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PubMed2003

Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7.

Reverse transcription of HIV-1 genomic RNA to double-stranded DNA by reverse transcriptase (RT) is a critical step in HIV-1 replication. This process relies on two viral proteins, the RT enzyme and nucleocapsid protein NCp7 that has well documented nucleic acid chaperone properties. At the beginning of the linear DNA synthesis, the newly made minus-strand strong-stop DNA ((-)ssDNA) is transferred to the 3'end of the genomic RNA by means of an hybridization reaction between transactivation response element (TAR) RNA and cTAR DNA sequences. Since both TAR sequences exhibit stable hairpin structures, NCp7 needs to destabilize the TAR structures in order to chaperone their hybridization. To further characterize the relationships between TAR stability and NC-mediated destabilization, the role of the A(49) and G(52) bulged residues in cTAR DNA stability was investigated. The stability of cTAR and mutants where one or the two terminal bulges were replaced by base-pairs as well as the NCp7-mediated destabilization of these cTAR sequences were examined. Thermodynamic data indicate that the two bulges cooperatively destabilize cTAR by reducing the stacking interactions between the bases. This causes a free energy change of about 6.4 kcal/mol and seems to be critical for NC activity. Time-resolved fluorescence data of doubly labelled cTAR derivatives suggest that NC-mediated melting of cTAR ends propagates up to the 10C.A(44) mismatch or T(40) bulge. Fluorescence correlation spectroscopy using two-photon excitation was also used to monitor cTAR ends fraying by NC. Results show that NC causes a very significant increase of cTAR ends fraying, probably limited to the terminal base-pair in the case of cTAR mutants. Since the TAR RNA and cTAR DNA bulges or mismatches appear well conserved among all HIV-1 strains, the present data support the notion of a co-evolutionary relationship between TAR and NC activity.

Amino Acid Sequence↗
PubMed1999

HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect.

The human immunodeficiency virus type 1 Vpr is a virion-associated protein that is incorporated in trans into viral particles, presumably via an interaction with the p6 domain of the Gag polyprotein precursor. Recently, several studies demonstrated that Vpr fusion proteins could be used as intravirion inactivating agents. In this study, we compared different Vpr-chloramphenicol acetyltransferase (CAT) fusion proteins for their virion incorporation ability and their effect on the infectivity of HIV viruses. Our deletion analysis indicates that both the N-terminal alpha-helical domain and the leucine/isoleucine-rich (LR) domain located in the middle region of Vpr are required for optimal virion incorporation of Vpr-CAT fusion proteins. The C-terminal basic region, associated with Vpr's ability to mediate cell cycle arrest in G2, was not required for virion incorporation, thus allowing the development of Vpr-based chimeric proteins devoid of any effect on cell growth. The fusion of Vpr at the N- or C-terminus of CAT targeted with equal efficiency the chimeric protein into virions. While the virion incorporation of most Vpr-CAT fusion proteins tested in this study was dependent on the presence of an intact p6 domain, fusion proteins containing only the N-terminal alpha-helical domain of Vpr (amino acid 1 to 42) were incorporated into virions in a p6-independent manner. Virion incorporation of Vpr-CAT fusion proteins was shown to decrease viral infectivity. Moreover, the insertion of HIV protease-cleavage sites between Vpr and CAT not only efficiently delivered and released the cleaved CAT product into HIV viral particles, but also greatly potentiated the inhibition of progeny virion infectivity. Overall, our study: (1) defines the Vpr sequence requirement and configuration necessary for the specific and optimal incorporation of Vpr fusion protein into HIV particles; (2) shows that Vpr fusion proteins have the ability to suppress HIV infectivity by targeting multiple steps of viral morphogenesis.

Amino Acid Sequence↗
PubMed1997

The active oligomeric state of the minimalistic influenza virus M2 ion channel is a tetramer.

The influenza A virus M2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes and also modulates the pH of the trans-Golgi network in virus-infected cells. The M2 protein is a homo-oligomer of 97 residues, and analysis by chemical cross-linking and SDS/PAGE indicates M2 forms a tetramer. However, a higher order molecular form is sometimes observed and, thus, it is necessary to determine the active form of the molecule. This was done by studying the currents of oocytes that expressed mixtures of the wild-type M2 protein (epitope tagged) and the mutant protein M2-V27S, which is resistant to the inhibitor amantadine. The composition of mixed oligomers of the two proteins expressed at the plasma membrane of individual oocytes was quantified after antibody capture of the cell surface expressed molecules and it was found that the subunits mixed freely. When the ratio of wild-type to mutant protein subunits was 0. 85:0.15, the amantadine sensitivity was reduced to 50% and for a ratio of 0.71:0.29 to 20%. These results are consistent with the amantadine-resistant mutant being dominant and the oligomeric state being a tetramer.

Amantadine↗
PubMed2000

Virology. Probing HIV's elusive activities within the host cell.

Once inside a host cell, HIV must make its way to the nucleus in order to replicate, but until now, its journey has largely been uncharted territory. As researchers begin to explore this landscape a common theme is emerging: Soon after viruses enter cells, they enlist components of their hosts to assist them in their journey. Now, in what is being called one of the most exciting frontiers in AIDS research, preliminary findings on how HIV enters cell nuclei are giving AIDS researchers new leads on possible drug targets to fight the ever-mounting epidemic.

Cell Membrane↗
PubMedDate not supplied

New epidemiological data on liver oncogenesis.

The purpose of this review is to address and discuss the following: a) the malignant transformation of hepatocytes may occur, irrespective of the etiologic agent, in the context of increased cellular turnover induced by chronic hepatic damage and regeneration, with genetic mutations being a frequent event prior to the development of this tumor; b) although it is clear that hepatitis B virus-induced chronic liver injury, regeneration and cirrhosis is a major risk factor for hepatocellular carcinoma development, there are also several reports of the direct carcinogenic effects of hepatitis B virus. For instance, integration of hepatitis B virus DNA can directly induce chromosomal rearrangements and viral gene product transactivating properties may influence cellular genes important in the control of the growth process; c) hepatitis C virus and other risk agents, including alcohol, environmental factors and metabolic diseases may operate, mainly through chronic liver damage that progress to liver cirrhosis, a major predisposing factor for the development of hepatocellular carcinoma, regardless of etiology; d) at the molecular level, the interaction between oncogenes, tumor suppressor genes (with or without aflatoxins and hepatitis B virus) and several growth factors may play an additional role in hepatocellular carcinoma development.

Animals↗
PubMed1980

Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.

Animals↗
PubMed2005

Integration targeting by avian sarcoma-leukosis virus and human immunodeficiency virus in the chicken genome.

We have analyzed the placement of sites of integration of avian sarcoma-leukosis virus (ASLV) and human immunodeficiency virus (HIV) DNA in the draft chicken genome sequence, with the goals of assessing species-specific effects on integration and allowing comparison to the distribution of chicken endogenous retroviruses (ERVs). We infected chicken embryo fibroblasts (CEF) with ASLV or HIV and sequenced 863 junctions between host and viral DNA. The relationship with cellular gene activity was analyzed by transcriptional profiling of uninfected or ASLV-infected CEF cells. ASLV weakly favored integration in active transcription units (TUs), and HIV strongly favored active TUs, trends seen previously for integration in human cells. The ERVs, in contrast, accumulated mostly outside TUs, including ERVs related to ASLV. The minority of ERVs present within TUs were mainly in the antisense orientation; consequently, the viral splicing and polyadenylation signals would not disrupt cellular mRNA synthesis. In contrast, de novo ASLV integration sites within TUs showed no orientation bias. Comparing the distribution of de novo ASLV integration sites to ERVs indicated that purifying selection against gene disruption, and not initial integration targeting, probably determined the ERV distribution. Further analysis indicated that ERVs in humans, mice, and rats showed similar distributions, suggesting purifying selection dictated their distributions as well.

Alpharetrovirus↗
PubMed1979

Integration of Rous-associated virus type O provirus in susceptible chicken cells.

The number of viral genome equivalents per haploid cell genome was determined in normal chicken embryos from three selected chicken lines and in cultured fibroblasts (CEF) from these embryos. The cellular concentration of endogenous proviral DNA is similar in embryos from chickens of lines SPAFAS, 7, 15, 7 x 15, and 100. The concentration of proviral DNA is not affected by in vitro cultivation in CEF from lines that do not spontaneously produce virus, nor in CEF from line 7, which lacks receptors for Rous-associated virus type 0 (RAV-0). There is, however, a restricted increase in the number of integrated proviral genome equivalents in CEF from line 7 x 15, which produces RAV-0 and can support replication of this virus, and in CEF from line 15 experimentally infected with RAV-0.

Animals↗
PubMed1994

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.

Base Sequence↗
PubMed2005

Kinetics of human immunodeficiency virus type 1 decay following entry into resting CD4+ T cells.

In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4(+) T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4(+) cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4(+) T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4(+) T cells. Decay processes dominate in resting CD4(+) T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4(+) T cells is highly labile.

CD4-Positive T-Lymphocytes↗
PubMed1994

Integration of multiple chicken retroviruses into multiple chicken herpesviruses: herpesviral gD as a common target of integration.

Integration of two different avian retroviruses, reticuloendotheliosis virus (REV) and avian leukosis virus (ALV), into the genome of two different avian herpesviruses, the herpesvirus of turkeys (HVT) and Marek's disease virus (MDV), was investigated. Integration events occurred by the fourth and sixth in vitro passage of cells coinfected with REV/HVT and ALV/MDV, respectively. In order to further characterize the integration events, integrated proviruses and surrounding herpesviral genetic material were cloned and analyzed. In the REV/HVT coinfection experiment, one of the three unique integrated proviruses was found to have integrated into the HVT gD gene, resulting in disruption of the coding region of this gene. The two additional unique integrations were localized to the UL and IRL border regions of HVT, two previously described common sites of REV integration into MDV. Interestingly, one of the integrated proviruses in the HVT genome appeared to be full length, was infectious when transfected into CEF cells, and therefore could potentially function to produce infectious REV from an HVT infectious platform. In the ALV/MDV coinfection experiment, one of two unique integrated proviruses was found to have integrated into the gD gene, resulting in disruption of the coding region of this gene. The second unique integration site was in the polyadenylation site of the SORF2 gene at the boundary of the IRS and US, once again a common site of REV integration into MDV. These results demonstrate that the U/IR-TR border regions of herpesviruses are common sites of retroviral integration. In addition gD, in the US region of the herpesvirus, is a common site of retroviral integration in multiple herpesvirus, indicating a possible selective advantage for disruption of this gene in the in vitro growth of a herpesvirus. Finally, this is the first instance of a full-length provirus found integrated into a herpesvirus genome, indicating that a retrovirus could alter its route of infection by being carried in a herpesvirus genome.

Amino Acid Sequence↗
PubMed1978

Sites of integration of infectious DNA of avian reticuloendotheliosis viruses in different avian cellular DNAs.

The pattern of integration for the infectious DNA of two avian reticuloendotheliosis viruses whose DNA is not inactivated by digestion with the restriction endonuclease, Eco RI was determined. High molecular weight DNA from infected chicken, turkey and pheasant cells was digested with Eco RI, electrophoresed through agarose gels and assayed for infectivity. The same patterns of integration of infectious viral DNA were found for these species of avian cells infected at high or low multiplicities with two reticuloendotheliosis viruses. There were multiple sites of integration in acutely infected cells with concomitant cell death. There was a single site of integration in chronically infected cells with no cell death. There were more integrated infectious viral DNA molecules per cell in acutely infected cells than in chronically infected cells. These results are consistent with the hypotheses that the cell death in the acute phase of infection is a result of the integration of the infectious viral DNA at multiple sites, and that only those cells survive that have the infectious viral DNA integrated exclusively at the single site.

Cell Line↗
PubMed1988

Integration of Friend murine leukemia virus into both alleles of the p53 oncogene in an erythroleukemic cell line.

The Friend virus-transformed erythroleukemic cell line DP16-9B4 has undergone a complex rearrangement of the p53 oncogene and lacks any detectable expression of the p53 protein. We report here characterization of both p53 alleles in this cell line and identify independent integrations of Friend murine leukemia virus sequences into the coding region of both alleles.

Alleles↗
PubMed2000

Site-specific integration of an adeno-associated virus vector plasmid mediated by regulated expression of rep based on Cre-loxP recombination.

Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the rep gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-loxP recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the rep coding sequence; in this configuration, rep expression is silent. However, Cre circularizes the rep expression unit, directly joining the p5 promoter to the 5' end of the rep78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.

Base Sequence↗
PubMed1997

Human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates Mg2+-dependent DNA integration in vitro.

The integrase (IN) protein of the human immunodeficiency virus mediates integration of the viral DNA into the cellular genome. In vitro, this reaction can be mimicked by using purified recombinant IN and model DNA substrates. IN mediates two reactions: an endonucleolytic cleavage at each 3' end of the proviral DNA (terminal cleavage) and the joining of the linear viral DNA to 5' phosphates in the target DNA (strand transfer). Previous investigators have shown that purified IN requires Mn2+ or Mg2+ to promote strand transfer in vitro, although Mg2+ is the likely metal cofactor in vivo. IN activity in the presence of Mg2+ in vitro requires high IN concentrations and low concentrations of salt. Here, we show that the viral nucleocapsid protein NCp7 allows efficient IN-mediated strand transfer in the presence of Mg2+ at low enzyme concentrations. This potentiating effect appears to be unique to NCp7, as other small DNA-binding proteins, while capable of stimulating integration in the presence of Mn2+, all failed to stimulate strand transfer in the presence of Mg2+.

Capsid↗
PubMed1997

Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.

Cell Line, Transformed↗