10th Conference on Retroviruses and Opportunistic Infections Boston, MA USA - February 10 -14, 2003

Conf Retroviruses Opportunistic Infect 2003 Feb 10-14;10th: abstract no. 25
A.H.Kaplan , S.C.Pettit
Univ of North Carolina Sch of Med, Chapel Hill

BACKGROUND: The HIV protease is translated as part of the 160 kDa GagPol precursor and is only functional as a dimer. The dimer interface of the 99 amino acid processed, mature protease includes a 4-stranded anti-parallel beta-pleated sheet comprised of the first 4 amino acids (PQIT) and the last 4 amino acids (TLNF) of the protease. Although the dimer interface has been well-characterized in the mature protease, the structure of the dimer interface within GagPol as well as the role that this structure plays in viral replication is less well understood. Further, the initial steps in protease activation and precursor processing, although critical for viral replication, have not been extensively evaluated.

Method: Using a rabbit reticulocyte system in which full length GagPol is expressed and auto-processed, we have evaluated the role of the dimer interface residues through an extensive mutational analysis. We have also evaluated the susceptibility of this protease embedded within GagPol to the inhibition by active site inhibitors of the enzyme (ritonavir and nelfinavir).

RESULTS: Out data demonstrate the following: 1) The initial cleavages within GagPol are accomplished as intra-molecular interactions in cis. (e.g., the protease dimer on one GagPol dimer processes the initial cleavage sites on the same dimer); 2) the protease embedded within GagPol is approximately 8,000-fold less sensitive to inhibition by either of these active site inhibitors than is the mature, 99 amino acid protease; 3) cleavage and release of the N-terminus of the protease is a late event in precursor processing; and 4) mutations that perturb the structure of the dimer interface B-sheet enhance cleavage of the N-terminus of the protease.

CONCLUSIONS: Overall, our data suggest a model for virus assembly in which the initial GagPol cleavages are accomplished in cis and indicate a structural mechanism by which release of the more active mature protease is delayed until later in virus assembly. We present a model that integrates protease activation and GagPol dimerization into virus particle assembly. Our data also suggest novel avenues for the design of protease inhibitors.

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