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1st International Workshop on HIV Drug Resistance & Treatment Strategies25-28 June 1997, St. Petersburg, Florida, USA |
COMPARISON OF GAG-POL PRECURSOR CLEAVAGE IN NATURALLY ARISING HIV-1 VARIANTS
Antiviral Therapy 1997;2 (Suppl 5):19 (abstract no. 29)
Gregory Bloom1, Elena Perez2, Shefal Parikh1, Maureen M Goodenow2 and Ben M Dunn1
1Department of Biochemistry and Molecular Biology; and 2Department of Pathology, University of Florida College of Medicine, Gainesville, Florida, USA
Despite the impressive progress made by including protease inhibitors in therapeutic intervention in HIV-infected patients, the problem of resistance development continues. This can limit the initial response to treatment or can lead to re-emergence of virus after weeks or months of therapy. Understanding how some viral variants are able to escape anti-protease drug treatment may contribute to the development of new strategies for anti-AIDS therapy. The purpose of this study was to explore the variation in self-processing of Gag-Pol polyproteins by variant forms of the viral protease.
A region of the Gag-Pol domain spanning five cleavage sites (B-F), including the complete protease, was cloned by PCR amplification from PBMCs isolated from HIV-infected mothers and their prenatally infected children into an Escherichia coli system that expresses 776 amino acids of the Gag-Pol precursor as a soluble protein. Processing of the initially formed polyprotein can be followed over time following IPTG induction by adding timed aliquots to Laemmli sample buffer. Using an antibody against p24 (CA or capsid), the resulting cleavage products containing that protein can be analysed by Western blotting from SDS-PAGE separations.
The results show that different rates of cleavage occur at sites A and C for different protease variants. By constructing chimeric Gag-Pol vectors, in which an efficient protease variant is paired with different Gag regions, it has been demonstrated that determinants outside of protease are critical to the efficiency of processing. This implies that the mechanism of drug resistance may include alterations in the polyprotein in general, as well as in the protease region.
Supported by NIH grant AI28571 to BMD and MMG
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1997-06-25
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