Transcriptional regulation of the adenovirus E1A gene.
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Adenovirus E1A Proteins↗
Biomedical subjects
K Fujinaga
Publications and source records attributed to K Fujinaga.
Immunohistochemical localization of the calcium/calmodulin-dependent protein phosphatase, calcineurin, in the mouse testis: its unique accumulation in spermatid nuclei.
Immunohistochemical localization of a calmodulin-dependent protein phosphatase, calcineurin, was studied in the mouse testis in relation to previous observations showing that calmodulin is unusually rich in spermatogenic stages from mid-pachytene spermatocytes to elongating spermatids. The antibodies raised against calcineurin from scallop testis reacted with subunit B, but not subunit A, of calcineurin isoforms from mouse brain and testis. Indirect immunofluorescence using these antibodies on the mouse testis revealed positive reactions only in the nuclei of round or elongating spermatids: calcineurin started to accumulate in nuclei from the acrosomal cap phase, peaked at the initial stage of nuclear elongation, and decreased thereafter. There was almost no signal in the cytoplasm; spermatogenic cells at other stages, including spermatogonia, spermatocytes, mature sperm, and other somatic cells in the seminiferous tubules were totally negative. Immuno-electron microscopy gave the same result, on the basis of measuring the density of immunogold particles. These results suggest a role for calcineurin in remodeling of the nuclear chromatin in metamorphosing spermatids.
Animals↗
Biologic activity of human papillomavirus type 16 E6/E7 cDNA clones isolated from SiHa cervical carcinoma cell line.
Three species of E6/E7 cDNAs of human papillomavirus type 16 (HPV16) for the full-length E6/E7 and spliced E6*I/E7 and E6*II/E7 mRNAs were synthesized by reverse transcriptase-(RT-)PCR from RNA of the cervical carcinoma cell line SiHa. Two cDNA mutants carrying point mutations in either a splice donor site or acceptor site within the E6 open reading frame were also constructed. These HPV16 E6/E7 cDNAs were cloned under the SV40 enhancer/promoter and the MMTV LTR to examine the activities of ras-collaborative transformation and induction of cellular DNA synthesis, both of which depend on the E7 gene product. The E6*II/E7 cDNA and two mutated cDNAs deficient in the spliced mRNA transcription showed lower levels of both activities than the full-length E6/E7 and the E6*I/E7 cDNA. The rat cell lines carrying each of the E6/E7 cDNAs contained the E6/E7 mRNA species expected. A small amount of E6*I/E7-sized mRNA was transcribed from a splice-donor site mutant of the E6/E7 cDNA, which turned out to be a transcript derived from a cryptic splice donor site six bases upstream from the conventional site. Among NIH3T3 cells carrying one of the above-mentioned E6/E7 cDNAs, the cells expressing E6*I/E7 mRNA [cells carrying cF(wt) and c*I] produced an amount of E7 protein comparable with those carrying the E7 or E6E7 region. These results suggest that the E6*I/E7 is the mRNA that is important for the efficient expression of E7 product from the HPV16 E6/E7 region.
3T3 Cells↗
Human papillomavirus type 18 DNA and E6-E7 mRNA are detected in squamous cell carcinoma and adenocarcinoma of the lung.
To provide an accurate evaluation of the association of human papillomavirus (HPV) with lung cancer, 36 cases of lung cancer were analysed for HPV DNAs by polymerase chain reaction (PCR) with dot-blot and Southern blot analyses, and for the transcripts from the E6-E7 transforming region by in situ hybridisation (ISH). HPV-18 DNA was detected in three (8%) of 36 specimens; histologically, in one (10%) of 10 squamous cell carcinomas and two (9%) of 22 adenocarcinomas. Neither HPV-16 nor -33 DNA was detected in any cases examined. Expression of E6-E7 mRNA was confirmed in the cases which contained, HPV-18 DNA. HPV-18 may play an important role in the development and progression of cancer in some cases of both squamous cell carcinoma and adenocarcinoma of the lung.
Adenocarcinoma↗
Variation of human papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world.
We examined the genomic diversity of human papillomavirus type 6 (HPV-6) and HPV-11 isolates from different parts of the world by comparing the nucleotide sequences of part of the long control region of three reference clones and 62 HPV-6 and 40 HPV-11 isolates from Africa, Europe, Asia, and North and South America. The genomic sequence of the HPV-6b reference type had to be amended by inclusion of a 94-bp segment, which is also present with minor differences in HPV-6a. Aside from two small inserts typical of all variants related to HPV-6a and three inserts found in HPV-11 variants, no major alterations to the size of the long control regions of these viruses were observed. This corrects the previous impression that these two HPV types are highly polymorphic. Altogether, 19 HPV-6 and 10 HPV-11 variant genomes could be distinguished, and most of the differences were due to point substitutions. The variants of either type were continuously connected in phylogenetic trees rather than clustered separately into subtype groups. Thirteen mutations, namely, the two HPV-6a inserts and 11 substitutions in HPV-6 or HPV-11 variants, reduced the dissimilarity between the types, but they bridged only a small fraction of the genetic distance between the two types. Genomes more obviously intermediate between HPV-6 and HPV-11 were not found and probably do not exist any more. A single HPV-11 variant was found in Africa, but otherwise, no significant correlations of HPV-6 or HPV-11 variants with geography or ethnicity of the patient cohort were found. Functional analysis of diverse enhancer variants showed activities that differed two-to threefold, and it must be considered that transcriptional differences may alter the biology or pathology of these viruses. Similar variants were found in lesions from anatomically different sites and in both benign and malignant lesions.
Base Sequence↗
[Overview: gene expression and gene therapy].
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Gene Expression↗
The carboxyl-terminal region of HIV-1 Nef protein is a cell surface domain that can interact with CD4+ T cells.
Our previous studies have shown that the HIV-1 Nef Ag is expressed, at least in part, on the surface of infected cells. We demonstrated this by using membrane immunofluorescence and flow cytometry with Nef murine mAbs. To identify the domain of Nef exposed on the cell surface, epitope mapping of these and a new mAb was performed by ELISAs by using several recombinant truncated Nef fusion proteins and synthetic peptides. The results showed that mAbs F1, E7, E9, and 4H4 recognized Nef epitopes located at amino acid residues 148-157, 192-206, 158-206, and 1-33, respectively. The intensity of cell surface Nef staining was stronger with mAbs E7 and E9 than with F1, and there was no staining by 4H4, which indicates that the carboxyl-terminal region of Nef is predominantly exposed on the surface of HIV-1-infected T cell lines and PBMC. This surface Nef domain displayed high affinity for the surface of uninfected CD4+ T cells, because the binding of a soluble form of recombinant Nef protein to the cell surface was specifically blocked by the E7 and E9 mAbs or by synthetic peptides that contained the carboxyl-terminal region of Nef. In addition, syncytium formation between infected and uninfected cells also was specifically reduced by the same mAbs or peptides. Thus, the cell surface domain of Nef seems to play an important role in the interaction between HIV-1-infected and CD4+ uninfected T cells.
Antibodies, Monoclonal↗
Nonsense mutations in the vpr gene of HIV-1 during in vitro virus passage and in HIV-1 carrier-derived peripheral blood mononuclear cells.
Long-term, persistent infection by HIV-1 is a prerequisite for the development of AIDS. However, little is known of the determinants required for HIV-1 to cause persistence. We have reported previously that persistent infection of a T cell line by a cytopathogenic strain of HIV-1 became increasingly likely with in vitro serial passage of the virus. DNA sequencing of the persistent strains revealed a nonsense mutation in the vpr gene in all isolates tested. Here, we report the development and use of a semi-quantitative PCR method to detect the vpr nonsense mutation within populations of virus. Our results show that vpr mutants also arise in cells during acute infection and increase progressively with serial passage of the virus. In addition, HIV-1-seropositive individuals were examined and found to carry the same vpr nonsense mutation at high frequency in virus-infected PBMC. These data are consistent with a mechanism of HIV-1 persistence in vivo and in vitro in which virus cytopathogenic potential is lost by the build up of nonsense mutations in vpr.
AIDS-Related Complex↗
Identification of functional domains of adenovirus tumor-specific transplantation antigen in types 5 and 12 by viable viruses carrying chimeric E1A genes.
The adenovirus (Ad) E1A gene induces in immunized animals a strong tumor transplantation (TSTA) immunity against Ad tumors. Such immunity with group-A and group-C viruses is highly group-specific and no cross-protection is detected between serotypes 5 and 12. This fact was used to map the domains of the Ad5 and Ad12 E1A gene products, respectively, which control the TSTA. We constructed a library of 8 recombinant viruses (H5sub1101 through H5sub1108) which carry chimeric Ad5/Ad12 E1A genes in the background of Ad5. The chimeric genes are functional and these viruses are viable. Some of these constructs induce strong and highly specific tumor syngraft immunity in immunized rats. The viruses carrying the 5' terminus of the first E1A exon derived from Ad12 (viruses H5sub1101, H5sub1102 and H5sub1103) induce strong protection against Ad12 tumors irrespective of the rest of their E1A sequence. The viruses which carry the second exon of the Ad5 E1A gene (viruses H5sub1101, H5sub1102 and H5sub1106) protect against group-C tumors, regardless of the origin of the rest of their E1A gene. The 2 viruses that carry the 5' E1A terminus of the first exon of Ad12 and the second exon of Ad5 (H5sub1101 and H5sub1102) are thus effective in inducing immunity against Ad12 tumors as well as against Ad2 tumors. The viruses which carry the 5' terminus of the first exon derived from Ad5 and the second exon of Ad12 (H5sub1107 and H5sub 1108) fail to induce immunity against either tumor. Expression of only the truncated 5' terminus of the Ad12 E1A gene (viruses H5sub1104 and H5sub1105) is sufficient for induction of Ad12 TSTA. Our results provide direct and unequivocal in vivo evidence that TSTA activities of adenovirus groups A and C are controlled by different domains of their respective E1A genes. The Ad12 TSTA is a function of the 5' terminus of the first E1A exon, while the Ad5 TSTA is coded for by the 3' exon of its E1A gene.
Adenovirus E1A Proteins↗
Unusual splice sites in the E1A-E1B cotranscripts synthesized in adenovirus type 40-infected A549 cells.
The adenovirus E1 DNA region consists of two transcription units, E1A and E1B. In this paper we report that the E1A-E1B cotranscripts containing sequences of both the E1A and E1B regions are synthesized during adenovirus type 40 (Ad40) infection of A549 cells. Cytoplasmic RNA was isolated from Ad40-infected A549 cells at 24, 72, and 100 h post infection (p.i.). The complementary (c) DNA was synthesized by reverse transcription using an oligo-dT primer and then amplified by the polymerase chain reaction (PCR) using primers derived from the E1A and E1B regions. The cDNAs thus amplified were sequenced either directly or after cloning into bacteriophage M13 vectors. Analysis of cDNA indicated that the E1A-E1B cotranscripts are synthesized at 72 h p.i., but not at 24 or 100 h p.i. Nucleotide sequences of three cDNAs of the E1A-E1B cotranscripts indicated that the cotranscripts originate from the E1A promoter and lack sequences for both the E1A poly(A) site and E1B cap site. The splices create open reading frames for E1A-E1B fused polypeptides around the E1A-E1B junctions in these mRNAs. Most interestingly, the sequence analysis showed that the 5' and 3' splice junctions in the two E1A-E1B cotranscripts do not conform to the splice consensus GT-AG rule. Our results thus suggest that factor(s) which lead to unusual splicing in the E1 mRNAs are present in Ad40-infected A549 cells.
Adenovirus E1A Proteins↗
Sequence variation of human papillomavirus type 16 E7 in preinvasive and invasive cervical neoplasias.
Variation in the nucleotide sequence of the HPV 16 E7 gene in preinvasive cervical intraepitherial neoplasia (CIN) and invasive cervical carcinoma specimens was analyzed. Direct DNA sequencing of PCR-amplified products with primers different from those used for PCR with 5'-end labeling generated distinct sequence ladders with a low background, even in specimens containing relatively low copy numbers of HPV. Of 14 cervical neoplasias, 11 cases showed sequence diversity from prototype HPV16, and a total of 22 nucleotide exchanges were detected. Nine of these led to single amino acid exchanges: [Thr5] to [Lys5] in one case and [Asn29] to [Ser29] in eight cases. The [Ser29] E7 was distributed uniformly among invasive carcinomas and precancerous legions, and was also found in a normal cervix. The [Lys5] E7 and [Ser29] E7 had transforming potential similar to the prototype E7 assessed by cooperation with the activated ras gene in rat embryo fibroblasts.
Amino Acid Sequence↗
Persistent infection of MT-4 cells by human immunodeficiency virus type 1 becomes increasingly likely with in vitro serial passage of wild-type but not nef mutant virus.
Our previous studies have shown that human immunodeficiency virus type 1 (HIV-1), with mutations in accessory genes such as vif, vpr or vpu, can generate persistent infection of MT-4 cells, whereas infection by wild-type or nef mutant HIV-1 causes extensive cell death. The possibility of generating a naturally attenuated form of HIV-1 with reduced cytopathogenicity in MT-4 cells was examined by in vitro serial passage of the wild-type and a nef mutant form of HIV-1, each derived from the infectious molecular clone pNL432. The ability to cause persistent infection was observed after four passages of wild-type HIV-1 with the frequency of persistence becoming progressively higher with serial passage. In contrast, persistent infection was not observed even after 50 passages of the nef mutant virus. Sequence analysis of the accessory gene loci in genomes recovered from the persistent infections caused by passaged virus revealed mutations in vif and vpr, but not in vpu. The processing of the Env precursor to mature forms was not modified in any of the passages of either wild-type or nef mutant HIV-1. However, when compared with acute infections caused by similarly passaged virus of both wild-type and nef mutant HIV-1, persistent infections by passaged wild-type HIV-1 showed a significant decrease in the cell surface expression and function of Env. Cell surface CD4 was only partially down-regulated on cells acutely infected with the passaged viruses, whereas on cells persistently infected with passaged wild-type HIV-1 it was completely down-regulated. These results suggest that, during serial passage of HIV-1, mutations accumulate at least in the accessory genes vif and vpr in parallel with a lesser interaction between cell surface Env and CD4 molecules, and lead to the generation of less cytopathogenic viruses capable of persistent infection. Our results also suggest an important role for the nef gene product in the generation of HIV-1 strains that are less cytopathogenic.
Amino Acid Sequence↗
Detection of human cytomegalovirus DNA in breast milk by means of polymerase chain reaction.
Three hundred and twenty-five breast milk samples were examined for the occurrence of human cytomegalovirus (HCMV) by cell culture method. Virus was isolated from the milk in 1 of 177 samples collected within 6 days after delivery, 2 of 115 samples collected during the period of 7 days to 1 month after delivery, 10 of 33 samples collected over 1 month after delivery. Next, we tried to amplify HCMV DNA from the breast milk samples from HCMV seropositive mothers and seronegative mothers at 1 month after delivery by polymerase chain reaction. HCMV DNA was detected in 12 of 13 samples from seropositive mothers and in none of 7 samples from seronegative mothers. It was thought that all women seropositive for HCMV principally shed the virus into their breast milk at 1 month after delivery.
Antibodies, Viral↗
[Molecular basis for HIV infection: implication for the therapy].
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AIDS Vaccines↗
Negative regulation of the gene for H-2Kb class I antigen by adenovirus 12-E1A is mediated by a CAA repeated element.
Both positive and negative regulatory elements responsive to the product of the adenovirus type 12 E1A gene are located in the promoter region of the gene for mouse H-2Kbm1 major histocompatibility complex (MHC) class I antigen (1). We have analyzed the negative regulatory element that is affected by E1A and identified a target CAA repeated motif, CAA(A)CAAA, within -1725 to -1705 and -1591 to -1568 in a 316-bp sequence located in the far upstream region of H-2Kb promoter (-1837 to -1522; +1 refers to the cap site). The extent of cell surface expression of the MHC class 1 antigen was significantly decreased in the case of transfectants obtained by introducing an expression plasmid that included MHC class 1 cDNA with the CAA repeated element, as compared with that of a plasmid with mutated CAA repeats. We have also characterized the nuclear proteins that bind to this motif. The analysis of the effects of mutations during competition assays of in vivo and gel retardation competition assays demonstrated that the CAA repeated element is essential not only for E1A-dependent repression of transcription but also for the cell surface expression of the product of the mouse H-2Kb class I gene, presumably through nuclear proteins that specifically bind to it.
Adenovirus E1A Proteins↗
Isolation of a cDNA encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncogene family.
The cDNA encoding adenovirus E1A enhancer-binding protein E1A-F was isolated by screening a HeLa cell lambda gt11 expression library for E1A-F site-specific DNA binding. One cDNA clone produced recombinant E1A-F protein with the same DNA binding specificity as that endogenous to HeLa cells. Sequence analysis of the cDNA showed homology with the ETS-domain, a region required for sequence-specific DNA binding and common to all ets oncogene members. Analysis of the longest cDNA revealed about a 94% identity in amino acids between human E1A-F and mouse PEA3 (polyomavirus enhancer activator 3), a recently characterized ets oncogene member. E1A-F was encoded by a 2.5kb mRNA in HeLa cells, which was found to increase during the early period of adenovirus infection. In contrast, ets-2 mRNA was significantly reduced in infected HeLa cells. The results indicate that E1A enhancer binding protein E1A-F is a member of the ets oncogene family and is probably a human homologue of mouse PEA3.
Adenovirus E1A Proteins↗
Detection of human papillomavirus deoxyribonucleic acid in penile carcinoma by polymerase chain reaction and in situ hybridization.
We investigated the prevalence of human papillomavirus types 16, 18 and 33 deoxyribonucleic acid (DNA) by polymerase chain reaction and the localization of human papillomavirus DNA by in situ hybridization using formalin-fixed, paraffin-embedded tissue specimens of penile carcinoma in Japan. Of 111 untreated penile carcinomas 70 (63.1%) were positive for human papillomavirus DNA by the polymerase chain reaction method. Human papillomavirus type 16 was identified in 68 penile carcinoma cases and type 18 in 2, whereas type 33 could not be detected. Of 12 treated penile carcinomas 2 (16.7%) were positive for human papillomavirus type 16. These data indicate that human papillomavirus type 16 DNA is the type most commonly associated with penile carcinoma. Human papillomavirus type 16 was also detected in lymph node metastasis of penile carcinoma, and that was the same as the human papillomavirus DNA type in the primary carcinoma. The in situ hybridization analysis found the human papillomavirus DNA to be localized in the nuclei of carcinoma cells.
Adult↗
A case of verrucous carcinoma associated with human papillomavirus type 16 DNA.
We report here a case of verrucous carcinoma which occurred on the penis of a 75-year-old male. The nodule was first noted six months earlier and was whitish, cauliflower-like, and 17 x 19 mm in size. The histopathological examination revealed hypertrophic epidermal proliferation with pale staining keratinocytes, extending into the deep dermis. Partial penectomy and inguinal lymph node dissection were done. No lymph node metastasis was recognized. DNA was isolated from the frozen tumor tissue and examined for the presence of human papillomavirus (HPV) 16, 18, and 33 DNA by the polymerase chain reaction (PCR), using common and specific primers. A 140 base pair (bp) band was amplified and finally determined to be the HPV16 sequence by dot-blot hybridization.
Aged↗