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D A Galloway

Publications and source records attributed to D A Galloway.

At least 91 records · Page 5Linked to original sources
PubMed1991

The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells.

The contribution of the E6 and E7 open reading frames of human papillomavirus type 6b (HPV6b) and HPV16 to immortalization of human keratinocytes was evaluated by using amphotropic recombinant retroviruses. The HPV16 E7 gene could immortalize primary human keratinocytes without the cooperation of the viral E6 gene; however, E6 was able to contribute significantly to the efficiency of the E7 immortalizing function. Infection of HFE cells with retroviruses carrying the 16E6, 6bE6, or 6bE6E7 open reading frame did not result in immortalization.

Cell Transformation, Neoplastic↗
PubMed1991

Characterization of human antibody-reactive epitopes encoded by human papillomavirus types 16 and 18.

We have previously reported that the most common human serum immunoglobulin G antibody reactivities to human papillomavirus type 16 and type 18 (HPV16 and HPV18)-encoded proteins are directed against the minor capsid proteins (HPV16 L2 and HPV18 L2) and to the E7 protein of HPV16 (S. A. Jenison, X.-P. Yu, J. M. Valentine, L. A. Koutsky, A. E. Christiansen, A. M. Beckmann, and D. A. Galloway, J. Infect. Dis. 162:60-69, 1990). In this study, the antibody-reactive segments of the HPV16 E7, HPV16 L2, and HPV18 L2 polypeptides were mapped by using nested sets of deleted recombinant proteins. A single major immunoreactive region was identified in the HPV16 E7 polypeptide between amino acids (aa) 21 and 34 (DLYCYE-QLNDSSEE). In contrast, three distinct immunoreactive regions of the HPV16 L2 polypeptide were present in the segment between aa149 and aa204, and three distinct immunoreactive regions of the HPV18 L2 polypeptide were present in the segment between aa110 and aa211. With the exception of one serum sample, serum immunoglobulin G antibodies which reacted with HPV16 L2 polypeptides or with HPV18 L2 polypeptides were not cross-reactive.

Amino Acid Sequence↗
PubMed1991

Characterization of murine polyclonal antisera and monoclonal antibodies generated against intact and denatured human papillomavirus type 1 virions.

Human papillomavirus type 1 (HPV1) virions, both as intact virion particles (IVP) and as detergent-denatured virions (DDV), were used to prepare polyclonal antisera and monoclonal antibodies (MAbs) in BALB/c mice. Anti-IVP antiserum contained type-specific HPV1 L2-reactive antibodies and no detectable HPV1 L1-reactive antibodies. Anti-IVP MAbs recognized a linear epitope between L2 amino acids 102 and 108 (PIDVVDP). Anti-DDV antiserum contained type-specific HPV1 L1-reactive and HPV1 L2-reactive antibodies. An anti-DDV MAb recognized a linear epitope between L1 amino acids 127 and 133 (AENPTNY). HPV1a L1- and L2-encoded polypeptides expressed in Saccharomyces cerevisiae and by in vitro translation were equivalent in size to the major and minor virion capsid proteins, respectively.

Amino Acid Sequence↗
PubMed1990

Alterations in the cellular phenotype induced by herpes simplex viruses.

Numerous studies have shown that herpes simplex virus types 1 and 2 (HSV-1, HSV-2) are able to transform the morphological phenotype of rodent cells. Unlike other DNA tumor viruses the transformed cells did not consistently retain or express a given set of viral genes. In fact, transformation could be obtained using fragments of viral DNA that did not wholly encode viral proteins. Of interest within the transforming fragments were sequences which could assume a secondary structure like that of insertion elements. The failure to detect viral DNA in transformed cells led to the hit-and-run hypothesis of HSV transformation. The mechanism by which HSV induces transformation is not understood. Various lines of investigation have shown that HSV is able to cause mutations--both point mutations and gene rearrangements. HSV is also able to induce gene amplification, particularly of sequences harboring an origin of replication such as SV40 or papillomaviruses. Other experiments have shown that HSV can activate the expression of endogenous type C retroviruses. More broadly, HSV has been shown to activate cellular transcription or to switch on the synthesis of host cell proteins not normally expressed in untransformed cells. The failure to detect viral DNA in a high proportion of human anogenital tumors made it difficult to implicate HSV in the etiology of those neoplasias, but it is consistent, however, with the observations on the mode of HSV transformation in vitro, and suggests that HSV could be involved in a multistage process of oncogenic transformation.

Animals↗
PubMed1990

Immunological cross-reactivity to laboratory-produced HPV-11 virions of polysera raised against bacterially derived fusion proteins and synthetic peptides of HPV-6b and HPV-16 capsid proteins.

Polysera raised in rabbits to bacterially derived fusion proteins and synthetic peptides of the L1 and L2 ORFs of HPV-6b and -16 were tested for cross-reactivity to laboratory-produced infectious HPV-11 virions. The polysera were analyzed in a series of five different immunological assays including immunoperoxidase staining of the koilocytotic nuclei in sections of formalin-fixed, paraffin-embedded as well as fresh frozen sections of HPV-11 experimental condylomas generated in the athymic nude mouse xenograft system, ELISA, Western blots, and neutralization of infectious HPV-11 virions. ELISA and Western blot assays were used to determine whether the polysera identified external or internal epitopes on HPV-11 virions, and whether there was cross-reactivity to BPV-1 or laboratory-produced CRPV virions. Seven of a total of 12 sera were positive for reactivity to HPV-11 in one or more assays, but none of the reactivity was directed to external epitopes on the intact virions as determined by ELISA. None of the L1 products generated group-specific antigen (GSA) antisera including a synthetic peptide spanning the GSA site. The combination of assays clearly demonstrated that apparent false positive and false negative reactivities of different antisera were obtained for each assay system tested. Thus, no single assay could be used reliably to determine the true antiviral reactivity of a given polysera.

Animals↗
PubMed1990

Comparison of Southern transfer hybridization and dot filter hybridization for detection of cervical human papillomavirus infection with types 6, 11, 16, 18, 31, 33, and 35.

A commercial dot filter hybridization kit (Virapap Kit) was compared with Southern transfer hybridization for the detection of seven types of human papillomavirus (HPV) in cervical specimens from 450 consecutive females attending a sexually transmitted diseases clinic. In comparison with Southern transfer hybridization, performed with the same probes used in the dot filter kit, the sensitivity, specificity, and positive and negative predictive values of dot filter hybridization were 90%, 94%, 74%, and 98%, respectively. Among patients with cervical cytologic dysplasia, HPV DNA was detected in 44% by dot filter hybridization and in 35% by Southern transfer hybridization. Although 26% of specimens positive by dot filter hybridization were not confirmed by Southern transfer hybridization, cervical dysplasia was detected in 5 (25%) of 20 with HPV DNA detected by dot filter hybridization alone, compared with 25 (8%) of those with no definitive evidence of HPV by either method (P = 0.009) and with 16 (30%) of 53 with HPV DNA detected by both methods (P = 0.7). The kappa statistic for interobserver and intraobserver reproducibility for interpretation of blots was similar for the two methods. The dot filter hybridization method evaluated appears to be a satisfactory alternative to Southern transfer hybridization for detection of HPV DNA.

Adolescent↗
PubMed1990

Evidence of prevalent genital-type human papillomavirus infections in adults and children.

Recombinant proteins encoded by the E2, E7, L1, and L2 open reading frames (ORF) of human papillomavirus (HPV) types 6b, 16, and 18 were used in Western blot assays to detect serum IgG antibodies in women attending a sexually transmitted diseases clinic (n = 92) and in hospitalized children (n = 81). Antibodies to late gene products (L1 or L2 ORF) were more common than antibodies to early gene products (E2 or E7), both in the adults and the children; overall, the antibody prevalences in the children and the sexually active adults were not significantly different. Human sera with high titers of antibodies to the HPV16 E7 recombinant protein immunoprecipitated the genuine HPV16 E7 protein from the cervical carcinoma cell line CaSki. As an independent measure of HPV infection, the polymerase chain reaction was used to detect HPV6b and HPV16 in oral mucosal scrapings from adults (n = 35) and preschool children (n = 21). In adults, HPV6b and HPV16 DNA were detected in 17% and 23% of oral mucosal samples, respectively. In preschool children, HPV6b and HPV16 DNA were found in 24% and 19% of oral samples, respectively.

Adolescent↗
PubMed1990

Characterization of the humoral immune response to genital papillomaviruses.

Human papillomaviruses (HPVs) have been detected in a wide range of proliferative lesions of squamous epithelium. A number of HPV types are associated with lesions of the genital tract. Some types (HPV types 6 and 11) are detected frequently in benign genital warts (condylomata acuminata); other types (HPV types 31, 33, 42 and others) are associated with dysplastic lesions of the uterine cervix; and certain HPV types (HPV types 16 and 18) are found in a high proportion of squamous cell carcinomas of the cervix and vulva. However, all of these HPV types have been detected also in normal epithelium. To date, investigators have relied primarily upon the detection of viral DNAs in clinical specimens as evidence of HPV infections. Such assays cannot determine whether past infections with HPVs have occurred which have subsequently resolved. Latent infections and current infections might also evade detection because of sampling errors or because of suboptimal sensitivity of DNA detection methods. Efforts to develop HPV serological assays have been hampered by the lack of appropriate viral antigens, since HPVs cannot be propagated in tissue culture and virions are not abundant in infected human tissues. Using HPV-encoded proteins expressed in Escherichia coli, we developed assays to measure human antibodies that react with HPV proteins. Human antibodies to late gene products (L1 and L2) of genital-type HPVs were more prevalent than antibodies to early gene products. However, approximately 15% of sera contained antibodies that reacted with the HPV16 E7-encoded protein, a gene product that has been implicated in HPV16-mediated cellular transformation. The human antibodies appeared to be type or "serotype" specific, because the antibodies did not cross-react with homologous proteins encoded by other HPV types. Antibodies to proteins encoded by HPV types 6 or 11 were detected in approximately 70% of adults, while antibodies to proteins encoded by HPV type 16 were found in approximately 50% of adults. Antibody prevalence was not associated with measures of sexual activity. There was also no significant difference between the prevalence of antibodies to HPV types 6 or 16 proteins in children when compared to the antibody prevalence in sexually active adults. These results suggest that infections by genital HPV types are widespread and frequently cause clinically inapparent infections. The viruses have a broad tropism for mucosal epithelium and are likely to be acquired by other modes, as well as by sexual transmission.

Antibodies, Viral↗
PubMed1989

Human papillomaviruses and carcinomas.

The recognition of multiple types of human papillomaviruses has resulted in remarkable progress in the detection of persisting viral nucleic acid sequences in carcinomas. The consistent transcription in tumors of two early open reading frames, E6 and E7, with few exceptions (Lehn et al., 1985), indicates a role for the products of these genes in the induction and/or maintenance of the transformed state. A number of studies have shown that in vitro transformation can be achieved by transfection of E6/E7 DNA, and proteins encoded by these DNA sequences can be demonstrated in primary human keratinocytes immortalized by this DNA (Kaur et al., 1989). Mutagenesis experiments are needed to determine the absolute requirement for and function of these genes in transformation. A preferential association of some types with benign lesions while others may be frequently found in malignant tumors has been observed. HPV types 5 and 8 in epidermodysplasia verruciformis patients and types 16, 18, 31, 33, etc. in genital lesions are most frequently associated with progression to malignancy, whereas other types, such as HPV-6,-10, -11, and -20, are regularly identified in benign warts. Such distinctions are not absolute but provide the initial steps toward establishing a causal role for some human papillomaviruses in carcinomas. The need for well-designed epidemiological studies in concert with optimum molecular and serologic evaluations is evident (Armstrong et al., 1988). The data from human and animal studies indicate that papillomaviruses contribute significantly to the development of many, if not all, carcinomas, but we do not yet have a clear understanding of the importance of other interacting viral, chemical, or cellular factors. The application of gene cloning and non-stringent hybridization (Law et al., 1979) has provided us with an apparently ever-increasing catalog of human papillomaviruses. More effort is now required to establish their prevalence, the natural history of infection, and the mechanism of neoplastic transformation.

Animals↗
PubMed1989

Prevalence of genital papillomavirus infection among women attending a college student health clinic or a sexually transmitted disease clinic.

We examined 454 women randomly selected from a Sexually Transmitted Disease (STD) Clinic and 545 consecutive college women undergoing annual examination. Patients were examined for visible genital warts, koilocytes, and human papillomavirus (HPV) antigen on cervical smears and for cervical HPV DNA types 6, 11, 16, 18, and 31. Genital warts were found in 11% of STD Clinic patients and 2% of students (P less than .001). Among those women without genital warts, HPV DNA or antigen was detected in cervical specimens from 10.6% of STD Clinic patients and 11.4% of students (P = .73), with HPV type 16, 18, or 31 being 9.8 times more frequent than cervical HPV 6 or 11 among students and 5.8 times more frequent among STD Clinic patients. Dysplasia was present in 53% of those with HPV type 6 or 11 and in 41% of those with HPV type 16, 18, or 31 DNA.

Adult↗
PubMed1989

Human antibodies react with an epitope of the human papillomavirus type 6b L1 open reading frame which is distinct from the type-common epitope.

Recombinant proteins encoded by the human papillomavirus type 6b (HPV6b) L1 open reading frame react with sera from patients with condylomata acuminata and also react with rabbit antiserum raised against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV1) virions. To map the immunoreactive epitopes, a series of procaryotic expression plasmids was made which contained a nested set of 3' to 5' deletions in the HPV6b L1 open reading frame. The deleted plasmids expressed a set of carboxy to amino terminus truncated fusion proteins. Regions containing the immunoreactive epitopes were mapped by determining which of the deleted fusion proteins retained reactivity with sera in Western immunoblot assays. The coding sequence for a human antibody-reactive linear epitope mapped between HPV6b nucleotide coordinates 7045 and 7087, and the rabbit anti-BPV1-reactive epitope coding sequence mapped between coordinates 6377 and 6454. Synthetic peptides derived from the epitope mapping were reacted with sera in enzyme-linked immunosorbent assay. Human sera reacted with synthetic peptide QSQAITCQKPTPEKEKPDPYK (HPV6b L1 amino acids 417 through 437). Rabbit anti-BPV1 and rabbit antisera raised against HPV16 L1 recombinant proteins reacted with the synthetic peptide DGDMVDTGFGAMNFADLQTNKSDVPIDI (HPV6b L1 amino acids 193 through 220). Human sera which reacted with HPV6b L1 fusion proteins cross-reacted with an HPV11 L1 fusion protein but did not react with fusion proteins encoded by HPV1a, HPV16, or HPV18. Rabbit anti-BPV1 reacted with L1 fusion proteins encoded by all of these HPV types. In contrast to the type-common (rabbit anti-BPV1-reactive) epitope, the human antibody-reactive epitope appears to be relatively HPV type specific.

Amino Acid Sequence↗
PubMed1989

The E7 protein of human papillomavirus type 16 is phosphorylated by casein kinase II.

The E7 protein of human papillomavirus type 16 (HPV16) transforms cultured cells and cooperates with the ras or fos oncogenes in the transformation of primary cells. In this study we have investigated the phosphorylation of E7. When we immunoprecipitated E7 from CaSki cells with a rabbit polyclonal antiserum to a bacterial fusion protein (trpE-E7), we found that E7 was phosphorylated at serine residues contained in five characteristic thermolysin peptides. Immunoprecipitated E7, and fusion proteins harboring the E7 protein from various HPV types, could all be specifically phosphorylated in vitro by the ubiquitous, growth factor-activated casein kinase II (CKII). Comparative peptide mapping showed that the sites of in vivo and in vitro phosphorylation are the same. CKII was shown previously to specifically phosphorylate serine or threonine residues within a cluster of acidic amino acids. The E7 protein contains such a sequence between amino acids 30 and 37. When a synthetic peptide corresponding to this region of E7 was phosphorylated by CKII in vitro, its thermolysin digestion products were the same as those in the phosphorylated E7 protein. We conclude that E7 is phosphorylated in vivo only at serines within the predicted CKII site and that CKII, or a CKII-like enzyme, participates in the reaction. Both the E1A and SV40 large T proteins contain similar CKII consensus sites proximal to the regions required for their associations with the retinoblastoma gene product (p105Rb). Thus it is conceivable that CKII phosphorylation can modulate the interaction between the transforming proteins and the retinoblastoma gene product.

Amino Acid Sequence↗
PubMed1988

Detection of human papillomavirus capsid antigens in various squamous epithelial lesions using antibodies directed against the L1 and L2 open reading frames.

HPV6 and HPV16 infect the squamous epithelium of the genital tract and are thought to be involved in the pathogenesis of benign and malignant lesions. HPV6 is primarily found in benign condylomas whereas HPV16 is present in dysplasias and in invasive squamous cell carcinomas. To examine the expression of the major and minor capsid proteins in these lesions polyclonal antisera directed against bacterially derived fusion proteins harboring different restriction fragments of the L1 and L2 ORFs of HPV6b and HPV16 were generated. L1 ORF-specific antisera were not type-specific and detected the major capsid antigen in lesions infected with related HPV types. Anti-L2 ORF antisera could distinguish among HPV1, HPV6, and HPV16 when the fusion protein used as the immunogen did not harbor the amino-terminus of the L2 ORF. The anti-L1 ORF antisera were employed to detect the major capsid protein in various lesions by immunohistochemical staining. Lesions harboring HPV16 were positive in a high percentage of cervical intraepithelial neoplasia I-II (87%), and less frequently in carcinomas in situ (29%) or invasive carcinomas (17%). In all cases capsid antigen expression was restricted to cells showing some differentiation at the surface or periphery of the lesion.

Antigens, Viral↗
PubMed1988

Identification of the E5 open reading frame of human papillomavirus type 16.

Sequencing of the E5 open reading frame (ORF) of human papillomavirus type 16 revealed an additional nucleotide, a thymidine residue, at position 3903 compared with the original sequence (Seedorf et al., Virology 145:181-185, 1985). The additional T had two effects; first, in reading frame 2, in which the original E5 ORF was predicted, the additional T changed the reading frame downstream of position 3903 to create an ORF, which we designated E5, that terminated at position 4018 and potentially encoded a 52-amino-acid polypeptide. Secondly, in reading frame 3, a new ORF was created (positions 3807 to 4097), which we propose is the authentic papillomavirus type 16 E5 ORF. It contained a methionine residue and encoded an additional 82 amino acids. Both ORFs have been cloned into bacterial expression vectors (pATH), and the fusion proteins have been used to generate polyclonal antibodies in rabbits.

Amino Acid Sequence↗
PubMed1988

Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins.

Human papillomavirus (HPVs) infect the genital epithelium and are found in proliferative lesions ranging from benign condylomata to invasive carcinomas. The immunological response to these infections is poorly understood because of the lack of purified viral antigens. In this study, bacterially derived fusion proteins expressing segments of all the major open reading frames (ORFs) of HPV type 6b (HPV-6b) have been used in Western blot (immunoblot) assays to detect antibodies directed against HPV-encoded proteins. The most striking reactivities present in sera from patients with genital warts were to the HPV-6b L1 ORF protein and, to a lesser extent, to the HPV-6b L2 ORF protein. Two cases of reactivity to HPV-6b E2 ORF were observed, but no reactivities were seen with other HPV-6b constructs. Two sera reacted with the HPV-16 L2 fusion protein, and two sera reacted with the HPV-16 E4 protein. The antibodies directed against the HPV-6b fusion proteins showed no cross-reactivity with comparable regions of the HPV-16 ORFs. This assay provides a useful approach for further studies of HPV serology.

Antibodies, Viral↗
PubMed1988

Sequence and structural requirements of a herpes simplex viral DNA replication origin.

Herpes simplex virus (HSV) types 1 and 2 contain two classes of origins of DNA replication, oriS and oriL, which are closely related. A series of plasmids was constructed which contained specifically altered versions of the HSV type 2 oriS replication origin. Their ability to replicate in an in vivo replicon assay allowed a core origin of 75 base pairs (bp) to be defined. It included both arms of a 56-bp palindrome and from 13 to 20 bp of sequence leftward of the palindrome. The AT-rich sequence at the center of the palindrome was essential. Sequences on either side of the core origin enhanced replication. When additional copies of the -AT-dinucleotide were introduced progressively into the center of the palindrome, an oscillating effect on origin function was observed. These and other data implicate a linear rather than a cruciform conformation of the oriS palindrome in the initiation of HSV replication.

Base Sequence↗
PubMed1987

Plasmid mediated mutagenesis of a cellular gene in transfected eukaryotic cells.

NIH3T3 cells are widely used in transformation assays and readily take up transfected DNA. A system has been devised using NIH3T3 cells to measure the mutagenic effect of transfected DNA on recipient cell genes. NIH3T3 cells can be mutated to 6-thioguanine resistance at a frequency which suggests that at least a portion of the cells have only one functional copy of the HGPRT gene. They have a low spontaneous background mutation frequency (approximately 1 X 10(-7)). Transfection of three different plasmids into NIH3T3 cells induced 6-thioguanine resistant mutants at frequencies ranging from 3 to 11 fold above background. The mutant phenotype is stable and reversion frequencies of several mutants are less than or equal to 1 X 10(-7). Southern blot analysis of the HGPRT gene in several mutants showed that 4 of 26 mutants (15.4%) had detectable alterations in the structure of the HGPRT gene. Interestingly 3 of the 4 mutants showing rearrangements were obtained by transfection of the HSV-2 morphological transforming region.

Animals↗