The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

Abstract

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

Importance of Protease Cleavage Sites within and Flanking Human Immunodeficiency Virus Type 1 Transframe Protein p6* for Spatiotemporal Regulation of Protease Activation

“…This implies that internal hydrolysis of p6* precedes carboxylterminal cleavage in virus particles, albeit the carboxyl-terminal site was processed at a 60-fold-higher hydrolysis rate in the in vitro cleavage assay. Hence, our data are concordant with a recently proposed model where p6* cleavage is initiated in cis at an internal site (P i ) that appears to be best accessible to the active site of the precursor-embedded PR (40,41). However, as mature RT and IN species together with an RT-IN intermediate were present in csM4 and csM7 virions where aminoterminal cleavage of the PR was blocked, our results also indicate that amino-terminal release of the PR is not a prerequisite for removal of the carboxyl-terminal RT-IN moiety.…”

“…These results indicate that the original cleavage site might be functionally replaced by the novel proximal site which has also been used in full-length GST-p6* proteins carrying the respective mutation. In a current model, the internal cleavage site is processed intramolecularly by the precursorembedded PR which exhibits a substrate specificity different than that of the mature free enzyme (30,40,41,61). Taking into account the fact that in vitro peptide cleavage was performed by a mature PR dimer in trans, we cannot completely exclude the possibility that the internal mutation might have different effects in the context of the Gag-Pol precursor.…”

“…Despite comprehensive analyses of PR-mediated processing events giving rise to infectious virions (40)(41)(42)(43)(44)57), the mechanism stringently controlling spatiotemporal PR activation during the late phase of viral replication is not yet fully understood. Correct folding of the enzyme within the dimerized Gag-Pol precursors is a prerequisite to allow formation of a functional active site (32,58).…”

“…Furthermore, p6* has been shown to contain a conserved internal PR cleavage site between Phe 8 and Leu 9 (see Fig. 1) hydrolyzed early during precursor maturation (1,7,29,40,41,64). Interestingly, natural polymorphisms of the p6* cleavage sites have been reported to impact PR release with considerable effects on susceptibility toward PR inhibitors (56).…”

Novel Cytotoxic T-Lymphocyte Escape Mutation by a Three-Amino-Acid Insertion in the Human Immunodeficiency Virus Type 1 p6 Pol and p6 Gag Late Domain Associated with Drug Resistance

“…The Pol NL8 epitope is the first and only epitope in the transframe region to appear in the HIV immunology database (www.hiv.lanl.gov). One of the cleavage products of the GagPol polyprotein located just downstream from the ribosomal frameshift site (18), p6 Pol is present in mature viral particles and affects protease activity (34,36,45,48). The Pol NL8 epitope corresponds to the consensus sequence for B clade isolates and is generally conserved in other clades, with the major difference in clade A, C, and D isolates being an arginine-to-serine change at the fifth position (http:www.hiv.lanl .gov).…”