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At least 289 records · Page 16Linked to original sources
PubMed2006

Chromosomal integration mechanism of infecting mu virion DNA.

DNA transposition is central to the propagation of temperate phage Mu. A long-standing problem in Mu biology has been the mechanism by which the linear genome of an infecting phage, which is linked at both ends to DNA acquired from a previous host, integrates into the new host chromosome. If Mu were to use its well-established cointegrate mechanism for integration (single-strand nicks at Mu ends, joined to a staggered double-strand break in the target), the flanking host sequences would remain linked to Mu; target-primed replication of the linear integrant would subsequently break the chromosome. The absence of evidence for chromosome breaks has led to speculation that infecting Mu might use a cut-and-paste mechanism, whereby Mu DNA is cut away from the flanking sequences prior to integration. In this study we have followed the fate of the flanking DNA during the time course of Mu infection. We have found that these sequences are still attached to Mu upon integration and that they disappear soon after. The data rule out a cut-and-paste mechanism and suggest that infecting Mu integrates to generate simple insertions by a variation of its established cointegrate mechanism in which, instead of a "nick, join, and replicate" pathway, it follows a "nick, join, and process" pathway. The results show similarities with human immunodeficiency virus integration and provide a unifying mechanism for development of Mu along either the lysogenic or lytic pathway.

Bacteriophage mu↗
PubMed2002

A sensitive, quantitative assay for human immunodeficiency virus type 1 integration.

Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.

CD4-Positive T-Lymphocytes↗
PubMed1979

Germ line integration of Moloney leukemia virus: identification of the chromosomal integration site.

The chromosomal integration site of the structural gene of Moloney murine leukemia virus (M-MuLV) in the genome of BALB/Mo mice was mapped genetically. These mice transmit the exogenous M-MuLV as an endogenous virus at a single Mendelian locus. Two independent experimental approaches were used: (i) Non-virus-producing fibroblasts prepared from homozygous BALB/Mo embryos were fused to Chinese hamster Wg3-h-o cells. In an analysis of 30 independent mouse-Chinese hamster cell hybrid clones, the segregation of the viral genome measured by molecular hybridization and enzymes assigned to 16 different mouse chromosomes were compared. We found a highly concordant segregation of M-MuLV sequences and the mouse enzyme triosephosphate isomerase (TPI, EC 5.3.1.1), whose gene has been assigned to chromosome 6. A further karyotype analysis of 9 clones, in which the chromosomes were identified cytochemically, supported this result. (ii) The segregation of the viral genome was studied in backcrosses of BALB/Mo with ABP/J mice. In the backcross ABP/Jx(ABP/JxBALB/Mo) a linkage of the M-MuLV genome to the morphological marker wa-1 on mouse chromosome 6 was found. This confirmed the conclusion that the M-MuLV genome is integrated in mouse chromosome 6. These experiments define the genetic locus Mov-1, denoting the genetically transmitted structural gene of M-MuLV in BALB/Mo mice.

Animals↗
PubMed1994

Chromosomal organization of viral integration sites in human papillomavirus-immortalized human keratinocyte cell lines.

The target specificity of viral integration is essential to determining the biologic significance of this integration to various pathologic conditions, including cancer. In this study the chromosomal features of several human papillomavirus (HPV)-16 integration sites mapped by in situ hybridization in human keratinocyte lines were visualized directly by G-banding and differential labeling with bromodeoxyuridine of later replicating domains. G-negative chromosomal bands exhibiting late replication were selectively targeted by HPV-16, suggesting that the structural and functional relationship of the state of chromatin condensation and replication is critical in accessibility to virus integration.

Attachment Sites, Microbiological↗
PubMed1996

A validatible porosimetric technique for verifying the integrity of virus-retentive membranes.

The verification of membrane integrity for filtration processes specifically designed for the removal of adventitious virus from biotherapeutics is of the utmost importance to both biomanufacturers and regulatory agencies. Although conventional bubble-point and air-diffusion tests are widely accepted for integrity testing of bacteria-retentive membranes, these tests have severe limitations in their ability to assess the integrity of virus-retentive membranes. A novel membrane integrity test based upon liquid-liquid porosimetric principles (CorrTest) has been specifically designed to correlate and predict the virus retention capabilities of Viresolve virus removing membranes. To optimize test sensitivity for both Viresolve/70 and Viresolve/180 membrane types, two distinct porosimetric correlations at different transmembrane pressures have been developed. Based upon an 80% prediction interval, an integrity test performed at either test pressure can reliably predict the ability of Viresolve membranes to remove the bacteriophage phi X174 to within 0.4 log removal value (LRV) units. To maintain test sensitivity and provide greater flexibility for conducting the liquid-liquid intrusion integrity test, appropriate pressure- and temperature-correction equations have been established. The two immiscible fluids employed in the developed technology are easily flushed from the membrane structure and are generally regarded as acceptable, non-toxic reagents for pharmaceutical applications. Consequently, the CorrTest integrity test can reliably and non-destructively measure both pre- and post-use membrane integrity to verify virus removal performance with the Viresolve module.

Bacteriophage phi X 174↗
PubMed1994

Antibodies of symptomatic human immunodeficiency virus type 1-infected individuals are directed to the V3 domain of noninfectious and not of infectious virions present in autologous serum.

The present study was designed to determine the antibody specificity for the human immunodeficiency virus type 1 (HIV-1) V3 domains of infectious and noninfectious virions present in the serum of AIDS patients. To accomplish this, HIV-1 was isolated in the presence of autologous antibodies from the serum samples of six AIDS patients in HIV-1-negative donor peripheral blood mononuclear cells by short-term cultivation. The isolated virus, defined as the infectious cell-free virus (iCFV), was characterized by sequence analysis of the proviral DNA coding for the third hypervariable (V3) region of the external glycoprotein gp120. This was carried out by amplifying and cloning the V3 region. In all six cases studied, 20 randomly selected V3 clones derived from the proviral DNA of the iCFV, 20 clones from patient cell-free virus, and 20 clones from cell-integrated virus were sequenced to study the distribution and frequency of the intrapatient virus population. The number of major virus variants in the six patients ranged from three to nine. The various V3 sequences found in the AIDS patients showed the typical amino acid pattern of the syncytium-inducing and non-syncytium-inducing viral phenotypes characteristic for the late stage of infection. However, only one patient-specific iCFV variant was detected within the 20 V3 clones analyzed per virus isolation. For the six patients a total of 34 V3-loop variants, either iCFV or non-iCFV, was observed. All 34 V3-loop sequences were expressed as glutathione-S-transferase fusion proteins (V3-GST). The autologous antibody response to the V3-GST fusion proteins was studied by Western immunoblot analysis. A strong antibody response to almost all non-iCFV V3-GST proteins was found in the sera of the six patients. In contrast, the autologous antibody response to the six iCFV V3 loops was undetectable (in four patients) or very faint (in two patients) compared with that to the non-iCFV V3 loops. Five of the six iCFV loops showed positively charged amino acids at positions strongly associated with the syncytium-inducing phenotype. These findings suggest that our in vitro isolation system selects for virions which are not recognized by V3-specific antibodies and are infectious both in vitro and in vivo.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Immunopathogenesis of pediatric acquired immunodeficiency syndrome.

Pediatric acquired immunodeficiency syndrome (PAIDS) results from infection with human immunodeficiency virus (HIV). The majority of infants with AIDS are infected in utero. In most instances, mothers are infected from intravenous drug abuse or bisexual partners. Infected individuals, both mothers and infants, may be asymptomatic for prolonged periods of time. Initially viral replication may be limited and the virus integrated into immune cells. Activation of the virus may result from several factors including infection with other viral agents, such as cytomegalovirus and Epstein-Barr virus. Variable progression of disease may be related to dose of virus, the degree of pre-existing immunodeficiency, and the presence of other infectious agents. Recent evidence suggests that individual HIV isolates vary in their capacity to infect cells. This may explain differences in clinical presentation and progression. A diagnosis of HIV infection may be difficult in infants who have persistence of maternal antibody. Viral isolation is often difficult and unreliable, even in infants who have clinical features of AIDS. Currently only azidothymidine has been shown to reduce viral replication and improve prognosis. However, azidothymidine does not eradicate HIV and patients require repeated or chronic therapy. The drug is not yet approved for use in children, although studies are being performed at several medical centers. Prevention of HIV infection is the single most important approach to controlling its rapid spread in infants. Screening of all blood products by using HIV antibody testing is essential. Increased effort in preventing drug abuse and in reducing pregnancy rates among infected women should be a priority of health care workers.

Acquired Immunodeficiency Syndrome↗
PubMed1976

Quantitative studies of integration of murine leukemia virus after exogenous infection.

Using a [3H]DNA probe prepared from AKR murine leukemia virus, we determined the number of copies of the AKR virus genome integrated into the cellular DNA after exogenous infection of NIH mouse, AKR mouse, and rat cells in tissue culture. NIH mouse cells, which lack a portion of the viral genome (referred to as Gross-AKR specific sequences), incorporated three to four copies of these sequences per haploid genome. AKR cells, in which the Gross-AKR specific sequences are already present as three to four copies per haploid genome, did not shwo any distinct change in copy number after infection. Rat cells, which lack DNA sequences homologous to murine leukemia virus, incorporated one copy of the viral genome per haploid genome. It is inferred that the presence of viral sequences may affect the efficiency of integration of exogenous provirus, and that there may be a limit to the number of copies that can be inserted.

AKR murine leukemia virus↗
PubMed1981

Chromosomal position and activation of retroviral genomes inserted into the germ line of mice.

The exogenous Moloney leukemia virus (M-MuLV) was inserted into the germ line of mice by exposing embryos to virus at different stages of embryogenesis. Mice derived from exposed embryos were mosaics with respect to integrated virus. Nine new substrains, designated Mov-5 to Mov-13, were derived, each of which carries a single M-MuLV genome at a different chromosomal position in its germ line. Four substrains, Mov-1 to Mov-4, were derived previously. Restriction enzyme analyses demonstrated that, with the exception of Mov-4 and Mov-6 mice, no major rearrangements or deletions have occurred in the integrated proviral genomes. Infectious virus is not activated in the majority of substrains (Mov-4 to Mov-8 and Mov-10 to Mov-12), whereas the other mice develop viremia. A detailed comparison between Mov-1 and Mov-13 mice demonstrated that the time of virus activation is different. Mov-13 mice activate infectious virus during embryogenesis, leading to a distinct pattern of virus expression in all tissues of the adult, but the viral genome in Mov-1 mice is activated only during the first two weeks after birth, leading to virus expression predominantly in lymphatic organs. Together with previous observations, at least four different phenotypes of virus expression-that is, early virus activation during embryogenesis, virus activation after birth, virus activation late in life and no expression of infectious virus at all-can be distinguished among the 13 substrains. Our results suggest that the chromosomal region at which a viral genome is integrated influences its expression during development and differentiation.

Animals↗
PubMed2001

In vivo CD40-CD154 (CD40 ligand) interaction induces integrated HIV expression by APC in an HIV-1-transgenic mouse model.

Because of their relative resistance to viral cytopathic effects, APC can provide an alternative reservoir for latently integrated HIV. We used an HIV-transgenic mouse model in which APC serve as the major source of inducible HIV expression to study mechanisms by which integrated virus can be activated in these cells. When admixed with transgenic APC, activated T lymphocytes provided a major contact-dependent stimulus for viral protein expression in vitro. Using blocking anti-CD154 mAb as well as CD154-deficient T cells, the HIV response induced by activated T lymphocytes was demonstrated to require CD40-CD154 interaction. The role of this pathway in the induction of HIV expression from APC in vivo was further studied in an experimental model involving infection of the HIV-transgenic mice with PLASMODIUM: chabaudi parasites. Enhanced viral production by dendritic cells and macrophages in infected mice was associated with up-regulated CD40 expression. More importantly, in vivo treatment with blocking anti-CD154 mAb markedly reduced viral expression in P. chabaudi-infected animals. Together, these findings indicate that immune activation of integrated HIV can be driven by the costimulatory interaction of activated T cells with APC. Because chronic T cell activation driven by coinfections as well as HIV-1 itself is a characteristic of HIV disease, this pathway may be important in sustaining viral expression from APC reservoirs.

Animals↗
PubMed1993

Novel RFLPs at protooncogene and cancer-related gene loci on mouse chromosomes.

DNA probes for the NRAS, HRAS, KRAS2, LCK, RAF1, MET, MYCL1, MYCN, MYB, ERBB2, FOS, CSF1R, and SRC protooncogene loci; the retinoblastoma gene locus (RB1); the tumor virus integration sites INT2, PVT1, and MLV12; and the locus of the tumor-specific antigen T1A were used to screen mouse genomic DNAs from RF/J, CAST/Ei, MOLF/Ei, Mus musculus musculus, M. m. poschiavinus, and M. spretus. Polymorphic DNA fragments for the 18 DNA probes have been identified using Southern blot hybridization and restriction fragment length polymorphism (RFLP) analysis.

Animals↗
PubMed2000

Phenotyping of Evi1, Evi11/Cb2, and Evi12 transformed leukemias isolated from a novel panel of cas-Br-M murine leukemia virus-infected mice.

Cas-Br-M murine leukemia virus (MuLV) is a slow-transforming retrovirus that potently induces leukemias in mice and therefore is well suited for retroviral insertional mutagenesis. We used Cas-Br-M MuLV in NIH/Swiss mice to establish a new panel of mainly myeloid leukemias. All tumors found in leukemic animals were classified by gross pathology, morphology, and immunophenotype, as well as the incidence of known common virus integration sites (VISs) in MuLV-induced myeloid malignancies (i.e., Evi1, Evi11/Cb2, Evi12, Fli1, and c-Myb). Interestingly, male mice were more susceptible than females to the induction of leukemia by Cas-Br-M MuLV. Seventy-four of the Cas-Br-M MuLV-inoculated mice developed a severe splenomegaly, sometimes in association with a thymoma. Although most of the immunophenotyped Cas-Br-M MuLV tumors were of myeloid origin (58%), numerous T-cell leukemias (21%) and mixed myeloid/T-cell leukemias (21%) were found. The myeloid leukemias and myeloid compartment of the mixed leukemias were further characterized by immunophenotyping with stem cell-, myeloid-, and erythroid-specific antibodies. The known Cas-Br-M MuLV common VISs (Evi1, Evi11/Cb2, and Evi12) were demonstrated in 19%, 12%, and 20% of the cases, respectively, whereas no Fli1 and c-Myb rearrangements were found. Integrations into Evi1 were restricted to myeloid leukemias, whereas those in Evi11/Cb2 and Evi12 were identified in myeloid as well as T-lymphoid leukemias. This panel of well characterized Cas-Br-M MuLV-induced hematopoietic tumors may be useful for the isolation and characterization of new proto-oncogenes involved in myeloid or T-cell leukemias.

3T3 Cells↗
PubMed1994

Interferon-gamma induces resistance in primary monocytes against human immunodeficiency virus type-1 infection.

Treatment of monocytes with interferon-gamma 1 day before, or at the time of infection with human immunodeficiency virus type-1 (HIV-1) induced complete resistance in monocytes against HIV-1 infection. There was no evidence of viral RNA, proviral DNA, p24 antigen, or reverse transcriptase activity through 2 weeks after inoculation. Ultrastructural examination of these cells showed no detectable virus particles. When interferon-gamma was added to monocytes 1 to 3 days post-infection, virus integration occurred, but the viral expression was either ablated (1 day post-infection) or significantly inhibited (3 days post-infection). Treatment of monocytes with interferon-gamma before or after infection with HIV-1 produced significantly higher levels of tumor necrosis factor-alpha and interleukin-8 than untreated or uninfected monocytes. These results suggest that altered regulation of cytokines may mediate antiviral activity of interferon-gamma in monocytes.

Cells, Cultured↗
PubMed2000

Physical state and expression of human papillomavirus in laryngeal carcinoma and surrounding normal mucosa.

Epidemiologic and biomolecular evidence suggests that human papillomavirus (HPV) infection may be associated with the development of head and neck cancers. To clarify the role of HPV in larynx carcinoma, 25 patients were studied for the presence of viral DNA, possible virus integration into the cellular genome, and viral expression both in neoplastic tissues and in neighbouring normal mucosa. Twelve of 25 patients with neoplasia (48%) showed negative results for HPV sequences, and 13 (52%) showed positive results. Among the latter group of patients, seven were HPV-16 positive, five were HPV-6, and one was HPV-45. No multiple infections were detected. The physical status of the HPV genome was analysed by three methods: polymerase chain reaction (PCR), bidimensional agarose gel electrophoresis, and in situ hybridisation. Viral integration into the host genome occurred in 43% of cases of HPV-16 and in 20% of cases of HPV-6. Viral RNA expression was detected by reverse transcription-PCR only in HPV-16-positive tumours. The pattern of expression was consistent with an active role of HPV in cellular transformation. In conclusion, the present work suggests that HPV infection may be involved in some cases of laryngeal carcinoma. However, the transformation mechanisms might be different from those currently accepted for anogenital cancers.

Adult↗
PubMedDate not supplied

The treatment of chronic hepatitis B.

The diagnosis of chronic hepatitis does not necessarily imply a treatment. This statement is endorsed by the fact that many forms of the disease are non-evolving or very slow evolving, and therefore development of cirrhosis or hepatocarcinoma is extremely unlikely in these minimal and mild forms of the disease. Furthermore, drugs used today occasionally lead to virus annihilation and, in any case, do not operate upon virus integration into the hepatocyte genome. However, at present there is a consensus regarding the opportunity of therapeutical intervention in chronic hepatitis B with major biological and histological signs of activity, although there remain two important issues to approach. The first one is the relatively low efficacy of therapy referred to the cost (i.e. a maximum of 40% of cases with long-lasting therapeutic response to interferon), while the second is the presence of various side effects, which can force the reducing of dosage or even the discontinuation of treatment. Antiviral therapies, which were using acyclovir, didanosine, zidovudine and vidarabine, have demonstrated their inefficiency. Therefore, two groups are still used nowadays: interferon and nucleosides analogues. Each of the medication enounced has defined dosages, treatment extent, indications and contraindications, side effects and therapy expectancies. Proper management in chronic hepatitis B can achieve the following end-points: obtaining HBeAg-to-anti-HBe seroconversion, prevention of the disease progression and quality-of-life improvement. The treatment of chronic hepatitis B is an ever-changing issue and our expectation for better results grows proportionally with the knowledge brought everyday by new research and clinical experience.

Antiviral Agents↗
PubMed1993

[Trans-activating function of integrated hepatitis B virus].

HBV DNAs are often found in integrated form in human hepatocellular carcinoma (HCC). Since transactivation of the X gene has been shown, surveys of collections of HBV integrants with flanking cellular sequence were performed to clarify whether they might exhibit a transactivation. The majority of integrants showed transactivation effect which may to be due to the virus-cell fusion products derived from the 3' truncated X gene. Additionally, it has been found that 3' truncated preS2/S gene in the integrant encodes a transactivator to which C terminal truncation is essential. These results suggest that the transactivating effect of integrated HBV DNAs plays a role in hepatocarcinogenesis by activating cellular genes.

Carcinoma, Hepatocellular↗