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At least 793 records · Page 44Linked to original sources
PubMed1994

Coexpression of exogenous and endogenous mouse mammary tumor virus RNA in vivo results in viral recombination and broadens the virus host range.

Mouse mammary tumor virus is a replication-competent B-type murine retrovirus responsible for mammary gland tumorigenesis in some strains of laboratory mice. Mouse mammary tumor virus is transmitted horizontally through the milk (exogenous or milk-borne virus) to susceptible offspring or vertically through the germ line (endogenous provirus). Exogenously acquired and some endogenous mouse mammary tumor viruses are expressed at high levels in lactating mammary glands. We show here that there is packaging of the endogenous Mtv-1 virus, which is expressed at high levels in the lactating mammary glands of C3H/HeN mice, by the virions of exogenous C3H mouse mammary tumor virus [MMTV(C3H)]. The mammary tumors induced in C3H/HeN mice infected with exogenous MMTV (C3H) virus contained integrated copies of recombinant virus containing a region of the env gene from an endogenous virus. This finding indicates that there was copackaging of the Mtv-1 and MMTV(C3H) RNAs in the same virions. Moreover, because Mtv-1 encodes a superantigen protein with a V beta specificity different from that encoded by the exogenous virus, the packaging of Mtv-1 results in an infectious virus with a broader host range than MMTV(C3H).

Animals↗
PubMed2002

Integrity of GH-loop of foot-and-mouth disease virus during virus inactivation: detection by epitope specific antibodies.

Vaccine against foot-and-mouth disease (FMD) is prepared after inactivating the virus produced in cell culture. Inactivation of the FMD virus (FMDV) was earlier done by formaline. However, several vaccine outbreaks, which occurred in Europe revealed that the formaline treatment is not highly effective for virus inactivation. Subsequently, binary ethyleneimine (BEI) was identified as an effective inactivation reagent for FMDV. However, these chemical reagents are likely to have effect on whole virus particle whose integrity is essential for vaccine potency. Therefore, a need is felt to develop non-chemical methods. We have studied induction of endonucleolytic activity as an alternative method for virus inactivation. This method of inactivation was compared with the chemical methods, and found to be highly effective for virus inactivation. The effects of endonucleolytic activity on the integrity of virus capsid was studied using antibodies raised against recombinant proteins, which elicited antibodies against major epitopes present on the surface of the virus. Further, the effect of the agents on the integrity of the virus capsid was studied by using antigen capture PCR (Ag-RT/PCR) which detects the whole virus. The studies showed that inactivation of the virus by induction of endonucleolytic activity is more effective besides maintaining virus integrity. The effect of various inactivating agents on four serotypes of FMDV has also been studied and found to have varying effects, depending on serotype.

Animals↗
PubMed1979

Analysis of cellular integration sites in avian sarcoma virus infected duck embryo cells.

The cellular sites of integration of avian sarcoma virus (ASV) have been examined in clones of duck embryo cells infected with the Bratislava 77 strain of ASV using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled ASV complementary DNA probes. DNA prepared from 11 clones of duck embryo cells infected with the Bratislava 77 strain of ASV was digested with the restriction enzymes HpaI, which cleaves once within the viral genome, and Hind III, which cleaves twice within the viral genome, and the virus-cell DNA juncture fragments were resolved by agarose gel electrophoresis. Analysis of the virus-cell junctures present in individual ASV-infected duck embryo clones revealed that all clones contain at least one copy of nondefective proviral DNA with some clones containing as many as 5 to 6 copies of proviral DNA. A comparison of the virus-cell juncture fragments present in different ASV-infected clones showed that each clone contains a unique set of virus-cell junctures. These data suggest that ASV DNA can integrate at multiple sites within the duck embryo cell genome and that these sites appear to be different as defined by digestion with the restriction enzymes HpaI and HindIII.

Animals↗
PubMed1985

[The use of the cationic detergent dodecyltrimethylammonium bromide for the isolation of influenza virus glycoproteins with subsequent integration of the protein into liposome membrane].

Glycoproteins were isolated from influenza virus with the use of cationic detergent dodecyltrimethylammoniumbromide and attached to preformed liposomes. Liposomal vesicles, thus, acquired their ability for hemagglutination and lysis of chicken erythrocytes. The possibility of using these liposomes for transfer of alien agents into eucaryotic cells is discussed.

Detergents↗
PubMed1996

Site-specific integration by adeno-associated virus.

Adeno-associated virus (AAV) has attracted considerable interest as a potential vector for gene delivery. Wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (AAVS1). Use of a functional model system for AAV DNA integration into AAVS1 has allowed us to conclude that the recombination event is directed by cellular DNA sequences. Recombinant junctions isolated from our integration assay were analyzed and showed characteristics similar to those found in latently infected cell lines. The minimal DNA signals within AAVS1 required for targeted integration were identified and shown to contain functional motifs of the viral origin of replication. A replication mediated model of AAV DNA integration is proposed.

Base Sequence↗
PubMed1984

Free and integrated recombinant murine leukemia virus DNAs appear in preleukemic thymuses of AKR/J mice.

We studied the appearance and structure of murine leukemia viral genomes in preleukemic AKR/J mice by Southern hybridization. Up to an average of one to two copies per thymocyte of unintegrated murine leukemia virus DNA appears in the thymuses of preleukemic mice beginning at 4 to 5 months of age and disappears in leukemic thymuses. The free viral genomes are absent in the spleens, livers, and brains of preleukemic mice. Using a series of ecotropic and nonecotropic murine leukemia virus hybridization probes, we showed that the unintegrated viral genomes are structurally analogous to those of recombinant mink cell focus-forming viruses that appear as proviruses in leukemic AKR thymocytes, suggesting that these free viral DNAs are the direct precursors to the leukemia-specific proviruses. The mosaic of ecotropic and nonecotropic sequences within these unintegrated viral DNAs varies from one preleukemic thymus to another but often appears structurally homogeneous within individual thymuses, indicating that often each thymus was being infected by a unique mink cell focus-forming virus. Analysis of high-molecular-weight DNA shows that recombinant proviruses reside in the chromosomal DNA of thymocytes within the preleukemic thymus, with the number rising to an average of several copies per thymocyte, but we do not detect any preferred integration sites. These results suggest that, in general, before the development of thymic leukemias in AKR mice there is a massive infection by a unique mink cell focus-forming virus which then integrates into many different sites of individual thymocytes, one of which grows out to become a tumor.

Animals↗
PubMed1980

Integration of the simian virus 40 genome into cellular DNA in temperature-sensitive (N) and temperature-insensitive (A) transformants of 3T3 rat and Chinese hamster lung cells.

We studied the pattern of integration of the simian virus 40 (SV40) genome into the cellular DNA of N-transformants (temperature sensitive) and A-transformants (temperature insensitive) derived from 3T3-Fisher rat and Chinese hamster lung cells. The SV40 DNA was covalently linked to the cellular DNA in both types of transformants. In the rat cells, most N-transformants contained SV40 sequences integrated at a single site; most A-transformants contained SV40 sequences integrated at two to five sites. In the Chinese hamster cells, no significant correlation between the number of integration sites and the phenotype of the transformant was found; one of three integration sites were observed for both the N- and A-transformants. Single copies and tandem repeats of SV40 sequences were observed in A- and N-transformants derived from rat cells. A-transformants arise neither by amplification of the SV40 genome nor by integration at a unique site.

Animals↗
PubMed2002

Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors.

Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At about 30 nM IN (20 min at 37 degrees C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of about 70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (about 125 microg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.

Cell Line↗
PubMed1982

Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.

We have asked whether oncogenesis by the mouse mammary tumor virus (MMTV), a slowly oncogenic retrovirus, involves integration of viral DNA within a certain region of the host genome. We first identified a C3H mouse mammary tumor bearing a single new MMTV provirus and cloned a 19 kilobase (kb) DNA restriction fragment containing a junction of viral and host sequences. Host sequences from this clone were used to retrieve 25 kb of the uninterrupted locus (termed MMTV int1) from a bacteriophage library of normal mouse DNA. Hybridization with subcloned DNA fragments of MMTV int1 detected abnormal restriction fragments in digests of DNA from 18 of 26 C3H mammary tumors. The rearrangements all appeared to be due to the insertion of an MMTV provirus, and the integration sites were located in at least five clusters over a total distance of 19 kb. A polyadenylated 2.6 kb RNA species transcribed from int1 was found in the few tumors tested, but not in lactating mammary glands from C3H mice. Of 12 tested viral oncogenes, none exhibited homology with cloned DNA from this locus. We propose that tumorigenesis by MMTV is strongly favored by proviral insertion within the int1 locus, perhaps as a consequence of enhanced expression of a novel cellular oncogene.

Animals↗
PubMed1997

Site-specific in situ amplification of the integrated polyomavirus genome: a case for a context-specific over-replication model of gene amplification.

The fate of the genome of the polyoma (Py) tumor virus following integration in the chromosomes of transformed rat FR3T3 cells was re-examined. The viral sequences were integrated at a single transformant-specific chromosomal site in each of 22 transformants tested. In situ amplification of the viral sequences was observed in 24 of 34 transformants analyzed. Large T antigen, the unique viral function involved in initiating DNA replication from the viral origin, was essential for the amplification process. There was an absolute requirement for a reiteration of viral sequences and the extent of the reiteration affected the degree of amplification. The reiteration may be important for homologous recombination-mediated resolution of in situ amplified sequences. Among 11 transformants harboring a 1 to 2 kb repeat, the degree of amplification was transformant-specific and varied over a wide range. At the high end of the spectrum, the genome copy number increased 1300-fold at steady state, while at the low end, amplification was below twofold. Some aspect of the host chromatin at the site integration that affected viral gene expression, also directly or indirectly modulated the amplification. Use of high-resolution electrophoresis for the analysis of the integrated amplified sequences revealed a recurring novel pattern, consisting of a ladder with numerous bands separated by a constant distance approximately the size of the Py genome. We suggest that this pattern was generated by conversion of the amplified viral genomes to head to tail linear arrays with cell to cell variations in the number of genome repeats at single, transformant-specific, chromosomal sites. In light of the known "out of schedule" firing of the Py origin, we propose an "onion skin" structure intermediate and present a homologous recombination model for the conversion from onion skins to linear arrays. The relevance of the in situ amplification of the Py genome to cellular gene amplification is discussed. Finally, these results clarify our understanding of the integration of the Py genome in rat cells. They suggest that, in most cases, the multiple bands previously described in Py-transformants are likely to reflect genome amplification rather than multiple independent integration events, as assumed in the past. This interpretation is congruent with the accepted view that the integration of the Py genome is a rare and rate-limiting event in transformation.

Animals↗
PubMed2004

LAP2alpha and BAF collaborate to organize the Moloney murine leukemia virus preintegration complex.

Integration of viral DNA into the host genome is an essential step in retroviral replication. The viral DNA made by reverse transcription is a component of the preintegration complex (PIC) that also contains the viral integrase protein, the enzyme that integrates the viral DNA. Several other viral and cellular proteins are present in the PIC, but their functional roles are less well established. Barrier-to-autointegration factor (BAF) is a cellular protein component of the PIC that blocks autointegration of the viral DNA and stimulates intermolecular integration. In uninfected cells, BAF interacts with members of the LEM family of inner nuclear membrane and nucleoplasmic proteins. Here, we demonstrate that one of the LEM proteins, lamina-associated polypeptide 2alpha (LAP2alpha), is a component of the PIC. LAP2alpha stabilizes the association of BAF with the PIC to stimulate intermolecular integration and suppress autointegration. To further understand the role of LAP2alpha, we established LAP2alpha-knockdown cell lines. Depletion of LAP2alpha significantly inhibited viral replication. Our results demonstrate a critical contribution of LAP2alpha to the nucleoprotein organization of the PIC and to viral replication.

Animals↗
PubMed1984

Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.

Adenine Phosphoribosyltransferase↗
PubMed2002

Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences.

BACKGROUND: In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. METHODS: Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. RESULTS: Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. CONCLUSIONS: Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA.

Binding Sites↗
PubMed1986

Molecular cloning and physical characterization of integrated equine infectious anemia virus: molecular and immunologic evidence of its close relationship to ovine and caprine lentiviruses.

Molecular clones of the integrated form of the genome of equine infectious anemia virus (EIAV), the etiologic agent of a naturally occurring, worldwide disease of horses, were obtained. The restriction map of a full-length genome was determined. Additional evidence for the close evolutionary relationship between EIAV and a prototype lentivirus (caprine arthritis encephalitis virus) was acquired by Southern blotting and immunological analyses. An interspecies radioimmunoassay was developed in which EIAV and ovine and caprine lentiviruses could be detected equally well. These studies make available precisely defined reagents to pursue the study of the mechanisms of pathogenesis of lentiviral induced diseases.

Animals↗
PubMed2006

Genomic sites of human immunodeficiency virus type 2 (HIV-2) integration: similarities to HIV-1 in vitro and possible differences in vivo.

Retroviruses have distinct preferences in integration site selection in the host cell genome during in vitro infection, with human immunodeficiency virus type 1 (HIV-1) integration strongly favoring transcriptional units. Additionally, studies with HIV-1 have shown that the genomic site of proviral integration may impact viral replication, with integration in heterochromatin associated with a block in viral transcription. HIV-2 is less pathogenic than HIV-1 and is believed to have a lower replication rate in vivo. Although differences in integration site selection between HIV-2 and HIV-1 could potentially explain the attenuated pathogenicity of HIV-2, no studies have characterized integration site selection by HIV-2. In this study, we mapped 202 HIV-2 integration sites during in vitro infection of peripheral blood mononuclear cells with a primary HIV-2 isolate. In addition, we assayed for in vivo proviral integration within heterochromatin in 21 HIV-1-infected subjects and 23 HIV-2-infected subjects, using an alphoid repeat PCR assay. During in vitro infection, HIV-2 displayed integration site preferences similar to those previously reported for HIV-1. Notably, 82% of HIV-2 integrations mapped to Refseq genes, and integration strongly favored regions of the genome with high gene density and high GC content. Though rare, the proportion of HIV-2 subjects with evidence of proviral integration within heterochromatin in vivo was higher than that of HIV-1-infected subjects. It is therefore possible that integration site selection may play a role in the differences in HIV-1 and HIV-2 in vivo pathogenesis.

Base Composition↗
PubMed1992

Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus.

In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.

Base Sequence↗
PubMed1979

Mode of integration of Epstein-Barr virus genome into host DNA in Burkitt lymphoma cells.

The ebv dna integrated into the host genome from cell of an African Burkitt lymphoma biopsy has been compared to the corresponding fraction of EBV DNA from the cell line SU-AmB-2 which is of American Burkitt lymphoma origin. It is shown that while, in the case of the African biopsy, large pieces of EBV DNA sequences are integrated into the host DNA, much smaller pieces of virus DNA are integrated into the DNA of the SU-AmB-2 cells. The possibility that this difference might be related to the fact that EBV is rarely associated with Burkitt lymphomas outside East Africa is discussed.

Biopsy↗