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D A Galloway

Publications and source records attributed to D A Galloway.

At least 73 records · Page 4Linked to original sources
PubMed1994

The E6 protein of human papillomavirus type 16 functions as a transcriptional repressor in a mechanism independent of the tumor suppressor protein, p53.

The E6 protein of human papillomavirus (HPV) type 16 displays a number of activities when transfected into cultured cells, including transcriptional activation of several viral promoters and targeting of p53 for degradation. HPV 16E6 was found to function as a transcriptional repressor of the moloney murine leukemia virus long terminal repeat and the cytomegalovirus immediate early promoter. Although the degree of transcriptional repression was low, a dose-dependent two- to threefold decrease in promoter activity was consistently seen in cells expressing 16E6. HPV 16E6-dependent transcriptional repression was observed in C33a cells, which express mutant p53, and in Saos-2 cells, which lack p53. These results indicate that 16E6-dependent repression of promoter activity is unlikely to be mediated by p53.

Adenoviruses, Human↗
PubMed1994

Three-dimensional structure of vaccinia virus-produced human papillomavirus type 1 capsids.

The capsid proteins of papillomavirus self-assemble to form empty capsids or virus-like particles that appear quite similar to naturally occurring virions by conventional electron microscopy. To characterize such virus-like particles more fully, cryoelectron microscopy and image analysis techniques were used to generate three-dimensional reconstructions of capsids produced by vaccinia virus recombinants (V capsids) that expressed human papillomavirus type 1 L1 protein only or both L1 and L2 proteins. All V capsids had 72 pentameric capsomers arranged on a T = 7 icosahedral lattice. Each particle (approximately 60 nm in diameter) consisted of an approximately 2-nm-thick shell of protein with a radius of 22 nm with capsomers that extend approximately 6 nm from the shell. At a resolution of 3.5 nm, both V capsid structures appear identical to the capsid structure of native human papillomavirus type 1 (T. S. Baker, W. W. Newcomb, N. H. Olson, L. M. Cowsert, C. Olson, and J. C. Brown, Biophys. J. 60:1445-1456, 1991), thus implying that expressed and native capsids are structurally equivalent.

Capsid↗
PubMed1994

The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest.

Functional p53 protein is associated with the ability of cells to arrest in G1 after DNA damage. The E6 protein of cancer-associated human papillomavirus type 16 (HPV-16) binds to p53 and targets its degradation through the ubiquitin pathway. To determine whether the ability of E6 to interact with p53 leads to a disruption of cell cycle control, mutated E6 proteins were tested for p53 binding and p53 degradation targeting in vitro, the ability to reduce intracellular p53 levels in vivo, and the ability to abrogate actinomycin D-induced growth arrest in human keratinocytes. Mutations scattered throughout the amino terminus, either zinc finger or the central region but not the carboxy terminus, severely reduced the ability of E6 to interact with p53. Expression of HPV-16 E6 or mutated E6 proteins that bound and targeted p53 for degradation in vitro sharply reduced the level of intracellular p53 induced by actinomycin D in human keratinocytes. A perfect correlation between the ability of E6 proteins to reduce the level of intracellular p53 and their ability to block actinomycin D-induced cellular growth arrest was observed. These results suggest that interaction with p53 is important for the ability of HPV E6 proteins to circumvent growth arrest.

Amino Acid Sequence↗
PubMed1994

Human papillomavirus vaccines: a warty problem.

Infection of the genital tract with human papillomaviruses (HPVs) is a common occurrence, and manifestations can include genital warts (condyloma acuminata), dysplasia, and invasive cancer. Approaches to diagnose and treat HPV infections are costly and are not fully effective. Even in populations at low risk for sexually transmitted diseases (STDs), HPV infection is severalfold more prevalent than all other STDs combined, and worldwide, uterine cervical cancer remains the most common cancer in women. It should be feasible to develop prophylactic vaccines to prevent HPV infection using the L1 and L2 capsid proteins or therapeutic vaccines to modulate the development or recurrence of disease based on the E6 and E7 oncoproteins or other viral proteins. In favor of success is (a) the relative simplicity of the HPV genome (only two proteins in the viral coat, and a small number of other genes), (b) the lack of genetic variability within types and stability of the genome, and (c) the encouraging results with vaccines against animal PVs. However, it is difficult to provide evidence of the efficacy of HPV vaccines because of the inability to propagate the virus in culture or in animal models and because of the incomplete understanding of the natural history of HPV infection.

Female↗
PubMed1993

Metastatic conversion of cells by expression of human papillomavirus type 16 E6 and E7 genes.

The human papillomavirus type 16 (HPV-16) is a DNA tumor virus highly associated with cervical carcinoma. Viral DNA from HPV-16 is found in primary tumors and their metastatic lesions. To investigate the role of HPV-16 oncoproteins in the development of cancer metastasis, the E6 and E7 genes from HPV-16 were inserted into retrovirus and introduced into nonmetastatic mouse cell lines. Expression of either of the viral genes from HPV-16 made the cells metastatic in nude mice. In contrast, expression of the E6 and E7 genes of HPV type 6 (HPV-6b), which is frequently found in nonmalignant HPV-associated diseases, did not. The metastatic ability of cells transduced with viral genes of HPV-16 did not correlate with their growth rate or sensitivity to destruction by natural killer cells. Our results demonstrate that expression of oncogenic proteins of HPV-16 can cause tumor metastasis and implicate HPV-16 in an important role regarding the progression of HPV-associated human cancers.

Animals↗
PubMed1993

HPV-1 capsids expressed in vitro detect human serum antibodies associated with foot warts.

Seventy-eight human serum samples were screened for their ability to immunoprecipitate the major (L1) and minor (L2) capsid proteins of HPV1. The L1 and L2 proteins expressed from a recombinant vaccinia virus were able to self assemble into capsids in the nuclei of infected cells. Twenty-eight of the sera precipitated the L1 protein. The L1 protein was only precipitated when the protein was native, denatured protein was not precipitated by the human sera. None of the sera precipitated the L2 protein. The assay demonstrated a significant association between the ability of sera to precipitate the L1 protein and a clinical history of foot warts (P = 0.001). The same serum samples were tested by immunoblots using L1 and L2-trpE bacterial fusion proteins. It was found that almost half of the sera reacted with the L2 fusion protein and few reacted with the L1 protein. Immunoblot results did not correlate well with a clinical history of foot warts (P = 0.7), suggesting that immune precipitation of capsid proteins may be superior to immunoblotting for serodiagnosis of HPV infections.

Adolescent↗
PubMed1993

Sequence variation in the noncoding region of human papillomavirus type 16 detected by single-strand conformation polymorphism analysis.

Human papillomavirus (HPV) type 16 variants were found by single-strand conformation polymorphism (SSCP) analysis of the noncoding region of the viral genome. Two sets of primers were used to analyze a 1018 bp region spanning nucleotides 7109-222. Twelve SSCP patterns were demonstrated among HPV 16 DNAs purified from 48 anal specimens from 24 homosexual men. Seven patterns were detected among 10 HPV 16 isolates from cervical carcinomas. In two pairs of sex partners, identical variants were recognized in each partner of the pair. Infection with two variants of HPV 16 from 2 specimens was observed in 1 of 21 subjects for whom there were multiple samples over time. DNA sequence analysis of 7 isolates confirmed that the SSCP technique showed polymorphisms only in fragments that had base substitutions and that all of these base substitutions resulted in detectable shifts in fragment mobility.

Anal Canal↗
PubMed1993

Sequence and antigenic diversity in two immunodominant regions of the L2 protein of human papillomavirus types 6 and 16.

To assess the extent of sequence and antigenic diversity in the minor capsid proteins (L2) of human papillomavirus (HPV) types 6 and 16, 24 clinical samples were obtained, and the regions encoding the immunodominant epitopes 6U3 and 16REx were amplified by polymerase chain reaction, sequenced, cloned into pATH plasmids, and tested for reactivity with human sera. Two of 11 HPV-6 DNAs were identical to the prototype strain in the 6U3 region, while 9 variants had a G to A transition at nt5020, changing a valine residue to isoleucine. Of 16 sera that did not react with the prototype HPV-6L2 fusion protein, 2 reacted with the 6U3-isoleucine variant, and all 8 sera that reacted with the prototype also reacted with the variant. Twelve of 13 HPV-16 DNAs were identical to the prototype strain in the 16REx region, while 1 variant had a C to G transversion at nt4825, changing a proline to an arginine, but not affecting antigenicity.

Amino Acid Sequence↗
PubMed1993

Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins.

Vaccinia virus vectors were used to express the major (L1) and minor (L2) capsid proteins of human papillomavirus type 1 (HPV-1) with the vaccinia virus early (p7.5K) or late (pSynth, p11K) promoters. All constructs expressed the appropriate-sized HPV proteins, and both L1 and L2, singly or in combination, localized to the nucleus. Capsids were purified by cesium chloride density gradient centrifugation from nuclei of cells infected with a vaccinia virus-L1 (vac-L1) recombinant or a vac-L1-L2 recombinant but not from vac-L2-infected cells. Electron microscopy showed that the particles were 55 nm in diameter and had icosahedral symmetry. Immunogold-labeled antibodies confirmed the presence of the L1 and L2 proteins in the HPV-1 capsids. Capsids containing L1 alone were fewer and more variable in size and shape than capsids containing the L1 and L2 proteins. The L1-plus-L2 capsids were indistinguishable in appearance from HPV-1 virions obtained from plantar warts. The ability to produce HPV capsids in vitro will be useful in many studies of HPV pathogenicity.

Base Sequence↗
PubMed1992

A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection.

BACKGROUND: Human papillomavirus (HPV) has been associated with cervical intraepithelial neoplasia, but the temporal relation between the infection and the neoplasia remains unclear, as does the relative importance of the specific type of HPV, other sexually transmitted diseases, and other risk factors. METHODS: We studied prospectively a cohort of 241 women who presented for evaluation of sexually transmitted disease and had negative cervical cytologic tests. The women were followed every four months with cytologic and colposcopic examinations of the uterine cervix and tests for HPV DNA and other sexually transmitted diseases. RESULTS: Cervical intraepithelial neoplasia grade 2 or 3 was confirmed by biopsy in 28 women. On the basis of survival analysis, the cumulative incidence of cervical intraepithelial neoplasia at two years was 28 percent among women with a positive test for HPV and 3 percent among those without detectable HPV DNA: The risk was highest among those with HPV type 16 or 18 infection (adjusted relative risk as compared with that in women without HPV infection, 11; 95 percent confidence interval, 4.6 to 26; attributable risk, 52 percent). All 24 cases of cervical intraepithelial neoplasia grade 2 or 3 among HPV-positive women were detected within 24 months after the first positive test for HPV. After adjustment for the presence of HPV infection, the development of cervical intraepithelial neoplasia was also associated with younger age at first intercourse, the presence of serum antibodies to Chlamydia trachomatis, the presence of serum antibodies to cytomegalovirus, and cervical infection with Neisseria gonorrhoeae. CONCLUSIONS: Cervical intraepithelial neoplasia is a common and apparently early manifestation of cervical infection by HPV, particularly types 16 and 18.

Adolescent↗
PubMed1992

Induction of cytotoxic T lymphocytes specific for a syngeneic tumor expressing the E6 oncoprotein of human papillomavirus type 16.

Human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical carcinomas, but it is unknown whether HPV-specific immunity can function in controlling the growth of HPV-associated carcinomas. We previously demonstrated that CD8+ T lymphocytes can inhibit the in vivo outgrowth of murine tumor cells transfected with the HPV-16 E7 gene and have now established a murine model to study the CTL responses to the E6 oncoprotein of HPV-16. Immunization of C3H/HeN mice with syngeneic fibroblasts expressing a transfected HPV-16 E6 gene induced regression of transplanted tumors expressing this gene. Populations of CTL isolated from the spleens of mice whose E6+ tumors had regressed were shown to specifically lyse E6+ target cells. The cytolytic activity was mediated by CD8+ CTL in a MHC restricted pattern. These data and our previous findings with transfected tumor cells expressing the E7 gene, support the conclusion that tumor cells associated with HPV-16 can be inhibited by CTL specific for molecules encoded by the HPV-16 E6 and E7 genes.

Animals↗
PubMed1992

Human antibodies recognize multiple distinct type-specific and cross-reactive regions of the minor capsid proteins of human papillomavirus types 6 and 11.

Human serum samples derived from a case-control study of patients with cervical carcinoma (n = 174) or condyloma acuminatum (n = 25) were tested for the presence of immunoglobulin G antibodies to human papillomavirus type 6 (HPV6) L2 and HPV11 L2 recombinant proteins in a Western immunoblot assay. Thirty-six samples (18%) were positive for HPV6 L2 antibodies alone, 25 (13%) were positive for HPV11 L2 antibodies alone, and 34 (17%) were positive for both HPV6 L2 and HPV11 L2 antibodies. Thirty samples that were positive for both antibodies were tested for the presence of HPV6-HPV11 L2 cross-reactive antibodies. Fifteen (50%) serum samples contained HPV6-HPV11 L2 cross-reactive antibodies, and 15 (50%) contained independent, type-specific HPV6 L2 and HPV11 L2 antibodies. Altogether, 82% of the HPV6 L2 and HPV11 L2 antibody reactivities were type specific and 18% were HPV6-HPV11 cross-reactive. There was no significant difference in the prevalence of antibody reactivities between samples from patients with cervical carcinoma and those with condyloma acuminatum. Deletion mapping identified five HPV6 L2 regions that reacted with HPV6 type-specific antibodies: 6U1 (amino acids [aa] 152 to 173), 6U2 (aa 175 to 191), 6U3 (aa 187 to 199), 6U4 (aa 201 to 217), and 6U5 (aa 351 to 367). Five HPV11 L2 regions that reacted with HPV11 type-specific antibodies were identified: 11U1 (aa 49 to 84), 11U2 (aa 147 to 162), 11U3 (aa 179 to 188), 11U4 (aa 180 to 200), and 11U5 (aa 355 to 367). Two HPV6-HPV11 cross-reactive regions were identified: 6CR1 (HPV6 L2 aa 106 to 128)/11CR1 (HPV11 L2 aa 103 to 127) and 6CR2 (HPV6 L2 aa 187 to 199)/11CR2 (HPV11 L2 aa 180 to 200).

Amino Acid Sequence↗
PubMed1992

The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.

Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation.

3T3 Cells↗
PubMed1992

Serological assays for the detection of HPV antibodies.

A variety of serological assays to detect antibodies to genital-type HPVs have been developed. Bacterially expressed fusion proteins, synthetic peptides and HPV 11 virus propagated in a xenograft system have been the most commonly used antigen targets in either Western blot assays or ELISAs. HPV antibodies have been readily detected and most studies suggest that they are type-specific. Primarily, antibodies appear to be directed against the capsid antigens. The presence or titre of antibodies to the HPV 16 E7 protein is strongly associated with cervical cancer in approximately 25% of cases. The significance of antibodies to other HPV antigens, or of antibodies which recognize conformational epitopes is less clear. Attempts to validate the sensitivity and specificity of serological assays are extremely preliminary, and are complicated by a lack of understanding of the natural history of papillomavirus infections.

Antibodies, Viral↗
PubMed1991

Expression of human papillomavirus proteins in yeast Saccharomyces cerevisiae.

The L1 and L2 proteins of human papillomavirus (HPV) types 1, 6, and 16 and the E6 and E7 proteins of HPV 16 were expressed in Saccharomyces cerevisiae. The yeast expressed proteins were readily detected by immune blotting and were generally intact. The HPV 1 L1 and L2 proteins expressed in yeast were indistinguishable from the major and minor capsid proteins purified from HPV 1 virions as judged by gel electrophoresis and immunoblotting. The HPV 6 and HPV 16 L2 proteins and HPV 16 E7 proteins were secreted from yeast by fusion to the yeast pre-pro-alpha-factor leader sequence. Following secretion of the HPV 16 E7 protein a rapid method of purification was developed. The yeast expressed proteins were used as antigen targets to study the human immune response in Western blot assay, ELISA, and immune precipitation. One human serum reacted with intact, but not denatured HPV 16 L2 proteins, suggesting that the yeast expressed proteins will be useful to detect antibodies reactive with conformational epitopes.

Antibodies, Viral↗
PubMed1991

Comparative virologic studies of condylomata acuminata reveal a lack of dual infections with human papillomaviruses.

Condylomata acuminata are epithelial proliferations caused by infection of the anogenital squamous epithelium with human papillomavirus (HPV). DNA-DNA hybridization techniques and the extremely sensitive polymerase chain reaction (PCR) were used to analyze biopsies from patients with clinically diagnosed condyloma acuminatum for the presence of HPV DNA. PCR analyses using primers and oligonucleotide probes specific for the E6/E7 region of HPV-6, -11, or -16 showed that 31 (93.9%) of 33 tissue biopsies contained HPV DNA: 22 contained type 6 DNA, 6 contained type 11 DNA, and 3 contained type 16 DNA. Eleven biopsies positive by PCR were Southern hybridization-negative or were considered inadequate for Southern analysis. In all 11, the presence of HPV DNA was corroborated by the observation of histopathologic evidence suggestive of HPV infection or by in situ hybridization. No evidence of multiple infections with HPV-6 or -11 and HPV-16 was seen.

Adult↗