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J S Butel

Publications and source records attributed to J S Butel.

At least 37 records · Page 2Linked to original sources
PubMed1998

Hepatitis B virus X protein interferes with cellular DNA repair.

The hepatitis B virus X protein (HBx) is a broadly acting transactivator implicated in the development of liver cancer. Recently, HBx has been reported to interact with several different cellular proteins, including our report of its binding to XAP-1, the human homolog of the simian repair protein UVDDB. In the present study, several HBx mutants were used to localize the minimal domain of HBx required for binding to XAP-1/UVDDB to amino acids 55 to 101. The normal function of XAP-1/UVDDB is thought to involve binding to damaged DNA, the first step in nucleotide excision repair (NER); therefore, we hypothesized that this interaction may affect the cell's capacity to correct lesions in the genome. When tested in two independent assays that measure NER (unscheduled DNA synthesis and host cell reactivation), the expression of HBx significantly inhibited the ability of cells to repair damaged DNA. Under the assay conditions, HBx was expressed at a level similar to that previously observed during natural viral infection and was able to transactivate several target reporter genes. These results are consistent with a model in which HBx acts as a cofactor in hepatocarcinogenesis by preventing the cell from efficiently repairing damaged DNA, thus leading to an accumulation of DNA mutations and, eventually, cancer. An adverse effect on cellular DNA repair processes suggests a new mechanism by which a tumor-associated virus might contribute to carcinogenesis.

Animals↗
PubMed1998

Natural isolates of simian virus 40 from immunocompromised monkeys display extensive genetic heterogeneity: new implications for polyomavirus disease.

Simian virus 40 (SV40) DNAs in brain tissue and peripheral blood mononuclear cells (PBMCs) of eight simian immunodeficiency virus-infected rhesus monkeys with SV40 brain disease were analyzed. We report the detection, cloning, and identification of five new SV40 strains following a quadruple testing-verification strategy. SV40 genomes with archetypal regulatory regions (containing a duplication within the G/C-rich regulatory region segment and a single 72-bp enhancer element) were recovered from seven animal brains, two tissues of which also contained viral genomes with nonarchetypal regulatory regions (containing a duplication within the G/C-rich regulatory region segment as well as a variable duplication within the enhancer region). In contrast, PBMC DNAs from five of six animals had viral genomes with both regulatory region types. It appeared, based on T-antigen variable-region sequences, that nonarchetypal virus variants arose de novo within each animal. The eighth animal exclusively yielded a new type of SV40 strain (SV40-K661), containing a protoarchetypal regulatory region (lacking a duplication within the G/C-rich segment of the regulatory region and containing one 72-bp element in the enhancer region), from both brain tissue and PBMCs. The presence of SV40 in PBMCs suggests that hematogenous spread of viral infection may occur. An archetypal version of a virus similar to SV40 reference strain 776 (a kidney isolate) was recovered from one brain, substantiating the idea that SV40 is neurotropic as well as kidney-tropic. Indirect evidence suggests that maternal-infant transmission of SV40 may have occurred in one animal. These findings provide new insights for human polyomavirus disease.

Amino Acid Sequence↗
PubMed1998

Sequence analyses of human tumor-associated SV40 DNAs and SV40 viral isolates from monkeys and humans.

SV40 DNA has been found associated with several types of human tumors. We now report a sequence comparison of SV40 DNAs from pediatric brain tumors and from osteosarcomas with viral isolates from monkeys and from humans. We analyzed the entire genomic sequences of five isolates, Baylor and VA45-54 strains from monkeys and SVCPC, SVMEN, and SVPML-1 recovered from humans, and compared them to the reference virus SV40-776. The viral sequences were highly conserved, but isolates could be distinguished by variations in the structure of the viral regulatory region and in the nucleotide sequence of the variable domain at the C-terminus of the large T-antigen gene. We conclude that multiple strains of SV40 exist that can be identified on the basis of sequences in these regions of the viral genome. The isolates were more similar to each other and to the Baylor strain than to the reference strain SV40-776. Human isolates SVCPC and SVMEN were found to be identical. The DNAs present in some human brain and bone tumors were authentic SV40 sequences. Many of the C-terminal T-ag sequences associated with human tumors were unique, but some sequences were shared by independent sources. There was no compelling evidence for human-specific strains of SV40 or for tumor type-specific associations, suggesting that SV40 has a relatively broad host range. The source of the viral DNA found in human tumors remains unknown.

Animals↗
PubMed1998

Detection of authentic SV40 DNA sequences in human brain and bone tumours.

This report summarizes our follow-up studies of SV40 DNA sequences in human brain tumors of early childhood and our confirmation of the presence of SV40 DNA in human osteosarcomas. We examined brain tumors and osteosarcoma samples by the polymerase chain reaction (PCR) using primers from four separated regions of the SV40 genome. Sequence analysis confirmed that authentic SV40 DNA was present. The regulatory region of each tumor-associated viral DNA was of archetypal length (non-duplicated enhancer); sequence variation was noted at the extreme C-terminus of the large T-antigen (T-ag) genes. Infectious SV40 was recovered from one brain tumor. We sequenced the entire early genomic region from three human isolates of SV40 and two laboratory strains originally recovered from monkeys. The predicted amino acid sequence of the large T-ags showed remarkable sequence conservation among isolates, except for a small variable region identified at the C-terminus of the protein. There were no human-isolate-specific changes detected that could serve to distinguish a human variant of SV40 nor were any tumor-type-specific viral markers observed. Based on these data, we conclude that authentic SV40 is associated with some human brain and bone tumors and that multiple SV40 strains can infect humans.

Amino Acid Sequence↗
PubMed1998

Consideration of PCR methods for the detection of SV40 in tissue and DNA specimens.

SV40 is capable of infecting humans, although its association with human diseases remains controversial. Recently, a subgenomic SV40 DNA sequence was detected by polymerase chain reaction (PCR) in certain types of human tumour tissue as well as in normal pituitary tissue. However, due to the limited DNA sequence information that was obtained in those experiments, SV40 could not be authenticated, and it was uncertain whether a related or hybrid virus (or endogenous DNA) accounted for the PCR-amplified DNA. To gain more insight into these observations, we are experimenting with PCR primers directed at various sites of the SV40 genome, as well as with various parameters of the PCR assay. In this communication, we describe methodology we currently use in our studies and discuss problems associated with the PCR detection of SV40 in various types of samples.

Bone Neoplasms↗
PubMed1998

Cytotoxic T lymphocyte recognition sequences as markers for distinguishing among tumour antigens encoded by SV40, BKV and JCV.

Simian virus 40 (SV40) has been shown to be associated with a number of human tumours. Two other human papova viruses, BKV and JCV, infect humans at a relatively high frequency and are activated upon immune suppression. The T antigens of both of these viruses share considerable homologies with the transforming protein T antigen of SV40. We have used SV40 T antigen specific cytotoxic T lymphocyte (CTL) clones to discriminate among the T antigens of SV40, BKV and JCV. These CTL clones directed to four distinct CTL epitopes serve as specific probes and can differentiate subtle alterations or deletions in the CTL epitopes relative to SV40 T antigen. Using this strategy, we have been able to authenticate three SV40 viruses isolated from humans as all four distinct CTL epitopes in the T antigens encoded by these three SV40 human isolates (SVCPC, SVMEN, and SVPML-1) were found to be identical to prototype SV40. We have further identified a 198 amino acid deletion T antigen variant of SVCPC. The finding of a deletion mutant in the SVCPC virus population suggests that the cellular immune response may play a role in the selection of antigenic loss variants.

Amino Acid Sequence↗
PubMed1997

SV40 DNA in human osteosarcomas shows sequence variation among T-antigen genes.

Authentic simian virus 40 (SV40) has been detected in association with human choroid plexus and ependymoma tumors, and SV40-like DNA sequences have been found in some human osteosarcomas. We report here an analysis of human osteosarcoma samples for the presence of SV40 DNA using PCR and primers directed at 4 distinct sites of the SV40 genome, coupled with sequence analysis. Authentic SV40 DNA sequences were detected in 5 of 10 osteosarcoma tumor samples. The SV40 regulatory region in each case was identical and of archetypal length (non-duplicated enhancer), as is usually found in natural isolates of SV40 from monkeys and in human brain tumors. A section of the gene that encodes a viral late gene product (VP1) was detected in 5 of 10 tumors and had an exact match with the known sequence of SV40. Two separated segments of the large T-antigen (T-ag) gene were found in the same 5 tumors. Analysis of the DNA sequences encoding the T-ag carboxy terminus revealed sequence variation among the tumors, as observed previously in viral DNA associated with human brain tumors. There does not appear to be a preferential association of a T-ag variable domain sequence with a given tumor type. No sequences from the regulatory region of human polyomaviruses JCV and BKV were detected in the bone tumors. We also noted less efficient recovery of SV40 DNA from tumor samples fixed in paraffin as compared to frozen tumors. Our results confirm the presence of SV40 DNA in human bone tumors and, based on the sequence variation observed for the carboxy terminus of the T-ag gene, suggest that there is not a specific SV40 strain associated with human osteosarcomas.

Adolescent↗
PubMed1997

Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus X associated protein-1 (XAP-1).

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located between positions -128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun. 220, 553-562]. The distal element (-128 to -85) appears to be liver specific because it shows positive activity in HepG2 cells and negative activity in HeLa cells. ApoB gene regulatory factor-2 (BRF-2) interacts with the sequence (-104 to -85). BRF-2 has been purified from rat liver nuclear extract, and its molecular weight has been determined to be approximately 120 kDa [Zhuang et al. (1992) Mol. Cell. Biol. 12, 3183-3191]. In this paper we report the isolation of two isoforms of BRF-2 by further purification using high-performance liquid chromatography. Both isoforms produced a single approximately 120-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis detected by silver stain. The amino acid sequences of two tryptic peptides derived from HPLC-purified heavier BRF-2 isoform were determined to be YLAIAPPIIK and ALYYLQIHPQELR. These two peptides were found to share 100% sequence homology with human hepatitis B virus X associated protein-1 (XAP-1) and monkey UV-damaged DNA-binding protein (UV-DDB). Anti-peptide antisera raised against two synthetic peptides of XAP-1 recognized a approximately 120-kDa polypeptide band in both BRF-2 isoforms in a western blot analysis. By using apoB promoter fragments containing various internal deletions and a substitution mutation as templates for gel mobility shift assays, we identified the region between -104 and -85 as crucial for binding by the high-molecular weight form. In contrast, the lower molecular weight isoform bound to all apoB mutants tested. Anti-peptide 2 antiserum directed against XAP-1 was found to inhibit in vitro transcription of the apoB gene in rat liver nuclear extracts by 50%. These results suggest that BRF-2 and XAP-1 are structurally and immunologically highly related trans-activators of the apoB gene. We propose that BRF-2 exists both as a monomer (BRF-2M) and as a homooligomer. probably a homodimer (BRF-2D), in solution; oligomerization appears to be an essential step for imparting sequence-specificity to BRF-2 protein and thereby facilitating its role as a trans-activator of the apoB gene.

Amino Acid Sequence↗
PubMed1997

High-fidelity PCR amplification of infectious copies of the complete simian virus 40 genome from plasmids and virus-infected cell lysates.

We describe here a long-polymerase chain reaction (PCR) method that can be used to amplify complete simian virus 40 (SV40) DNA with high fidelity, and we show that authentic, viable virus can be produced from molecular clones of the PCR-amplified viral DNAs. A commercial long-PCR kit that employed a combination of Taq and GB-D polymerases was used, together with a pair of overlapping primers that recognized a unique EcoRI site in the SV40 genome. Efficient amplification required linearization of the circular SV40 genomic DNAs with EcoRI. Entire SV40 genomes were successfully PCR-amplified from an SV40 plasmid and from two different SV40-infected cell lysates and were cloned into pUC-19. Three separate segments of the cloned viral genomes were DNA sequenced, and no nucleotide changes relative to the parental virus were detected, suggesting that the viral DNAs had been amplified with high fidelity. Each PCR clone was infectious, and no differences were detected in the growth characteristics of viruses derived from these clones as compared to the original viral strain. The procedure we utilized shortens and simplifies the molecular cloning of small double-stranded DNA viruses and will be useful for viral diagnostic tests and for recovery of virus from clinical samples. The results of these experiments have broad implications, as the methodology is applicable to many systems.

Base Sequence↗
PubMed1997

Unscheduled DNA replication precedes apoptosis of photoreceptors expressing SV40 T antigen.

Expression of simian virus 40 T antigen (Tag) in the rod photoreceptors of transgenic mice leads to cell death that is completed by the end of the third week of postnatal development. To understand the mechanistic link between Tag expression and the death of the expressing photoreceptors, cell cycle activity was followed in a transgenic mouse family that expresses Tag directed by the mouse opsin promoter. Tag-expressing photoreceptors also expressed rhodopsin suggesting that these cells were differentiated. The presence of Tag in the photoreceptors induced the expression of both proliferating cell nuclear antigen (PCNA) and thymidine kinase (TK). The abnormally high levels of PCNA and TK continued until the complete disappearance of the cells expressing Tag. Photoreceptor cell death was also associated with continued DNA synthesis that ceased shortly after postnatal day 16. The specific loss of the rod photoreceptors that re-entered the cell cycle accounted entirely for the loss of photoreceptors from the outer nuclear layer. The antiproliferative nature of the mature retina is directly involved in the apoptotic death of photoreceptors expressing Tag.

Animals↗
PubMed1997

A coupled PCR and restriction digest method for the detection and analysis of the SV40 regulatory region in infected-cell lysates and clinical samples.

The polymerase chain reaction (PCR) is an increasingly popular analytical tool for the detection of virus sequences in laboratory preparations as well as in human clinical samples. In studies involving papovaviruses SV40, BK virus (BKV), and JC virus (JCV), one of the primary targets for analysis is the viral regulatory region, as that section of the papovavirus genome is distinct. A primary concern with PCR-based studies is whether amplified DNA sequences may be derived from laboratory contaminants. Recognizing that common sources of PCR contamination are the positive control templates, we devised a facile method to distinguish between real and false-positive PCR-amplified SV40 regulatory region DNAs. SV40 constructs that had been engineered to contain different combinations of 72-basepair (bp) enhancer elements and 21-bp repeats, as well as two introduced unique restriction enzyme sites, were used as positive control templates for PCR amplification. Cleavage of PCR-amplified DNA identifies products from the engineered control plasmids. The procedure is rapid, simple and cost-effective. We also report that primer sets predicted to be specific for the SV40 regulatory region can be used to amplify BKV and JCV regulatory region sequences under conditions of reduced stringency.

BK Virus↗
PubMed1997

Loss of T-antigen sequences allows SV40-transformed human cells in crisis to acquire a senescent-like phenotype.

Normal human cells transfected with SV40 DNA exhibit an extended proliferative potential compared with controls, but they eventually enter a phase known as "crisis." During crisis, extensive cell death occurs and the cells exhibit some gene expression changes similar to senescent cells. This article presents results which indicate that crisis most likely depends on expression of the viral gene T-antigen. We have obtained a unique subpopulation of cells that have deleted the T-antigen gene and, rather than dying as cells do in crisis, remain viable and exhibit some senescent-like characteristics. We also found that the SV40 promoter is poorly expressed in senescent versus young cells. We hypothesize that decreased activity of the viral promoter may result in decreased expression of T-antigen, which is challenged by over-expression of the cell cycle inhibitors such as p21Sdi1. Conflicting signals to proceed/halt cells cycle progression result in the cell death associated with crisis.

Antigens, Polyomavirus Transforming↗
PubMed1997

Tissue culture adaptation of natural isolates of simian virus 40: changes occur in viral regulatory region but not in carboxy-terminal domain of large T-antigen.

The regulatory region of natural isolates of simian virus 40 (SV40) is different from that of laboratory-adapted strains of the virus. The latter have a nucleotide sequence duplication within the enhancer region which varies slightly with each strain, whereas the duplication is lacking in fresh isolates of SV40, which contain an 'archetypal' regulatory region. Many isolates also display nucleotide differences in the DNA encoding the carboxy terminus of large tumour antigen (T-ag). To determine whether genetic changes in these two regions of the SV40 genome were detectable during laboratory adaptation and long-term passage, low-passage virus stocks of two laboratory strains which had detailed passage histories spanning more than 25 years (Baylor strain and VA45-54) were analysed using PCR, cloning and sequencing assays. Both laboratory and archetypal regulatory regions were present in low-passage stocks. Following duplication in the regulatory region, no additional changes were detectable. The variable region at the T-ag carboxy terminus did not undergo any change with tissue culture passage and may serve as a useful site for taxonomic classification of different strains of SV40. Cloned genomes containing single or duplicated enhancers derived from both SV40 strains were viable in CV-1 cells. Attempts to induce regulatory region duplications by 14 serial passages of SV40 archetypal strains in monkey cells were not successful. The results are compatible with tissue culture adaptation of SV40, reflecting either selection of a rare variant pre-existing in the original sample or generation of a rare regulatory region duplication in infected cells.

Adaptation, Physiological↗
PubMed1997

Interaction of the UV-damaged DNA-binding protein with hepatitis B virus X protein is conserved among mammalian hepadnaviruses and restricted to transactivation-proficient X-insertion mutants.

We carried out a comparative analysis of several proposed host protein partners of the human hepatitis B virus X protein (HBx) using both the GAL4- and the LexA-based yeast two-hybrid system. We showed that the interaction of HBx with the UV-damaged DNA-binding protein (UVDDB) is positive in both yeast systems, detectable in cotransfected human cells, conserved by rodent hepadnavirus X proteins (known to transactivate in human cells), and tightly correlated with the transactivation proficiency of X-insertion mutants. Taken together, our results strongly suggest that UVDDB is involved in X-mediated transactivation.

Binding Sites↗
PubMed1996

Activation of the HTLV-I long terminal repeat by the hepatitis B virus X protein.

The human T-cell leukemia virus type I (HTLV-I) Tax protein and the hepatitis B virus (HBV) X protein have each been shown to activate transcription of their respective viral promoters as well as a subset of cellular gene promoters. Here we show that the HTLV-I long terminal repeat (LTR) is responsive to HBV X transactivation. Maximum levels of X-mediated transactivation of the LTR were 8-fold. An X-responsive-region (XRR) of the LTR is located between nucleotides -355 and -276 and contains an AP-2 binding site, a previously recognized X-responsive element. We demonstrated that Tax and X synergize to activate transcription from the HTLV-I LTR, although the AP-2 binding site was not required for this synergy. These results raise the possibility that the HBV X protein may affect the level of HTLV-I gene expression in co-infected individuals.

Base Sequence↗
PubMed1996

Identification of a variable region at the carboxy terminus of SV40 large T-antigen.

The entire early regions of three human isolates of simian virus 40 (SV40), as well as two laboratory strains recovered from monkeys, were sequenced. The early coding region of each isolate contains a number of nucleotide differences when compared to the reference strain SV40-776. These differences result in some changes in the predicted amino acid sequence of the unique region of small t-antigen and in the carboxy (C) terminus of large T-antigen. The amino acid sequence of the remainder of large T-antigen was absolutely conserved among all isolates. Thus, SV40 large T-antigen contains a variable domain at the C-terminal end of the molecule.

Animals↗
PubMed1996

Increased sensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus X gene.

The role of the hepatitis B virus (HBV) X protein in liver tumorigenesis is unresolved. Transgenic mice harboring the X gene (nt 1376-1840 under the control of the human alpha-1-antitrypsin regulatory elements) (ATX mice) display only minor histopathologic alterations of the liver. To determine if ATX mice are more susceptible to the effects of hepatocarcinogens, 12- to 15-d-old male ATX and control littermate mice were injected with a single dose (2 microgram/g body weight) of diethylnitrosamine (DEN). The animals were killed 6-10 mo after exposure and were analyzed for histological changes in the liver. One hundred percent of the DEN-treated AXT mice developed abnormal liver lesions. Then their liver tissues were compared by stereological analysis with those of non-transgenic animals, the ATX mice had a relative twofold increase in the total number of focal lesion and a twofold increase in the incidence of hepatocellular carcinoma. Elevated levels of X protein and p53 protein were not detected in carcinogen-induced nodules or tumors. These results are consistent with a model in which the expression of the HBV X protein potentiates the induction of DEN-mediated liver disease.

Age Factors↗