Placement of p6pol between tandem repeat HIV-1 protease domains reduces Gag cleavage efficiency

Background

HIV-1 protease (PR) is encoded by pol, which is initially translated as a Pr160^gag-pol polyprotein by a ribosomal frameshift event [1]. Pr160^gag-pol is incorporated into virions via interactions with assembling Pr55^gag. The PR-mediated proteolytic cleavage of Pr55^gag and Pr160^gag-pol, known as virus maturation, is essential for the acquisition of viral infectivity. Within the Gag-Pol, the p6gag is truncated and is replaced by a transframe domain referred to as p6* or p6pol. Removal of p6pol improves Gag-Pol autoprocessing, suggesting that p6pol is involved in regulation of PR activation [2]. However, overlapping of p6gag/p6pol reading frame hampers generic approach to studying p6pol biological function. To assess the p6pol contribution to PR-mediated virus maturation without affecting p6gag reading frame, we introduced an extra copy of p6pol-PR or PR coding sequence at the PR C-terminus.

Materials and methods

PCR-amplified p6pol-PR or PR fragments were inserted at the PR C-terminus of an env-deleted HIV-1 proviral vector. Each of the constructs was transiently expressed in 293T cells, and virus assembly and processing were analyzed by Western blot. Virus infectivity was determined by a single-cycle infection assay.

Results

HIV-1 mutants containing tandem repeat PR domains were severely defective in virus particle production due to enhanced Gag cleavage. Inactivation of the proximal PR affects Gag cleavage efficiency at a greater extent than inactivation of the distal PR. Placement of p6pol between the tandem repeat PR domains resulted in diminished Gag cleavage efficiency.

Conclusions

Our study indicates that the Gag cleavage enhancement effect incurred by over-expressed HIV-1 PR is reduced following the placement of p6pol between the tandem repeat PR domains. This supports the proposal that p6pol plays a negative role in the process of PR activation.