Characterization of chimeric enzymes between caprine arthritis–encephalitis virus, maedi–visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli

Berger, Nicola; Heller, Astrid; Störmann, Klaus D.; Pfaff, Eberhard

doi:10.1099/0022-1317-82-1-139

Cited by 7 publications

(4 citation statements)

References 52 publications

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“…Viral elements, such as integrase (IN) and the terminal structures of viral DNA, that are required for retroviral integration have been extensively characterized. It has also been shown that selection of the proviral integration targets is nonrandom (15, 29-31, 37, 47) and that the central domain of IN plays a role in determining the target specificity (3,36). The efficiency of chromosomal sites to become a preferred integration target appears to be affected by several factors, such as transcriptional activity (35,46), DNase I hypersensitivity (9,32,33,45), methylation (15), GC content (5,16,34), nuclear scaffold attachment (20), nucleosome structure (27,28,30,31), and DNA structure of a higher order (14,22,24,27,28).…”

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confidence: 99%

Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR

Jin

Ishibashi

Nomoto

et al. 2002

J Virol

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Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virusbased retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.The RNA genome of retroviruses is reverse transcribed into a double-stranded DNA copy, which is then integrated into the host chromosome as a provirus. Viral elements, such as integrase (IN) and the terminal structures of viral DNA, that are required for retroviral integration have been extensively characterized. It has also been shown that selection of the proviral integration targets is nonrandom (15, 29-31, 37, 47) and that the central domain of IN plays a role in determining the target specificity (3, 36). The efficiency of chromosomal sites to become a preferred integration target appears to be affected by several factors, such as transcriptional activity (35, 46), DNase I hypersensitivity (9,32,33,45), methylation (15), GC content (5, 16, 34), nuclear scaffold attachment (20), nucleosome structure (27,28,30,31), and DNA structure of a higher order (14,22,24,27,28). However, these results were obtained by in vitro studies using artificial target DNA or analysis of a small number of in vivo integration sites, and it is still unclear why proviral integration takes place at certain target sites more often than others. To elucidate the mechanism of target selection for proviral integration, it may be useful to compile and analyze a large number of nucleotide sequences corresponding to proviral integration sites in the context of actual cellular chromosome. Traditionally, cellular flanking DNA of a provirus was cloned by the time-consuming method o...

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confidence: 99%

Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR

Jin

Ishibashi

Nomoto

et al. 2002

J Virol

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“…Previous analyses of chimeras between HIV-1 and other lenti-retroviruses, including feline immunodeficiency virus [56], [57], Visna [58], [59], caprine arthritis encephalitis virus [58], and PFV [60] have used similar divisions of IN into NTD, CCD, and CTD domains to map viral substrate and local target DNA specificity. These studies, consistent with more recent insights from PFV structures, largely agree that retroviral cDNA substrate specificity is not affected by the NTD and is determined by the CCD with contributions from the CTD.…”

Section: Discussionmentioning

confidence: 99%

Directed DNA Shuffling of Retrovirus and Retrotransposon Integrase Protein Domains

Pino-Vargas

Larsen

et al. 2013

PLoS ONE

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Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR. This method easily and accurately shuffles multiple DNA gene sequences simultaneously at specific base-pair positions, such as protein domain boundaries. It produced all 27 properly-ordered combinations of the amino-terminal, catalytic core, and carboxyl-terminal domains of the integrase gene from human immunodeficiency virus, prototype foamy virus, and Saccharomyces cerevisiae retrotransposon Ty3. Retrotransposons can display dramatic position-specific integration specificity compared to retroviruses. The yeast retrotransposon Ty3 integrase interacts with RNA polymerase III transcription factors to target integration at the transcription initiation site. In vitro assays of the native and chimeric proteins showed that human immunodeficiency virus integrase was active with heterologous substrates, whereas prototype foamy virus and Ty3 integrases were not. This observation was consistent with a lower substrate specificity for human immunodeficiency virus integrase than for other retrovirus integrases. All eight chimeras containing the Ty3 integrase carboxyl-terminal domain, a candidate targeting domain, failed to target strand transfer in the presence of the targeting protein, suggesting that multiple domains of the Ty3 integrase cooperate in this function.

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“…Previous studies have established that HIV and SIV integrases (IN) are highly specific to their “ att ” sequences located in the terminal regions of U3 and U5 sequences [33]. Furthermore it has been demonstrated that the recombinant IN protein of CAEV does not allow in vitro integration of HIV LTR DNA [34, 35]. From these data we hypothesized that a SHIV genome, in which the SIV LTRs are replaced with those of CAEV, and used as DNA vaccine, will generate an integration-deficient viral genome but will retain the capacities to undergo one cycle of replication during which viral proteins will be expressed from the episomal unintegrated DNA.…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity of a lentiviral-based DNA vaccine driven by the 5′LTR of the naturally attenuated caprine arthritis encephalitis virus (CAEV) in mice and macaques

Arrode-Brusés¹,

Hegde²,

Jin³

et al. 2012

Vaccine

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Increasing the safety and the efficacy of existing HIV vaccines is one of the strategies that could help to promote the development of a vaccine for human use. We developed a HIV DNA vaccine (Δ4-SHIVku2) that has been shown to induce potent polyfunctional HIV-specific T cell responses following a single dose immunization of mice and macaques. Δ4-SHIVku2 also induced protection when immunized macaques were challenged with homologous pathogenic viruses. In the present study, our aim was to examine whether a chimeric HIV DNA vaccine (CAL-Δ4-SHIVku2) whose genome is driven by the LTR of the goat lentivirus, caprine arthritis encephalitis (CAEV) expresses efficiently the vaccine antigens and induces potent immune responses in animal models for HIV vaccine. Data of radioimmunoprecipitation assays clearly show that this chimeric genome drives efficient expression of all HIV antigens in the construct. In addition, evaluation of the p24 Gag protein in the supernatant of HEK-293-T cells transfected in parallel with Δ4-SHIVku2 and CAL-Δ4-SHIVku2 showed no difference suggesting that these two LTRs are inducing equally the expression of the viral genes. Immunization of mice and macaques using our single dose immunization regimen resulted in induction of similar IFN-γ ELISPOT responses in Δ4-SHIVku2- and CAL-Δ4-SHIVku2-treated mice. Similar profiles of T cell responses were also detected both in mice and macaques when multiparametric flow cytometry analyses were performed. Since CAEV LTR is not dependent of Tat to drive viral gene expression and is not functional for integration with HIV integrase, this new vector increases the safety and efficacy of our vaccine vectors and vaccination strategy.

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Characterization of chimeric enzymes between caprine arthritis–encephalitis virus, maedi–visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli

Cited by 7 publications

References 52 publications

Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR

Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR

Directed DNA Shuffling of Retrovirus and Retrotransposon Integrase Protein Domains

Immunogenicity of a lentiviral-based DNA vaccine driven by the 5′LTR of the naturally attenuated caprine arthritis encephalitis virus (CAEV) in mice and macaques

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