Construction of Infectious Molecular Clones of HIV-1 Containing Defined Mutations in the Protease Gene
| Autor: | Helen Scarnati, Dean L. Winslow, Radonna Tritch, R.J. Zagursky, R.A. Horlick, K. Ackerman, Lee T. Bacheler, Elizabeth D. Anton |
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| Rok vydání: | 1994 |
| Předmět: |
Genes
Viral medicine.medical_treatment Genetic Vectors Molecular Sequence Data Biophysics Biology Recombinant virus Polymerase Chain Reaction Biochemistry Cell Line law.invention Plasmid HIV Protease Shuttle vector law medicine Humans Coding region Cloning Molecular Molecular Biology Gene DNA Primers Genetics Protease Base Sequence Cell Biology Virology Restriction site DNA Viral HIV-1 Mutagenesis Site-Directed Recombinant DNA Deoxyribonucleases Type III Site-Specific |
| Zdroj: | Biochemical and Biophysical Research Communications. 205:1651-1657 |
| ISSN: | 0006-291X |
| DOI: | 10.1006/bbrc.1994.2857 |
| Popis: | A DNA clone of HIV-1 containing the full-length infectious viral sequence was cleaved at a unique Nco I restriction site within the viral genome, and DNA fragments containing the 5' and 3' portions of the HIV genome were subcloned into separate plasmid vectors. The 5' 'half-virus' construct was further modified by incorporating a class IIS restriction site, Esp3I, near the 3' end of the protease gene of HIV. This site, in combination with a natural ApaI site near the 5' end of the protease gene, creates a convenient cassette shuttle vector in which the protease coding region can be easily replaced. Recombinant viruses containing protease genes either altered by site-directed mutagenesis or amplified from clinical or laboratory isolates can be reconstructed. The DNA fragment containing the protease gene is first subcloned into the 5' half-virus shuttle vector plasmid. Infectious recombinant virus is subsequently recovered by cotransfecting 5' and 3' half-virus plasmids linearized at their common Nco I sites into mammalian cells. This method was successfully applied to constructing viruses containing various substitutions in protease. |
| Databáze: | OpenAIRE |
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