Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration

Fujiwara, T; Craigie, R

doi:10.1073/pnas.86.9.3065

Cited by 109Virus-based gene therapy researchT-cell and Retrovirus StudiesHIV Research and Treatment

Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration

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Fujiwara, T, Craigie, R·1989·Proceedings of the National Academy of Sciences

DOI 10.1073/pnas.86.9.3065PMC PMC287065

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Abstract

After retroviral infection of a permissive cell, the viral RNA is reverse-transcribed to make a DNA copy of the viral genome. Integration of this DNA copy into the host genome is a necessary step for efficient viral replication. We have developed a cell-free system for integration of exogenous mini-retroviral DNA. The termini of this linear mini-Moloney murine leukemia virus (MoMLV) DNA are designed to mimic the ends of authentic unintegrated MoMLV DNA. The viral proteins required for integration can be provided either as a cytoplasmic extract of MoMLV-infected NIH 3T3 cells or as disrupted MoMLV particles. Phage lambda DNA serves as the target for integration. Genetic markers present on the mini-MoMLV DNA enable integration events to be detected, and the recombinants recovered, by selection in Escherichia coli. Integration, which occurs at heterogeneous locations in the target DNA, is absolutely dependent on the presence of a source of viral proteins and a divalent cation in the reaction mixture. The fidelity of the integration reaction was confirmed by sequencing the junctions between the integrated MoMLV DNA and adjacent lambda DNA sequence. In each case, as expected for authentic MoMLV DNA integration, a 4-base-pair duplication of target DNA sequence flanked the integrated MoMLV DNA.

Integrity signals

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Crossref · 10.1073/pnas.86.9.3065

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PIC-containing reverse-transcribed dsDNA can be integrated into heterologous DNA in vitro.
“These studies demonstrated that PIC-containing reverse-transcribed dsDNA could be integrated into a heterologous DNA (Fujiwara and Mizuuchi 1988, Fujiwara and Craigie 1989, Bushman et al. 1990, Craigie et al. 1990, Katz et al. 1990, Lee and Coffin 1990, Engelman et al. 1991, Brown 1997).”
From Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication (2026) · cites this paper as Fujiwara and Craigie 1989

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Authors: Fujiwara, T., & Craigie, R.
Affiliations: National Institute of Diabetes and Digestive and Kidney Diseases (US)
ORCID: Craigie, R
Venue: Proceedings of the National Academy of Sciences
Publisher: National Academy of Sciences
Published: 1989-05
Volume: 86
Issue: 9
Pages: 3065-3069
ISSN: 0027-8424 (ISSN-L)
1091-6490
DOI: 10.1073/pnas.86.9.3065
PMC: PMC287065
OpenAlex: W1971994381
Cited by: 109
References: 13
Citations by year: 2025: 1 · 2023: 2 · 2019: 1 · 2018: 1 · 2016: 3 · 2015: 1 · 2014: 2 · 2013: 1 · 2012: 1
FWCI: 9.02
Type: journal article
Language: en
Open access: green
europepmc.org
Locations: Proceedings of the National Academy of Sciences (publishedVersion)
Europe PMC (PubMed Central) (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Life Sciences › Immunology and Microbiology › Immunology › T-cell and Retrovirus Studies
Life Sciences › Immunology and Microbiology › Virology › HIV Research and Treatment
Keywords: retroviral integration, Moloney murine leukemia virus, reverse transcription, target site duplication, divalent cations
MeSH terms: Animals, Base Sequence, Cell Line, Cell-Free System, Chromatography, Gel, Cytoplasm, DNA Nucleotidyltransferases, DNA, Viral, Moloney murine leukemia virus, Bacteriophage lambda, Recombination, Genetic, Viral Proteins, Virus Replication, Integrases, Mice

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Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration

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