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PubMed1994

Establishment and characterization of synovial cell clones with integrated human T-cell leukemia virus type-I.

Human T cell leukemia virus type-I (HTLV-I)-integrated synovial cell clones (SCCs) were established from four HTLV-I-infected patients with chronic proliferative arthropathy. Extracted DNAs of these SCCs were examined for the existence of HTLV-I proviral DNA. Electron microscopic analysis and immunohistochemical staining were also performed to examine the nature of the HTLV-I integrated SCCs. SCCs derived from all four patients consisted of either HTLV-I integrated or nonintegrated cells. HTLV-I integrated SCCs also produced viral messenger RNA which was detected by reverse transcriptase-polymerase chain reaction as well as viral protein which was detected by immunohistochemical staining. Phenotypically, these SCCs containing HTLV-I proviral DNA were shown to be all cell types as observed by electron microscopic analysis and immunohistochemical analysis. Moreover, expression of HTLV-I glycoprotein was greater in SCCs with a monocyte marker. These findings suggest that SCCs harboring HTLV-I have an important role in synovial hyperplasia.

Aged↗
PubMed1994

Papillomavirus genomes in human cervical carcinoma: analysis of their integration and transcriptional activity.

Eighty-four biopsies derived from cervical tissues were analyzed for the presence of human papillomavirus (HPV) DNA types 6, 16 and 18 using Southern blot hybridization. HPV 6 was found in none of the cervical biopsies, and HPV types 16 and 18 were found in 44% of them. The rate of HPV 16/18 positive samples increased proportionally to the severity of the lesion. In normal tissue there were no positive samples, in mild and moderate dysplasias HPV 16/18 was present in 20% and in severe dysplasias and invasive carcinomas in 37 and 50%, respectively. In biopsies from 13 cases with squamous cell carcinoma of the uterine cervix and CIN III lesions HPV 16 was integrated within the host genome. It was concluded that the virus could be integrated at variable, presumably randomly selected chromosomal loci and with different number of copies. Transcription of HPV 16 and 18 was detected in one cervical cancer and in HeLa cells, respectively. These results imply that HPV types 16 and 18 play an etiological role in the carcinogenesis of human cervical epithelial cells.

DNA, Viral↗
PubMed1993

Photochemical inactivation of cell-associated human immunodeficiency virus in platelet concentrates.

Photochemical decontamination (PCD) of platelet concentrates, with adequate preservation of platelet function, has been shown using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA). To further evaluate this technique, models for the inactivation of pathogenic human cell-associated viruses and integrated proviral sequences are required. We have assessed the ability of the PCD technique to inactivate cell-associated human immunodeficiency virus 1 (HIV-1) in platelet concentrates. We correlated PCD inhibition of HIV-1 infectivity with 8-MOP-DNA adduct formation in contaminating nucleated cells, and measured the inhibition of polymerase chain reaction (PCR)-mediated amplification of cellular DNA sequences as a surrogate for inactivation of integrated proviral nucleic acid sequences. After PCD treatment (8-MOP 300 micrograms/mL, UVA 17 mW/cm2) for 60 minutes, 0.5 x 10(6) plaque-forming units (PFU)/mL of cell-associated HIV-1 were inactivated and no virus was detectable by infectivity assay. After 60 minutes of PCD, 15 8-MOP-DNA adducts per 1,000 bp were formed, while in the absence of UVA, no adducts were formed. PCR-mediated amplification of a 242-bp cellular DNA sequence (HLA-DQ-alpha) was inhibited when greater than eight psoralen-DNA adducts per 1,000 bp were present. These studies indicate that high titers of cell-associated HIV-1 in platelet concentrates were inactivated by PCD, and the numbers of 8-MOP-DNA adducts in nucleated cells were sufficient to inhibit amplification of DNA segments that encode for as few as 80 amino acids. Based on the frequency of 8-MOP-DNA adducts, for the 10-kb HIV-1 genome, the probability of an integrated genome without at least one 8-MOP adduct after 60 minutes of PCD was 10(-33).

Blood Component Transfusion↗
PubMed1982

Crown gall: tumor as a result of oncogenic DNA transfer.

It has been well documented that some viruses can cause neoplasmic transformations in animal cells because particular segments of the virus genome integrated in the animal cell DNA alter the differentiated state of these transformed cells. Therefore, the DNA of these viruses is oncogenic. Recent studies on so-called crown gall tumors in plants have shown that oncogenic DNA's need not always be of viral origin. Indeed, it was shown that these plant tumors result from the integration, maintenance, and expression in the plant cell nucleus of a specific DNA segment introduced in the plant cells by some pathogenic bacteria. Thus crown gall neoplastic transformation is a natural example of "genetic engineering" because it was shown that the growth of the tumor-inducing bacteria is stimulated by organic compounds (so-called "opines") excreted by the transformed plant cells. The bacterial DNA transferred to and expressed in the plant cells is directly responsible for the synthesis of the opines and for the tumorous character of the transformed plant cells. The bacterial oncogenic DNA (T-DNA) is a segment of an extrachromosomal element (Ti-plasmid) carried by the tumor-inducing bacteria. A complicated set of genes on the Ti-plasmid control the various steps involved in the tumorous transformation and are thus responsible for the transfer of the oncogenic T-DNA. A molecular study of the products of the T-DNA will allow us to have a better understanding of the control of growth and differentiation in eukaryotic cells. Furthermore, the Ti-plasmid can be used as a gene vector and thus allow the transfer of selected genes into plants.

Cell Transformation, Viral↗
PubMed2004

Mutational analyses of the core domain of Avian Leukemia and Sarcoma Viruses integrase: critical residues for concerted integration and multimerization.

During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme.

Alpharetrovirus↗
PubMed2003

Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins.

Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.

Animals↗
PubMed1996

Clinical implication of the integration patterns of human T-cell lymphotropic virus type I proviral DNA in adult T-cell leukemia/lymphoma.

In this review, we discuss the possible relationship between the clinical characteristics and the integration patterns of human T-cell lymphotropic virus type I (HTLV-I) proviral DNA in patients with adult T-cell leukemia/ lymphoma (ATL). Some ATL patients show unusual integration patterns such as multiple or defective HTLV-I and have clinical characteristics unlike those of most ATL patients who have the characteristic integration pattern of one complete provirus. Multiple HTLV-I integrations can be detected as two or more bands using the standard Southern blotting method when the tumor cellular DNA is digested with an endonuclease that does not cleave within the provirus. This includes cases of one tumor cell clone carrying two or more copies of the provirus, or alternatively two or more cell clones, each carrying one copy of the provirus. The former group of patients always manifest severe dyspnea and hypoxemia with unusual organ infiltrations including the retina and muscle and an extremely aggressive clinical course. On the other hand, the latter group of patients have an indolent course with skin lesions or small T lymphocytes with cleaved or lobulated nuclei. A solitary defective HTLV-I in some ATL patients can be detected as one smaller band after digestion of cellular DNA with an endonuclease that does not cleave within the provirus. These patients generally have a favourable clinical course with small cleaved or bilobulated T lymphocytes without lymphadenopathy or skin lesions. These findings suggest that there are clinical implications for the integration patterns of HTLV-I and this may be one of the explanations for the heterogeneous behaviour of the disease. Such studies may provide information on the relationship between virus integration and the clinical manifestations and also improve our understanding of the pathogenesis of ATL.

Adult↗
PubMed2004

A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase.

The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.

Animals↗
PubMed1986

Transforming potential of DNA of the human PLC/PRF/5 hepatoma cell line.

DNA of the human PLC/PRF/5 hepatoma cell line can induce the appearance of colonies on soft agar after transfection of BK-BK cells (BALB/c mouse kidney cells partially transformed by BK virus). Blot hybridization analyses indicate that the transformants contained the hepatitis B virus DNA sequence. This sequence disappeared during serial passage of transformants. The amplification and rearrangement of the integrated BK virus genome appears to be specifically associated with transformation.

Animals↗
PubMed1980

Isolates of avian sarcoma virus Bratislava 77 (B77) oncogenic for mammals do not contain an excess of transformation-defective mutants.

Several isolates of the avian sarcoma virus Bratislava 77 (B77), used in tumor induction in rats, hamsters and mice, were tested for the excess of spontaneously segregated transformation-defective mutants (tdB77). The question was asked whether these td mutants could interfere with transforming sarcoma viruses at tumor induction in mammals. It was found that the B77 virus isolates used in successful sarcoma induction in mammals did not contain an excess of td mutants. One virus isolate which had an excess of td mutants did not induce tumors in mammals. The further characterization of the rescued viruses from virogenic mammalian cells showed that all rescued viruses had the same sub-group C specificity as the original isolate of B77 virus. The integration of the viral genome into mammalian cellular genome did not alter transforming ability of the rescued viruses on duck embryo cells. It seems that propagation of B77 virus in conditions in vivo did not support the segregation, and accumulation of an large excess of td mutants in stocks of B77 virus.

Animals↗
PubMed1999

Mouse mammary tumor virus carrying a bacterial supF gene has wild-type pathogenicity and enables rapid isolation of proviral integration sites.

Mouse mammary tumor virus (MMTV) has frequently been used as an insertional mutagen to identify provirally activated mammary proto-oncogenes. To expedite and facilitate the process of cloning MMTV insertion sites, we have introduced a bacterial supF suppressor tRNA gene into the long terminal repeat (LTR) of MMTV, thus allowing selection of clones containing it in lambda vectors bearing amber mutations. The presence of supF in the LTR should circumvent the screening process for proviral insertion sites, since only those lambda clones with supF-containing proviral-cellular junction fragments should be able to form plaques on a lawn of wild-type Escherichia coli (i.e., lacking supF). The resulting virus (MMTVsupF) induced mammary tumors at the expected rate in infected mice, deleted the appropriate T-cell population by virtue of its superantigen gene, and stably retained the supF gene after passage via the milk to female offspring. To test the selective function of the system, size-selected DNA containing two proviral-cellular junction fragments from an MMTV supF-induced mammary tumor was ligated into lambdagtWES.lambdaB, packaged, and plated on a supF-deficient bacterial host for selection of supF-containing clones. All plaques tested contained the desired cloned fragments, thus demonstrating the utility of this modified provirus for the rapid cloning of MMTV insertion sites.

Animals↗
PubMed1992

Comparison of detergents for extraction and ion-exchange high-performance liquid chromatography of Sendai virus membrane proteins.

The integral membrane proteins of Sendai virus, haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with a non-ionic and two zwitterionic detergents, i.e., pentaethylene glycol monolauryl ether (C12E5), lauryldimethylamine oxide (LDAO) and dodecyldimethylammoniopropane-1-sulphonate (SB12), respectively. The extracts were subjected to ion-exchange high-performance liquid chromatography (HPIEC) using 0.1% of the detergent in the eluent on four different columns (MA7Q, Zorbax BioSeries SAX, Mono Q and PL-SAX) with a quaternary amine as interacting ligand and with different pore sizes: non-porous and 30, 80 nm and 400 nm, respectively. The relative recoveries of protein were similar for all the columns. The highest recovery of HN and F protein and the best separation were obtained with C12E5. Analysis of HPIEC fractions with monoclonal antibodies directed against conformational epitopes showed that C12E5 had less effect on the conformation than the other two detergents.

Animals↗
PubMed1983

Analysis of the primary translational product and integrated DNA of a new feline sarcoma virus, GR-FeSV.

The Gardner-Rasheed strain of feline sarcoma virus (GR-FeSV), is a recent isolate of a naturally occurring cat sarcoma. The primary translational product of GR-FeSV (GR P70) was shown to be a phosphoprotein with associated tyrosine-specific protein kinase activity. The relationship between the GR-FeSV provirus and once genes of other transforming retroviruses known to code for tyrosine kinases was examined by molecular hybridization. Probes repesenting onc genes of Snyder-Theilen and McDonough strains of feline sarcoma virus, Rous sarcoma virus, and Abelson murine leukemia virus did not detectably hybridize integrated GR-FeSV. These findings suggest that GR-FeSV contains a distinct tyrosine kinase-coding onc gene.

Animals↗
PubMed1995

Structure of the int-5, a novel MMTV integration genomic locus containing mouse early transposon LTR homology region.

Previous studies have reported the cloning and identification of the int-5, a novel mouse mammary tumor virus (MMTV) integration locus involved in mammary tumorigenesis. Here we report the characterization of the 5.5 kb region from this novel MMTV integration site. Our results show that the region after the MMTV integration site, a 258 bp sequence is homologous (100%) to the mouse early transposon (mETn) long-terminal repeat and other sequences of the this transposon are not present in the 5.5 kb region. Our results also show for the first time the tumor specific expression of mETn and expression of the region downstream of the MMTV integration site that is homologous to mETn-LTR in D2 mammary tumors.

Animals↗
PubMed1975

Studies on characterization of the integration sites of avian RNA tumor virus-specific DNA.

A sequential hybridization procedure is described which allows the integration sites of viral-specific DNA to be characterized according to their reassociation kinetics. In addition, this approach enables us to estimate the size of the integrated viral DNA. Endogenous virus sequences in normal cells appear to be associated with cell sequences reiterated 1200 times, and each integration unit is approximately equal to one 35S RNA subunit. In AMV-infected cells, the additional AMV-specific DNA sequences reassociate as if they were integrated adjacent to unique cellular DNA or in tandem with the endogenous viral DNA sequences.

Animals↗
PubMed1993

Enhanced incorporation of transgenic DNA into zebrafish chromosomes by a retroviral integration protein.

Manufacture of lines of fish containing specific transgenes is difficult because most fish that hatch from embryos injected with foreign DNA are mosaic; few have the transgenic DNA integrated in germ-line cells. To determine whether the process of integration of exogenously supplied DNA into fish genomes could be accelerated, we examined the ability of the Moloney murine leukemia virus (MoMLV) integration protein (IN) to function in embryonic zebrafish cells. We used partially purified IN from a baculovirus/insect cell expression system and unpurified IN from extracts of psi-2 mouse cells that carry a MoMLV provirus. Both forms of IN were able to enhance expression in zebrafish 10 days after fertilization. At day 14 of development, fish injected with IN had higher levels of transgenic DNA than control fish. The ability of IN to enhance integration of transgenic constructs was demonstrated by a ligation-mediated polymerase chain reaction procedure, which was employed to detect junction fragments of foreign and host genomic DNA, generated by IN-mediated integration.

Animals↗