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PubMed1989

Integrated proviral human immunodeficiency virus type 1 is present in CD4+ peripheral blood lymphocytes in healthy seropositive individuals.

Evidence of a latent human immunodeficiency virus type 1 (HIV-1) infection in healthy, seropositive individuals who do not have viral antigens in their sera and from whom virions cannot be rescued in cocultivation experiments was examined. Proviral DNA was detected by amplification by the polymerase chain reaction procedure. In each of 10 seropositive individuals, the presence of HIV-1 proviral sequences was demonstrated in their peripheral blood mononuclear cells. By using fluorescence-activated cell sorting, we obtained highly enriched subpopulations of peripheral blood mononuclear cells and found that the CD4+ T-cell subset is the cell subset that consistently harbors the HIV-1 proviral sequences. The number of HIV-1-infected CD4+ T cells was variable among the 10 healthy individuals, ranging from 1 in 100 to 1 in 40,000. While in vitro infection of CD4+ T cells causes down regulation and eventual loss of CD4 surface molecules, this is not true in vivo where it is only the CD4+ population that harbors the virus. This disparity may reflect differences between a latent infection in vivo with the lytic response of cells infected in vitro.

Antibodies, Monoclonal↗
PubMed2003

An amino acid in the central catalytic domain of three retroviral integrases that affects target site selection in nonviral DNA.

Integrase can insert retroviral DNA into almost any site in cellular DNA; however, target site preferences are noted in vitro and in vivo. We recently demonstrated that amino acid 119, in the alpha2 helix of the central domain of the human immunodeficiency virus type 1 integrase, affected the choice of nonviral target DNA sites. We have now extended these findings to the integrases of a nonprimate lentivirus and a more distantly related alpharetrovirus. We found that substitutions at the analogous positions in visna virus integrase and Rous sarcoma virus integrase changed the target site preferences in five assays that monitor insertion into nonviral DNA. Thus, the importance of this protein residue in the selection of nonviral target DNA sites is likely to be a general property of retroviral integrases. Moreover, this amino acid might be part of the cellular DNA binding site on integrase proteins.

Amino Acid Sequence↗
PubMed1987

Hepatitis B virus (HBV)-DNA-positive hepatocellular carcinoma following hepatitis B virus infection in a child.

Integrated hepatitis B virus (HBV) DNA sequences were found in neoplastic liver tissue of a hepatitis B surface antigen (HBsAg)-negative child who had previously suffered from HBsAg-positive chronic active hepatitis and was anti-HBs and anti-hepatitis B core (HBc) positive at the time of tumor development. Reintegration pattern was consistent with the presence of a single integration site of the HBV genome into cellular DNA, and clonal proliferation of such infected cells. A normal liver, tested in the same experiment with the same amount of total DNA, was negative for viral DNA sequences. These findings support the possible oncogenic role of HBV in the development of liver cancer, not only in adults, but also in children, even in patients who are negative for HBsAg at the time of tumor diagnosis.

Carcinoma, Hepatocellular↗
PubMed1994

Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration.

Adeno-associated virus type 2 (AAV2)-based vectors are capable of stable expression in the airway epithelium and may be useful for gene therapy for human diseases, such as cystic fibrosis. Certain virus vectors, such as retroviruses, require active cell division for integration and expression, but this has not been formally evaluated in the case of AAV2. The cystic fibrosis bronchial epithelial cell line, IB3-1, which can be transduced by AAV2 vectors, was shown to undergo a decrease in DNA synthesis to undetectable levels when grown to confluence. Cultures in which < 0.1% of cells were dividing could still be efficiently transduced with AAV-lacZ or AAV-neo vectors, with a linear dose response, up to 91% with a multiplicity of 3,000 vector particles per cell. The fate of vector DNA in nondividing target cells was investigated by Southern blotting of both low molecular weight, nonintegrated DNA and high molecular weight, genomic DNA fractions. Detectable levels of vector DNA were only seen in the nonintegrated state. These results indicate that AAV2-based vectors, unlike retrovirus vectors, do not require active cell division or integration for expression to occur and thus possess a unique profile of biologic properties.

Bronchi↗
PubMed1981

The site of integration of the herpes simplex virus type 1 thymidine kinase gene in human cells transformed by an HSV-1 DNA fragment.

To analyze the site of integration of the herpes simplex virus type I (HSV-I) thymidine kinase (TK) gene in biochemically transformed human cells, TK-HeLa-(BU25) cells were transformed to the TK+ phenotype by a cloned, 2 kbp Pvull fragment of HSV-I DNA. The transformed cells [HeLa(BU25)/TF pAGO PP3] were fused with mouse LM(TK-) cells, and human-mouse somatic cell hybrid clones (LH PP3 clones 1, 2, 3, 5 and 6) were isolated in HATG-ouabain selective medium. The HeLa(BU25)/TF pAGO PP3 cells and the LH PP3 hybrid clones expressed HSV-I specific TK activity and a herpesvirus-associated nuclear antigen, and contained herpesvirus nucleotide sequences. Molecular hybridization experiments were carried out to map the HSV-I and flanking cellular nucleotide sequences in the biochemically transformed cells. These experiments demonstrated that the HSV-I nucleotide sequences were integrated at a single site, and that the same cellular nucleotide sequences flanked the viral DNA in transformed HeLa(BU25)/TF pAGO PP3 and LH PP3 clone 5 cells. TK- revertant subclones isolated by growing the LH PP3 clone 5 cells in BrdUrd (and diphtheria toxin) failed to form colonies in HATG medium, but retained HSV-I nucleotide sequences. Isozyme analyses on 21 gene-enzyme systems representing 21 human chromosomes revealed that all of the LH PP3 clonal lines expressed human hexosaminidase B, which has been assigned to chromosome 5, and all were sensitive to diphtheria toxin, which is also a marker for chromosome 5. Chromosome analyses showed that chromosome 5 was the nly human chromosome present in mitoses of LH PP3 clone 5 cells and that human chromosome 5 was present in most of the mitoses of LH PP3 clone 1, 2, 3, and 6 cells. The latter clones also contained 1 or 2 additional human chromosomes in some of the cells. As expected from the molecular hybridization analyses, TK- revertants of LH PP3 clone 5 cells retained portions of chromosome 5 and expressed human hexosaminidase B. The results indicate that HSV-I nucleotide sequences were stably integrated in the biochemically transformed cells, most likely in human chromosome 5.

Animals↗
PubMed1999

The open reading frame 3 of equine arteritis virus encodes an immunogenic glycosylated, integral membrane protein.

Open reading frame 3 (ORF 3) of equine arteritis virus (EAV) is predicted to encode a glycosylated membrane protein (GP3) that is uncharacterized. ORF 3 of the American Type Culture Collection strain of EAV was in vitro transcribed and the encoded GP3 protein was in vitro translated with and without canine microsomal membranes. The GP3 protein was approximately 17 kDa after in vitro translation without canine microsomal membranes whereas the glycosylated form, after translation with microsomal membranes, was a diffuse band of 36-42 kDa, indicating that the GP3 protein is extensively glycosylated. Deglycosylation reduced the GP3 protein to approximately 17 kDa, the same size as that translated without microsomal membranes, indicating that the signal sequence was not cleaved. The EAV GP3 protein was membrane associated and not released as a soluble protein, in marked contrast to the ORF 3-encoded proteins of some other arteriviruses. The GP3 protein was protected from protease digestion in closed membrane vesicles, suggesting that the protein extends into the membrane vesicles and is anchored by the N-terminal signal sequence, a C-terminal hydrophobic domain, or both, but does not span the membrane three times. A GP3 protein lacking the C-terminal transmembrane domain remained membrane associated, indicating that this terminus is not a necessary membrane anchor. Sera from stallions persistently infected with EAV and horses immunized repeatedly with the modified live EAV vaccine contained antibodies specific for the GP3 protein. The data indicate that the GP3 protein is an extensively glycosylated membrane protein that is immunogenic during some EAV infections.

Animals↗
PubMed1988

Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma.

The structure of integrated viral DNA in a hepatocellular carcinoma of a duck from Chi-tung county in China was analyzed. Three different clones of integrated viral DNA, lambda DHS 6-1, lambda DHS 6-2, and lambda DHE 6-2, were obtained from the neoplastic portion of the liver by molecular cloning. One of the three clones, lambda DHS 6-1, showed inverted repetition of integrated viral DNA with chromosomal flanking sequences. Another clone, lambda DHS 6-2, showed a head-to-head configuration of the core and surface gene regions of duck hepatitis B virus (DHBV) DNA. The virus-chromosome junctions were often located near direct repeat 1 or 2 of DHBV DNA in three independent clones. Nucleotide sequences at the virus-virus junctions in two clones, lambda DHS 6-1 and 6-2, indicated the possible rearrangement of chromosomal DNA and recombination of viral DNA. DHBV DNA appears to be integrated into the genome of hepatocytes in a manner similar to that of human and woodchuck hepatitis viruses. Thus, the duck system may serve as a useful animal model for the study of human hepatocarcinogenesis.

Animals↗
PubMed2004

Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration.

Integrated hepadnaviral DNA in livers and tumors of chronic hepatitis B patients has been reported for many years. In this study, we investigated whether hepatitis B virus DNA integration occurs preferentially at sites of cell DNA damage. A single I-SceI homing endonuclease recognition site was introduced into the DNA of the chicken hepatoma cell line LMH by stable DNA transfection, and double-strand breaks were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. Alteration of the target cleavage site by imprecise nonhomologous end joining occurred at a frequency of approximately 10(-3) per transfected cell. When replication of an avian hepadnavirus, duck hepatitis B virus, occurred at the time of double-strand break repair, we observed integration of viral DNA at the site of the break with a frequency of approximately 10(-4) per transfected cell. Integration depended on the production of viral double-stranded linear DNA and the expression of I-SceI, and integrated DNA was stable through at least 17 cell divisions. Integration appeared to occur through nonhomologous end joining between the viral linear DNA ends and the I-SceI-induced break, because small deletions or insertions were observed at the sites of end joining. The results suggest that integration of hepadnaviral DNA in infected livers occurs at sites of DNA damage and may indicate the presence of more widespread genetic changes beyond that caused by viral DNA integration itself [corrected].

Animals↗
PubMed1995

[Effect of erythroid enhancer on the expression of beta-globin gene in mice erythroleukemia (MEL) cells].

Our previous works have verified that the beta-globin gene carrying large fragments of erythroid enhancer transferred by retrovirus vector caused the unstable provirus integration and low virus titer in infected cells, but the 36bp enhancer had not this negative effect. In order to circumvent this problem, we inserted the intact beta-globin gene (beta) or partially IVS II deleted beta-globin gene (delta beta) and truncated erythroid enhancer (36bp, 292bp and 341bp) into the N2A retrovirus vector. Recombinants were transfected into psi-2 ecotropic pachaging cells first, then the produced virus were used to infect PA317 amphotropic packaging cells. Virus supernatent from PA317 clonies with high virus titer and intact provirus integration was used to infect MEL cells. RNase protection assay was used to detect the expression of beta-globin gene. Results showed that not only the stable provirus integration and high virus titer of the transferred genes, but also the high levels expression of beta-globin gene carrying 292bp or 341bp erythroid enhancer were got.

Animals↗
PubMed2002

Characterization of integration patterns and flanking cellular sequences of hepatitis B virus in childhood hepatocellular carcinomas.

Hepatitis B virus (HBV) DNA integration into host chromosomes is detected in more than 80% of HBV-related hepatocellular carcinomas (HCC), yet its significance in tumor development remains obscure. In this study, we re-examined the integration pattern of HBV in childhood HCC tissues, which has less environmental confounding factors than adult HCC. The HBV junctions and flanking cellular sequences were amplified from five childhood HCC patients by the inverse polymerase chain reaction (IPCR) method using primers located near HBV direct repeats (DR) 1 and 2. The viral junctions in nine of the ten obtained IPCR clones were demonstrated to be located near HBV DR1, and their patterns were classified to type I integrants. Southern blot analyses demonstrate that the cellular junctions derived from two of the five HCC tissues were male specific and contained sequences homologous to human long interspersed DNA elements (LINE-1). HBV integrant of one HCC tissue (1217T) was integrated into a RNA binding motif Y chromosome (RBMY) gene. The expression of RBMY, which is normally found only in male germ cells, was detected in HCC tissue 1217T by RT-PCR but not in the corresponding non-tumor liver tissue. The prevalence of RBMY expression in liver tissues from the tumor and non-tumor parts of ten other HCC children and seven biliary atresia (BA) children was studied by RT-PCR. No RBMY transcripts were detected in the non-tumor parts of HCC patients or the cirrhotic livers of BA children, whereas 30% (three of ten) of HCC tissues specifically expressed RBMY. The results indicate that HBV integration and activation of RBMY gene expression in liver cells may be associated with the development of childhood HCC.

Base Sequence↗
PubMed2004

Integrated cell culture/PCR for detection of enteric viruses in environmental samples.

Recently, an integrated cell culture/polymerase chain reaction (ICC/PCR) technique has been developed for the detection of viruses in environmental samples providing a reliable method for practical analysis and direct monitoring of environmental samples for viral pathogens. CC/PCR allows for detection of infectious viruses in hours to days compared with the days or weeks necessary with cell culture alone. Bacterial indicator organisms are commonly used to evaluate environmental samples with respect to fecal contamination and potential public health impacts. These organisms do not correlate well with the presence of viruses, but a rapid, reliable method was not previously available for direct virus testing. Using ICC/PCR, environmental samples may be directly surveyed for pathogenic viruses, in a timely manner. Direct virus analysis will lead to better assessment of the presence and risk of human enteric viruses in the environment, so that control measures may be developed with true virus occurrence data. The ICC/PCR approach combines two previously applied virus detection methods, conventional cell culture and PCR amplification, utilizing the major advantages and overcoming the major limitations of each methodology when used alone. Cell culture assay is the standard method for the detection of viable human viruses (i.e., poliovirus, coxsackievirus, echovirus, adenovirus, hepatitis A virus, reovirus, and rotavirus) in environmental samples, serving as the method against which all newer technologies are evaluated. Although cell culture is theoretically capable of detecting a single viable virus in relatively large volumes of sample, the time required for confirmed results with conventional cell culture makes it an impractical method for routine monitoring of environmental samples. Furthermore, cell culture does not detect noncytopathogenic viruses (viruses that are viable, infecting cells, and continually spreading to neighboring cells but that do not cause a visible cytopathogenic effect [CPE] on the cell monolayer). Rotavirus and most wild-type hepatitis A viruses (HAV) are infectious to cell cultures but do not produce a clear CPE.

Animals↗
PubMedDate not supplied

Target integration by a chimeric Sp1 zinc finger domain-Moloney murine leukemia virus integrase in vivo.

A specificity protein 1 (Sp1) zinc finger domain containing two tandem zinc fingers was fused to the C terminus of the integrase (IN) protein of the Moloney murine leukemia virus (MuLV). The integrity of the MuLV IN was completely preserved, since the fusion was conducted at the last amino acid residue of the protein. The vector pMIN-Sp1, which carried the fused MuLV IN-Sp1 zinc finger domain gene, was cotransfected with a wild-type MuLV vector pMLV-K to NIH/3T3 cells. A nonradioactive reverse transcriptase assay was performed on culture supernatants collected from the cotransfected cells to confirm the production of recombinant viruses. The expression of the fusion protein and the integration of the MuLV genome by the fusion protein were confirmed by a Northern and then a Southern hybridization analysis on the total RNA or genomic DNA extracted from cells infected by viruses collected from the supernatants of the cotransfected cells. Regions of the host chromosome that were selected by the fusion protein as the integration targets were sequenced using the TOPO(TM) cloning method on a series of PCR products generated with a nested set of primers. The percentage of positive clones screened that contained the DNA-binding sequence of the fused Sp1 zinc finger domain was around 13% (5 out of 39 clones). It was found that the Sp1 DNA-binding sequence was only present in regions that were proximal to one of the long terminal repeats of the integrated viral genome, suggesting that the fusion protein could select a target sequence for integration. The host flanking sequences determined for all the positive clones were also used as queries to perform a BLAST search on the GenBank mouse EST entries. Although matching scores for sequences of some of the clones computed were more significant than others, it was difficult to judge whether or not the integration in these clones had been targeted to some gene sequences. Most of the integration sites might exist in the introns, since we found that the probability of the gene sequences containing an Sp1 DNA-binding site was low.

3' Flanking Region↗
PubMed2025

HIV-1 inhibits IFITM3 expression to promote the infection of megakaryocytes.

Despite an undetectable plasma viral load as a result of antiretroviral therapy, HIV-1-infected individuals with poor immune reconstitution harbor infectious HIV-1 within their platelets. Megakaryocytes, as platelet precursors, are the likely cellular origin of these HIV-1-containing platelets. To investigate the mechanisms that allow megakaryocytes to support HIV-1 infection, we established in vitro models of viral infection using hematopoietic stem cell-derived megakaryocytes and the megakaryocytic MEG-01 cell line. We observed HIV-1 DNA provirus integration into the megakaryocyte cell genome, self-limiting virus production, and HIV-1 protein and RNA compartmentalization, which are hallmarks of HIV-1 infection in myeloid cells. In addition, following HIV-1 infection of megakaryocyte precursors, the expression of interferon-induced transmembrane protein 3 (IFITM3), an antiviral factor constitutively expressed in megakaryocytes, was inhibited in terminally differentiated HIV-1-infected megakaryocytes. IFITM3 knockdown in MEG-01 cells prior to infection led to enhanced HIV-1 infection, indicating that IFITM3 acts as an HIV-1 restriction factor in megakaryocytes. Together, these findings indicate that megakaryocyte precursors are susceptible to HIV-1 infection, leading to terminally differentiated megakaryocytes harboring virus in a process regulated by IFITM3. Megakaryocytes may thus constitute a neglected HIV-1 reservoir that warrants further study in order to develop improved antiretroviral therapies and to facilitate HIV-1 eradication.

Humans↗