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At least 757 records · Page 42Linked to original sources
PubMed1990

Integration of human immunodeficiency virus type 1 DNA in vitro.

A highly efficient cell-free system for the integration of human immunodeficiency virus type 1 DNA is described. Linear viral DNA synthesis occurs in the cytoplasm of newly infected cells, reaching peak levels 4 hr after infection. The linear viral DNA molecules present in cytoplasmic extracts are capable of integrating into heterologous DNA targets in vitro. The viral DNA resides in a high molecular weight nucleoprotein structure that can be separated from the bulk of cellular protein and nucleic acid without a detectable decrease in the ability to integrate in vitro.

Blotting, Southern↗
PubMed1998

Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection.

The presence of latently infected, resting CD4(+) T cells carrying replication-competent HIV-1 has been demonstrated in chronically infected individuals who are antiretroviral therapy naive as well as in those who are receiving highly active antiretroviral therapy (HAART). It is not clear, however, whether the establishment of a pool of latently infected CD4(+) T cells can be blocked by early initiation of HAART after primary infection. The present study demonstrates that initiation of HAART in infected individuals as early as 10 days after the onset of symptoms of primary HIV-1 infection did not prevent generation of latently infected, resting CD4(+) T cells carrying integrated HIV-1 DNA as well as infectious HIV-1 despite the successful control of plasma viremia shortly after institution of HAART. Furthermore, there was no correlation between either the duration of HAART at the time of study (range: 0.2-17 months) or the time of initiation of HAART after the onset of symptoms of primary HIV-1 infection (range: 0.3-4 months) and the frequencies of resting CD4(+) T cells carrying either integrated HIV-1 DNA or infectious virus. These results underscore the rapidity with which latent reservoirs are established in primary HIV-1 infection and indicate that it is unlikely that early treatment during primary infection can prevent establishment of a pool of latently infected, resting CD4(+) T cells as long as treatment is initiated after plasma viremia becomes evident.

Anti-HIV Agents↗
PubMed2005

Differential integration rates of hepatitis B virus DNA in the liver of children with chronic hepatitis B virus infection and hepatocellular carcinoma.

BACKGROUND AND AIM: Integration of hepatitis B virus-DNA (HBV-DNA) into the host genome, a phenomenon found frequently in hepatocellular carcinomas (HCC) and causally linked to oncogenesis, has not been well characterized in children. The aim of the present study was to determine the prevalence of HBV integration more accurately and to decide whether the integration rate varies at different stages of chronic HBV infection in children. METHODS: Of 13 children with chronic hepatitis, 14 liver biopsy tissues were analyzed. One liver tissue with pure liver cirrhosis, nine non-tumor, and nine tumor liver tissues from children with HCC were analyzed by a very sensitive method, inverse polymerase chain reaction (IPCR). RESULTS: Thirteen genuine viral-host junctional sequences from 23 patients were successfully isolated and proved that IPCR is a useful method in this context. The results also indicated that the detection rate of HBV-DNA integration increased in parallel with the progress of liver histology towards the neoplastic transformation, with 0% in the liver of chronic hepatitis, 22.2% in non-tumor livers of HCC patients, and 66.7% in tumor liver tissues of HCC patients. CONCLUSION: The present results indicate that integration of HBV-DNA into the host genome was rarely confirmed at the early stage of chronic hepatitis in children until the stage of HCC formation.

Carcinoma, Hepatocellular↗
PubMed1991

The isopycnic, compartmentalized integration of Rous sarcoma virus sequences.

Rous sarcoma virus (RSV) can cause tumors in hamsters, which harbor complete or partially deleted RSV sequences, in their genomes. Here we have studied the localization of RSV sequences integrated into the genome of cell lines derived from six independent hamster tumors. We have found that integration occurred in the isochores richest in guanine + cytosine, of the host genome, as it had been previously observed for bovine leukemia and hepatitis B viral sequences. The integration of RSV proviral sequences is, therefore, 'isopycnic' (i.e., it takes place in host genome sequences which compositionally match the viral sequences) and compartmentalized (i.e., it occurs in a small compositional compartment of the host genome). The hamster genome compartment hosting RSV sequences precisely corresponds to a compartment of the human genome which is the most active in both transcription and recombination. The notion of a compartmentalized, isopycnic integration of RSV proviral sequences fits, therefore, with the viral integration into transcriptionally active and recombinogenic regions of the host genome observed by other authors, but is broader, in that it includes, in addition, the requirement for a compositional match between host genome sequences and expressed viral sequences.

Animals↗
PubMed1991

Targeted integration of adeno-associated virus (AAV) into human chromosome 19.

A key feature in adeno-associated virus (AAV) replication is efficient integration of the viral genome into host cell DNA to establish latency when helper virus is absent. The steps involved in this process remain largely uncharacterized, even though AAV integration was first documented 20 years ago. Using a protein--DNA binding method we isolated AAV--cellular junction DNA sequences. The cellular component hybridized to a single restriction fragment in the virus-free parental cell line, and also co-migrated with AAV-specific sequences in numerous latently infected cell lines. Analysis of somatic cell hybrids indicated that this cellular sequence maps to the distal portion of the q arm of human chromosome 19. In situ hybridization of AAV DNA to chromosomes from latently infected cells confirms the physical location of AAV integrations to be q13.4-ter of chromosome 19. Sequence analysis of several independent integration sites shows breakpoints occurring within a 100 bp cellular region. This non-pathogenic parvovirus thus appears to establish viral latency by integrating its DNA specifically into one chromosomal region. Such specific integration is so far unique among the eukaryotic DNA viruses. The incorporation of site-specific integration into AAV vector schemes should make this vector system attractive for human gene therapy approaches.

Base Sequence↗
PubMed1979

Genetic transmission of Moloney leukemia virus: mapping of the chromosomal integration site.

Mice genetically transmitting the exogenous Moloney leukemia virus (Balb/Mo) have been previously derived. These animals carried one copy of Moloney virus DNA (M-MuLV) in their germ line and transmitted the virus as a single Mendelian gene to the next generation. Homozygous BALB/Mo mice were used to genetically map the M-MuLV locus. Embryo fibroblasts were fused to established Chinese hamster cells and somatic cell hybrids were selected. Segregation of mouse chromosomal markers in the hybrids was correlated to the loss of M-MuLV-specific sequences as detected by molecular hybridization. Of 15 isozymes located on different mouse chromosomes only triosephosphate isomerase segregated syntenic with the M-MuLV gene, suggesting that the virus was integrated on chromosome No. 6. This was confirmed by sexual genetic experiments analyzing segregation of Moloney viremia and two markers on chromosome 6 and 15, respectively. The results show that M-MuLV expression is linked to wa-1 on chromosome 6 at a distance of about 30 map units. These data define a new genetic locus, Mov-1, representing the structural gene of M-MuLV in BALB/Mo mice.

Animals↗
PubMed1998

Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid repeats are a disfavored target.

Integration of retroviral cDNA into host chromosomal DNA is an essential and distinctive step in viral replication. Despite considerable study, the host determinants of sites for integration have not been fully clarified. To investigate integration site selection in vivo, we used two approaches. (i) We have analyzed the host sequences flanking 61 human immunodeficiency virus type 1 (HIV-1) integration sites made by experimental infection and compared them to a library of 104 control sequences. (ii) We have also analyzed HIV-1 integration frequencies near several human repeated-sequence DNA families, using a repeat-specific PCR-based assay. At odds with previous reports from smaller-scale studies, we found no strong biases either for or against integration near repetitive sequences such as Alu or LINE-1 elements. We also did not find a clear bias for integration in transcription units as proposed previously, although transcription units were found somewhat more frequently near integration sites than near controls. However, we did find that centromeric alphoid repeats were selectively absent at integration sites. The repeat-specific PCR-based assay also indicated that alphoid repeats were disfavored for integration in vivo but not as naked DNA in vitro. Evidently the distinctive DNA organization at centromeres disfavors cDNA integration. We also found a weak consensus sequence for host DNA at integration sites, and assays of integration in vitro indicated that this sequence is favored as naked DNA, revealing in addition an influence of target primary sequence.

Binding Sites↗
PubMed1988

Large scale production of hepatitis A virus in cell culture: effect of type of infection on virus yield and cell integrity.

Approaches to cell culture propagation of hepatitis A virus (HAV) have used either acute infection by passage of infected cell lysates or supernatants into uninfected cells or the passage of persistently infected cells. The findings presented here demonstrate that the growth and recovery of purified virus from foetal rhesus monkey kidney (FRhK4) cells persistently infected with HAV isolate HAS-15 decreased over a 2 to 3 month period. In contrast, high multiplicity acute infection of FRhK4 cells with purified HAS-15 HAV resulted in degeneration of the cell monolayer 2 to 3 weeks later. Large scale propagation of acutely infected cells followed by traditional picornavirus purification procedures reproducibly yielded milligram amounts of purified virus.

Animals↗
PubMed1990

Plus-strand origin for human immunodeficiency virus type 1: implications for integration.

The start site for human immunodeficiency virus type 1 plus strands within the polypurine tract was mapped by an in vitro analysis to the sequence 5'-ACTG....From this result, it can be inferred that integration of human immunodeficiency virus type 1 must be accompanied by the loss of two base pairs from the polypurine tract-primed long terminal repeat end.

Avian Sarcoma Viruses↗
PubMed2000

Crystal structure of an active two-domain derivative of Rous sarcoma virus integrase.

Integration of retroviral cDNA is a necessary step in viral replication. The virally encoded integrase protein and DNA sequences at the ends of the linear viral cDNA are required for this reaction. Previous studies revealed that truncated forms of Rous sarcoma virus integrase containing two of the three protein domains can carry out integration reactions in vitro. Here, we describe the crystal structure at 2.5 A resolution of a fragment of the integrase of Rous sarcoma virus (residues 49-286) containing both the conserved catalytic domain and a modulatory DNA-binding domain (C domain). The catalytic domains form a symmetric dimer, but the C domains associate asymmetrically with each other and together adopt a canted conformation relative to the catalytic domains. A binding path for the viral cDNA is evident spanning both domain surfaces, allowing modeling of the larger integration complexes that are known to be active in vivo. The modeling suggests that formation of an integrase tetramer (a dimer of dimers) is necessary and sufficient for joining both viral cDNA ends at neighboring sites in the target DNA. The observed asymmetric arrangement of C domains suggests that they could form a rotationally symmetric tetramer that may be important for bridging integrase complexes at each cDNA end.

Amino Acid Sequence↗
PubMed2003

The replication strategy of foamy viruses.

The replication strategy of foamy viruses diverges in many aspects from what is commonly accepted as the rules of retroviral replication. Although many questions on the details of the replication pathway are still unanswered, it appears that foamy viruses have adopted a strategy which functionally bridges the retroviral and the hepadnaviral replication pathways. A number of experimental findings in favour of the view that foamy viruses are reverse transcribing DNA viruses which integrate into the host cell genome are discussed.

Animals↗
PubMed1998

Relationship of stable integration of herpes simplex virus-2 Bg/II N subfragment Xho2 to malignant transformation of human papillomavirus-immortalized cervical keratinocytes.

Transfection of the right end Xho2 subfragment of Bg/II N of herpes simplex virus-2 (HSV-2) into human genital keratinocytes immortalized by human papillomavirus (HPV) type 16 or 18 resulted in invasive and noninvasive indolent cystic squamous carcinomas when cells were injected into immunocompromised mice. Retention and expression of the right end portion of the Bg/II N fragment correlated with malignancy, as the corresponding HSV-2 sequences were integrated and transcribed in the tumorigenic cell lines. HPV-immortalized cells alone were not tumorigenic. In contrast, previous results have shown that using the entire Bg/II N region can malignantly transform HPV-immortalized cells, although HSV2 DNA was not retained. Together, these observations localize the transforming activity of Bg/II N to Xho2 and suggest that the remaining sequences have an inhibitory effect on stable integration. The Xho2 sequence is 2480 bp long and contains an open reading frame (ORF) extending from nucleotides 559 to 1797. The ORF encodes a putative protein of 412-aa with a m.w. of 42-43 kDa and is highly homologous to UL43 of HSV-I. The correlation of tumorigenicity with stable integration and expression of Xho2 DNA in HPV-immortalized cells indicates that HSV-2 should be investigated further for a possible role in cervical cancer.

Amino Acid Sequence↗
PubMed1993

Down-regulation of integrated Epstein-Barr virus nuclear antigen 1 and 2 genes in a Burkitt lymphoma cell line after somatic cell fusion with autologous EBV-immortalized lymphoblastoid cells.

Epstein Barr virus (EBV) latent gene expression was analyzed in somatic cell hybrids between an EBV-positive Burkitt lymphoma (BL) cell line (BL 60) and an autologous EBV-immortalized lymphoblastoid cell line (LCL, IARC 277). The EBV genomes carried by the parental cell lines differ in sequence and in their physical state. The BL 60 EBV genome is integrated into the host cell genome whereas the LCL IARC 277 carries exclusively episomal EBV molecules. The hybrid cells contain both EBV genomes and display the differentiation phenotype of the parental LCL with regard to growth characteristics and cell-surface antigen expression in vitro and in vivo. While the EBNA 1 and EBNA 2 gene expression of the LCL-derived EBV is maintained in these hybrid cells, the BL-60-derived EBNA 1 and EBNA 2 genes are transcriptionally down-regulated. Mapping of the genomic region surrounding the latent Cp promoter of the BL-60-derived EBV revealed an extensive deletion upstream of the Cp promoter including the enhancer element in the ori P region, the origin of latent viral replication (ori P), the coding sequences for the EBV latent membrane protein (LMP) and the EBV terminal protein (TP), and suggested that one viral-cellular junction sequence is located near the Cp promoter. Integration of EBV into the host cell genome together with the extensive deletion might be causally related with the altered latent gene expression pattern after introduction of a lymphoblastoid host-cell background by somatic cell fusion. Down-regulation of the BL-60-derived EBNA genes could be due to loss of regulatory sequences in the BL-derived EBV necessary for EBNA 1 and EBNA 2 transcription in the lymphoblastoid hybrid cells, but not in the parental BL cells.

Antigens, Viral↗
PubMed1995

Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome.

A model system using an episomal Epstein-Barr virus shuttle vector was recently developed to study the adeno-associated virus (AAV) site-specific integration event in chromosome 19q13.3-qter (C. Giraud, E. Winocour, and K.I. Berns, Proc. Natl. Acad. Sci. USA 91:10039-10043, 1994). In this study, we analyze the recombinant junctions generated after integration of the AAV genome into an Epstein-Barr virus shuttle vector carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration sequence (AAVS1 locus). In most of the recombinants, one end of the viral genome was joined to a portion of the AAVS1 DNA previously shown to be a minimum target for AAV integration. Within this AAVS1 segment, the AAV insertion points were strikingly clustered around a binding site for the AAV regulatory protein. In all cases, the second junction with AAV occurred with vector DNA outside of the AAVS1 segment. With respect to the viral genome, one junction with the shuttle vector DNA occurred either within the AAV inverted terminal repeat (itr), or near the P5 promoter, approximately 100 nucleotides distal to a modified itr. The modified itr in 5 of 11 recombinants involved a head-to-tail organization. In one such instance, the AAV insert contained slightly more than one genome equivalent arranged in a head-to-tail manner with a junction close to the P5 promoter; the AAV insert in this recombinant episome could be rescued by adenovirus infection and replicated to virus particles. The significance of the head-to-tail organization is discussed in terms of the possible circularization of AAV DNA before or during integration.

Adenoviruses, Human↗
PubMed1988

Isolation of an integrated provirus of Moloney murine leukemia virus with long terminal repeats in inverted orientation: integration utilizing two U3 sequences.

We have previously described the construction of a mutant of Moloney murine leukemia virus, in594-2, which carries a 2-base-pair insertion in the U5 region of the genome and is partially defective in forming the integrated proviral DNA. We have now recovered a cloned copy of an unusual provirus from rat cells infected with this mutant. The viral genome is flanked by long terminal repeats in inverted orientation, with U3 sequences joined to cellular DNA at both of the outer edges. In addition, the provirus is a recombinant, containing a segment of a VL30 element in inverted orientation in place of the Moloney murine leukemia virus env region. The recovery of this provirus indicates that two U3 regions can be used for viral integration and suggests that there may be no absolute requirement in the reaction for those U5 sequences outside the 13-base-pair inverted repeats.

Amino Acid Sequence↗
PubMed1984

DNA sequence studies of simian virus 40 chromosomal excision and integration in rat cells.

Cell fusion between simian CV1 cells and the simian virus 40-transformed rat cell line 14B, which contains a single copy of integrated simian virus 40 DNA, results in chromosomal excision of viral DNA. A heterogeneous population of circular molecules containing both viral and cellular DNA is detected in the replicating pool. We present the DNA sequences across six novel junctions created by these excision events and use this information to define the parental genomic sequences involved in this form of "illegitimate" recombination. The data were analyzed to discover whether any common structural feature(s) could be detected at these sites. In each case a redundancy of either two or three base-pairs was found at the precise points of cross-over in both parental DNA molecules. The cross-over points were further distinguished by the presence of at least one copy of the sequence 5'Pyr-T-T3' in either of the homologous sequences that define the cross-over points. Additional stretches of homology are found extending from the homologous cross-over points. To explore the possibility that the selection of the cross-over sites is determined by the free energy of base-pairing, we have used the program of Zuker & Stiegler (1981) to form model heteroduplexes between single-stranded parental DNA molecules. In some cases model heteroduplexes were formed that paired the cross-over points, although these structures were of dubious thermodynamic stability. We therefore conclude that, while the redundancies at the cross-over points must play some role in these processes, other factors aside from simple base-pairing across replicating structures must also be involved. In order to expand our analysis of the recombination events that accompany transformation of rat cells by simian virus 40, we determined the DNA sequences across one of the sites on the rat genome that served as the target for the integration event that engendered the 14B line. Our analysis of this DNA showed that: (1) viral and chromosomal DNA share three base-pairs of homology at the site of cross-over; (2) the cross-over site in the rat genome is adjacent to the trinucleotide 5'Pyr-T-T3'; and (3) the homology shared by the virus and chromosome does not resemble the homology reported at another integration locus, but is similar in that it is flanked on one side by alternating purine and pyrimidine nucleotides.

Animals↗