminated material as the alditol of Gal beta 1-4GlcNAc, establishing that the human cytomegalovirus virion basic phosphoprotein contains single O-linked residues of N-acetylglucosamine.

Acetylglucosamine↗
PubMed1988

Location, transcript analysis, and partial nucleotide sequence of the cytomegalovirus gene encoding an early DNA-binding protein with similarities to ICP8 of herpes simplex virus type 1.

The results presented here locate the gene encoding an early, nonvirion, single-stranded DNA-binding protein of human and simian strains of cytomegalovirus (CMV) [HCMV(Towne) DB140 and SCMV(Colburn) DB129, respectively] and provide additional evidence that this protein is the CMV homolog of the herpes simplex virus type 1 (HSV-1) major DNA-binding protein (ICP8), as proposed earlier (D. G. Anders, A. Irmiere, and W. Gibson, J. Virol. 58:253-262). The ICP8 gene was used as a probe in Southern analyses done at moderate stringency as an approach to locating similar sequences in the CMV genome. The BamHI K and EcoRI V fragments from the center of the long unique segment of HCMV(Towne) hybridized with the ICP8 probe and were in turn used to identify corresponding sequences in the EcoRI D fragment of SCMV(Colburn). RNA prepared from SCMV(Colburn)-infected cells directed the in vitro synthesis of DB129. If the RNA was first hybridized with the cloned 12.5-kilobase EcoRI D fragment, in vitro synthesis of DB129 was specifically inhibited. Additional hybrid-arrested in vitro translation experiments with subclones spanning the EcoRI D fragment demonstrated that the DB129 gene is located in the left half of that fragment, approximately bisected by a SalI site. RNA analyses identified 3.9-, 8.9-, and 10.0-kilobase RNA species expressed from this region. A partial nucleotide sequence of the Colburn region mapping within the boundaries of the 3.9-kilobase transcript, suspected to be the primary coding species, showed significant sequence similarity to the major DNA-binding protein gene homolog identified in B95-8 Epstein-Barr virus.

Animals↗
PubMed1988

Homosexual and heterosexual sex-role orientation on six sex-role scales.

A comparison was made of the sex roles of homosexual and heterosexual men and women on the Bem Sex Role Inventory, Personality Attributes Questionnaire, Personality Research Form Androgyny Scale, Adjective Checklist Masculinity and Femininity Scales, Extended Personality Attributes Questionnaire and Undesirable Characteristics Scale. The results indicated that homosexuals and heterosexuals differ in their response to different aspects of sex roles. The most consistent difference was the greater femininity of male homosexuals in respect to male heterosexuals. Other differences were scale-specific and the low interscale comparability indicated such scales should not be used interchangeably. Differences between results of studies comparing sex roles of the homosexuals and heterosexuals appear attributable to sample heterogeneity and distinctions between sex-role scales.

Adult↗
PubMed1987

Immunological characterization of an early cytomegalovirus single-strand DNA-binding protein with similarities to the HSV major DNA-binding protein.

Monospecific polyclonal antisera were prepared against the 129-kDa, early, single-strand DNA-binding protein (DB129) of strain Colburn cytomegalovirus (CMV), and used to study its distribution in infected cells and its relatedness to a proposed human CMV (HCMV) counterpart (DB140). Indirect immunofluorescence of fixed, infected human fibroblasts showed DB129 to be localized within the intranuclear inclusions characteristic of replicating CMV. Treatment of infected cells with 50 to 100 micrograms phosphonoformic acid per milliliter resulted in the overproduction of DB129 and its accumulation within nuclei, both inside the inclusions and in surrounding areas of the nucleoplasm, whereas treatment with 500 micrograms/ml prevented inclusion formation, and DB129 was localized at discrete points throughout the infected-cell nuclei. The sera cross-reacted an estimated 10% with HCMV DB140 in an indirect immunoassay, and their use in immunofluorescence localized DB140 to the nuclear inclusions of HCMV-infected cells. Their immunological cross-reactivity, as well as their similar biochemical properties and intracellular distribution, support the likelihood that DB129 and DB140 are the protein products of homologous genes. The relationship of these proteins to the herpes simplex major DNA-binding protein is discussed.

Cell Compartmentation↗
PubMed1986

Identification and characterization of a major early cytomegalovirus DNA-binding protein.

We characterized a DNA-binding protein with an approximate molecular weight of 129,000 (DB129) which is present in the nuclei of cytomegalovirus- (strain Colburn) infected cells, but not in virus particles. Results of two types of experiments demonstrated that DB129 is a member of the early class of herpesviral proteins. First, time course pulse-labeling experiments showed that its synthesis begins after that of the immediate-early protein IE94, but prior to the appearance of late viral proteins, and was reduced at late times. Second, in the presence of inhibitors of viral DNA replication, DB129 continued to be made and accumulated to elevated levels. A second set of experiments showed that DB129 bound to single-stranded DNA in vitro and was eluted by a NaCl gradient in two peaks, one at about 0.2 M and the second at about 0.6 M. A similar pattern of release was observed when infected-cell nuclei were serially extracted with increasing NaCl concentrations. In addition, treatment of nuclei with DNase I selectively released DB129, along with a small but significant fraction of another DNA-binding protein, DB51. These results suggest that DB129 is associated with DNA in vivo and that it interacts directly with single-stranded DNA. It was also shown that cells infected with human cytomegalovirus (strain Towne) contain a slightly larger counterpart to DB129, which was designated DB140. Similarities between these proteins and the major DNA-binding protein of herpes simplex virus are discussed.

Cell Nucleus↗
PubMed1986

Primate cytomegalovirus glycoproteins: lectin-binding properties and sensitivities to glycosidases.

The lectin-binding properties and glycosidase sensitivities of the virion glycoproteins of primate cytomegaloviruses (CMVs) were examined. Three simian CMV (SCMV) strains, including Colburn, and four human CMV (HCMV) strains were compared. Their proteins were separated in denaturing polyacrylamide gels and electrotransferred onto nitrocellulose, and the glycosylated species were visualized with iodinated concanavalin A or wheat germ agglutinin (WGA). Virions of both HCMV and SCMV strains contained six principal and several minor lectin-reactive bands. Neuraminidase treatment abolished WGA binding and reduced the charge and charge heterogeneity of the SCMV (i.e., Colburn) virion glycoproteins and had a similar, although less dramatic, effect on those of HCMV. The specificities of concanavalin A and WGA in these assays were evaluated with endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F, and a combination of lectins and glycosidases was used to demonstrate that many of the primate CMV glycoproteins contain both high-mannose and complex, N-linked oligosaccharides. Results suggest that the HCMV virion glycoproteins are more extensively glycosylated or have more completely processed carbohydrate side chains, or both, than their SCMV counterparts.

Acetylglucosaminidase↗
PubMed1986

Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.

Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions separated the protein kinase activity into two fractions: one that phosphorylated exogenous casein and another that phosphorylated primarily the endogenous virion proteins.

Cytomegalovirus↗
PubMed1985

Anti-complement immunofluorescence establishes nuclear localization of human cytomegalovirus matrix protein.

A monospecific, polyclonal antiserum to the 69-kDa matrix protein of human cytomegalovirus (HCMV) was prepared in a guinea pig and used to determine the intracellular distribution of this viral antigen. The resulting antiserum was specific for infected cells as tested by immunofluorescence, and specific for the HCMV matrix protein as determined by "nitrocellulose immunoassay" of electrophoretically separated, infected-cell proteins. Antibodies were reacted with fixed, infected human fibroblasts, and visualized by the anti-complement immunofluorescence procedure to avoid complications arising from the strong IgG Fc binding activity of the infected-cell-specific cytoplasmic inclusion. Results establish that the matrix protein is located in the nucleus, and indicate that it is concentrated in the nucleoplasm rather than within the intranuclear inclusions.

Animals↗
PubMed1985

Further analysis of intraspecific variation in Trypanosoma brucei using restriction site polymorphisms in the maxi-circle of kinetoplast DNA.

We have compared the maxi-circle kinetoplast DNA of 21 Trypanosoma brucei sp. stocks by analysis of restriction sites for nine restriction endonucleases. The analysis shows most of these stocks to have a maxi-circle sequence similar to that of 11 previously analysed stocks, with a difference of less than 3% between any two stocks. However, seven stocks stand out from the rest with at least two sites lost or gained for six of the nine restriction enzymes used. These seven distinctive stocks fall into two groups with some shared and some unique polymorphisms. One group had already been designated the kiboko group on the basis of isoenzyme patterns, but the relationship between nuclear markers and maxi-circle type is less clear-cut for the other group, designated sindo. Both groups seem to be in a wild animal-tsetse fly transmission cycle, with occasional infections in domestic stock, and may be reproductively isolated from the main T. brucei sp. population. The existence of the kiboko and sindo sub-groups shows that the maxi-circle is not shielded from evolutionary change. The lack of difference observed between the maxi-circles of the majority of T. brucei sp. stocks, including the gambiense and rhodesiense variants, must therefore reflect their close homology. Two geographical trends occur in T. brucei as a whole: (a) a trend in maxi-circle size, with increasing length of the variable region from West to East Africa, and (b) a greater frequency of certain restriction enzyme polymorphisms in East African stocks as compared to West African stocks.

Animals↗