Activity of human immunodeficiency virus type 1 protease inhibitors against the initial autocleavage in Gag-Pol polyprotein processing

Abstract

Inhibitors of HIV protease have proven to be important drugs in combination anti-HIV therapy. These inhibitors were designed to target mature protease and prevent viral particle maturation by blocking Gag and Gag-Pol processing by mature protease. Currently there are few data assessing the ability of these protease inhibitors to block the initial step in autoproteolytic processing of Gag-Pol. This unique step involves the dimerization of two Gag-Pol polyproteins and autocleavage of the Gag-Pol polyprotein by the embedded dimeric protease. We developed a plasmid encoding a modified form of Gag-Pol that can undergo autoprocessing only at the initial cleavage site between p2 and nucleocapsid. Using an in vitro transcription/translation system, we assessed the ability of six different approved protease inhibitors (darunavir, indinavir, nelfinavir, ritonavir, saquinavir, and tipranavir) to block this initial autocleavage step. Of these inhibitors, darunavir and saquinavir were the most effective. Darunavir and saquinavir were also the most effective at blocking the initial autoprocessing of full-length Gag-Pol in HIV-1-infected T cells. Thus, we have identified at least two HIV-1 protease inhibitors that have activity against the primary autocatalytic step of the embedded HIV-1 protease in Gag-Pol at concentrations that may be attained in HIV-1-infected patients. Due to unique aspects of the initial processing step, it may be possible to develop inhibitors with greater potency against this step, thus halting viral maturation at the earliest stages. The transcription/translation assay could be used to develop more potent inhibitors of this essential first step in viral maturation.

Fig 1

Effect of ritonavir and darunavir on WT Gag-Pol processing. In vitro transcription/translation was carried out in the presence of [³⁵S]methionine with pGPfs WT plasmid. The reaction was run in the presence of increasing concentrations of each drug at 0, 0.01, 0.05, 0.10, 0.50, and 1.0 μM as described in Materials and Methods. Radiograms of Gag-Pol processing in the presence of increasing concentrations of ritonavir (A) or darunavir (B). The location for Gag-Pol (Gag-Pol and Gag-Pol internal initiation band), the products of the first step of Gag-Pol processing (NC-Int and MA-p2), and the product of the second step of Gag-Pol processing (TF-Int) are indicated to the right. Shown is a representative of two experiments with similar results.

Fig 2

Schematic diagram for Gag-Pol polyprotein precursor (WT), one-cut Gag-Pol polyprotein precursor containing active endogenous protease, and Gag-Pol precursor containing inactive protease. (A) The WT Gag-Pol contains nine major cleavage sites as shown. The initial processing site is indicated with an arrow at the p2/NC junction, and the processing site within the transframe protein (the second cleavage site) is indicated with a dashed line since it is located within the transframe protein). (B) The one-cut Gag-Pol substrate (S1) retains the initial p2/NC cleavage site and an active embedded HIV-1 protease. Cleavage by the embedded protease yields two major products, P1 and P2 (MA-p2 and NC-Int, respectively) as shown. The substrate and products are indicated in the radiogram to the right (lane 1). As shown, in the presence of 500 nM ritonavir there is little effect on the embedded protease activity as shown previously (lane 2). (C) Schematic diagram of the one-cut Gag-Pol substrate (S1) that retains the initial p2/NC cleavage site but with a mutation (D25A) rendering the embedded HIV-1 protease inactive (PR⁻). In the absence of protease only unprocessed Gag-Pol is produced by the PR⁻ plasmid construct (lane 1). Cleavage by exogenously added (trans) mature protease yields the same two major products, P1 and P2 (MA-p2 and NC-Int, respectively) (lane 2), as shown in panel B. In the presence of 500 nM ritonavir the activity of the exogenous protease is substantially inhibited (lane 3). The other minor bands in both radiograms are inherent in the in vitro translation system.

Fig 3

Inhibition of Gag-Pol cleavage (cis and trans) by HIV-1 protease inhibitors. (Top) For the endogenous protease assay, in vitro transcription/translation was carried out in the presence of [³⁵S]methionine with the one-cut Gag-Pol plasmid encoding active endogenous protease as described in Materials and Methods. The reactions were run in the presence of increasing concentrations of each drug at 0, 0.1, 0.5, 1.0, 5.0, 10, 50, and 100 μM as described in Materials and Methods. (Bottom) For the exogenous assay, in vitro transcription/translation was carried out for 1.5 h in the presence of [³⁵S]methionine with the one-cut Gag-Pol plasmid encoding inactive endogenous protease. HIV-1 protease was then added to each reaction and terminated 10 min later. The reactions were run in the presence of increasing concentrations of each drug at 0, 0.5, 1.0, 5.0, 10, 50, 100, and 500 nM. (G) Graphical overlay for the dose-response curves for each drug in the two assays. The percentage of inhibition for endogenous and exogenous assays was calculated as [(percentage of processing without drug − percentage of processing with drug)/(percentage of processing without drug)] × 100. The percentage of processing for each lane was calculated as [(sum of products)/(substrate + sum of products)] × 100. Each value represents the mean of results of three separate experiments except for that for tipranavir (exogenous), which represents the mean of results of two separate experiments. The individual dose-response curves are shown in Fig. S2 in the supplemental material. The resultant curves were obtained using the sigmoidal nonvariable slope dose-response equation in Prism 4 and represent the mean ± the standard deviation of results of three separate experiments. For tipranavir (exogenous), the mean of results of two experiments and the range are shown.

Fig 4

Inhibition of HIV-1 p165 Gag-Pol polyprotein processing and HIV-1 p55 Gag processing in the presence of HIV-1 protease inhibitors. H9 cells chronically infected with HIV-1 were treated for 3 days with control (Ctl) vehicle (0.05% DMSO) or 10 μM the indicated inhibitor. The supernatant was collected and virus pelleted by ultracentrifugation. The viral pellets were analyzed by Western blotting with the following monoclonal antibodies. (A) Monoclonal antibody to HIV-1 reverse transcriptase (RT); shown is one representative blot from two experiments. (B) Monoclonal antibody to HIV-1 integrase (Int); shown is a representative blot from three experiments. (C) Monoclonal antibody to HIV-1 p24 capsid protein; shown is a representative blot from three experiments with similar results. The locations for p165 Gag-Pol, Nc-Int, TF-Int, RT subunits (p64 and p51), p55 Gag, p47 Gag, and p24 Gag and Int (integrase) are indicated. Below the blots in panels A and B is the quantification of the percent Gag-Pol over the total bands detected [(pixels for Gag-Pol/total pixels) × 100].