HIV-1 Nef inhibits Protease activity and its absence alters protein content of mature viral particles

Abstract

Nef is an important player for viral infectivity and AIDS progression, but the mechanisms involved are not completely understood. It was previously demonstrated that Nef interacts with GagPol through p6*-Protease region. Because p6* and Protease are involved in processing, we explored the effect of Nef on viral Protease activity and virion assembly. Using in vitro assays, we observed that Nef is highly capable of inhibiting Protease activity. The IC50 for nef-deficient viruses in drug susceptibility assays were 1.7- to 3.5-fold higher than the wild-type counterpart varying with the type of the Protease inhibitor used. Indicating that, in the absence of Nef, Protease is less sensitive to Protease inhibitors. We compared the protein content between wild-type and nef-deficient mature viral particles by gradient sedimentation and observed up to 2.7-fold reduction in the Integrase levels in nef-deficient mature particles. This difference in levels of Integrase correlated with the difference in infectivity levels of wild type and nef-deficient viral progeny. In addition, an overall decrease in the production of mature particles was detected in nef-deficient viruses. Collectively, our data support the hypothesis that the decreased infectivity typical of nef-deficient viruses is due to an abnormal function of the viral Protease, which is in turn associated with less mature particles being produced and the loss of Integrase content in these particles, and these results may characterize Nef as a regulator of viral Protease activity.

Figure 1. Nef effects in PR activity in vitro.

(A) SDS-PAGE of purified GST-Nef fusion (lane 1) and GST (lane 2) proteins stained with Coomassie Blue. Molecular weights (MW) are shown on the left. (B) WB of the lysate of E. coli expressing HIV-1 Protease (PR) (lane 1) and a lysate control (LC) (lane 2). *Denotes the detection of two nonspecific bands only in the LC. (C) Protease activity measured by the cleavage of a specific FRET substrate over a 2-hour interval. Substrate cleavage allows emission of light and is represented by the y-axis. All conditions were tested in triplicate. RLU – Relative light units.

Figure 2. HIV-1ΔNef is less sensitive to Protease Inhibitors.

HIV-1 and HIV-1ΔNef viruses were produced in Hek-293T cells treated with increasing concentrations of LPV or in MOLT4 cells treated with increasing concentrations of DRV. The infectivity of the viral progeny was measured in TZM-bl cells, and dose-response curves and IC50 values were fitted using Hill 4-parameter non-linear regression. (A) The concentration-response curves for HIV-1 and HIV-1ΔNef in the presence of LPV. (B) The concentration-response curves for HIV-1 and HIV-1ΔNef in the presence of DRV. Representative of three experiments.

Figure 3. HIV-1 and HIV-1ΔNef mature particles have different protein content and distribution on a density gradient.

HIV-1 and HIV-1ΔNef virions were produced in Hek-293T cells and separated using a 30–70% continuous sucrose density gradient. Ten fractions were collected from top to bottom and numbered #1 through #10 accordingly. The fractions were analyzed to determine the content and distribution of IN (top panel) and CA (lower panel). (A) Protein content of soluble proteins (fractions #1–4), immature particles (#5–7) and mature particles (#8–10) for the HIV-1 (left panels) and HIV-1ΔNef viruses (right panels). (B) The CA content of cell-free supernatants before fractionation. (C) Quantification of the amount of CA in fractions #8–10. (D) Quantification of the amount of IN in fraction #8–10. *p = 0.061, **p = 0.036. The WB and ELISA results presented are representative of three experiments, CA and IN values are triplicates.

Figure 4. Distribution and protein content differ between HIV-1 and HIV-1ΔNef viral particles produced in MOLT cells.

HIV-1 and HIV-1ΔNef virions were produced in MOLT cells and separated using a 30–70% continuous sucrose density gradient. Twelve fractions were collected from top to bottom and numbered #1 through #12 accordingly. The fractions were precipitated with 20% TCA and analyzed by WB. (A) Infectivity levels of the viral progeny produced in MOLT cells. (B) Lysates of HIV-1- and HIV-1ΔNef-transfected cells, showing equivalent levels of viral protein expression. (C) Cell-free supernatants of HIV-1- and HIV-1ΔNef-transfected cells, showing equivalent levels viral release. (D) CA content of cell-free supernatants before separation by the density gradient as measured using a p24-ELISA. (E) CA (top panel), IN (middle pannel) and MA (bottom pannel) protein content of mature particles. The top three panels represent the fractions for HIV-1, and the bottom three panels represent the fractions for HIV-1ΔNef. (F) Quantification of the amount of CA in mature fractions after densitometry, *p = 0.002. (G) Quantification of the amount of MA in mature fractions after densitometry, **p = 0.059 (H) Quantification of the amount of IN in mature particles after densitometry. The results presented are representative of three experiments.

Figure 5. CA, MA and IN levels differ between mature HIV-1 and HIV-1ΔNef viral particles produced in MOLT cells.

HIV-1 and HIV-1ΔNef virions were produced in MOLT cells and separated using a 9.6–18% continuous optiprep density gradient. Twelve fractions were collected from top to bottom and numbered #1 through #12 accordingly. The fractions were precipitated with 20% TCA and analyzed by WB to determine the contents and distributions of CA (top panel), MA (middle panel) and IN (bottom panels). Left panels represent the fractions for HIV-1, and the right panels represent the fractions for HIV-1ΔNef. (A) The protein contents of the mature particles for the HIV-1 (left) and HIV-1ΔNef viruses (right). (B) Numbers of blue-foci of non-fractionated supernatant (sn) and non-preciptated fractions for each virus. *Denotes the detection of a nonspecific band. The result presented is the mean of three experiments.