3rd International Workshop on HIV Drug Resistance 2-5 August 1994, Kauai, Hawaii, USA

A METHOD FOR CONSTRUCTION OF INFECTIOUS MOLECULAR CLONES OF HIV-1 CONTAINING DEFINED MUTATIONS IN THE HIV PROTEASE GENE

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:11 (abstract no. 10)

E.D. Anton, L.T. Bacheler, R.A. Horlick, R.J. Zagursky, R.J. Tritch and D.L. Winslow
DuPont Merck Pharmaceutical Company, Glenolden, Pennsylvania and Wilmington, Delaware (USA)

HIV protease inhibitors are promising class of antiretroviral agents that have recently entered clinical trials. Otto et al. and others have reported several amino acid alterations in the protease gene which may be associated with reduced susceptibility to various protease inhibitors. The creation of defined protease gene mutations in an isogenic viral background is desirable in order to assess the contribution to drug susceptibility of alterations created in vitro or selected during virus replication in cell culture or in patients. A DNA clone of HIV-1 (pHxB2gpt) containing the full-length infectious viral sequence was divided at a unique Ncol 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 llS restriction site, Esp 31, near the 3´ end of the protease gene of HIV. This site, in combination with a natural Apal 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 by first subcloning the DNA fragment containing the protease gene into the 5´ half-virus shuttle vector plasmid. Infectious recombinant virus is then recovered by cotransfecting 5´ and 3´ half-virus plasmids linearized at their common Ncol sites into mammalian cells. This method was successfully applied to constructing viruses containing various substitutions in HIV protease.

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1994-08-02
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