GS-CA1 and lenacapavir stabilize the HIV-1 core and modulate the core interaction with cellular factors

Abstract

The HIV-1 capsid is the target for the antiviral drugs GS-CA1 and Lenacapavir (GS-6207). We investigated the mechanism by which GS-CA1 and GS-6207 inhibit HIV-1 infection. HIV-1 inhibition by GS-CA1 did not require CPSF6 in CD4⁺ T cells. Contrary to PF74 that accelerates uncoating of HIV-1, GS-CA1 and GS-6207 stabilized the core. GS-CA1, unlike PF74, allowed the core to enter the nucleus, which agrees with the fact that GS-CA1 inhibits infection after reverse transcription. Unlike PF74, GS-CA1 did not disaggregate preformed CPSF6 complexes in nuclear speckles, suggesting that PF74 and GS-CA1 have different mechanisms of action. GS-CA1 stabilized the HIV-1 core, possibly by inducing a conformational shift in the core; in agreement, HIV-1 cores bearing N74D regained their ability to bind CPSF6 in the presence of GS-CA1. We showed that GS-CA1 binds to the HIV-1 core, changes its conformation, stabilizes the core, and thereby prevents viral uncoating and infection.

Keywords: Biological sciences; Immunology; Virology.

Graphical abstract

Figure 1

GS-CA1 does not require the expression of CPSF6 in CD4⁺ T cells to inhibit HIV-1 infection (A) Loss of CPSF6 expression using the CRISPR/Cas9 system in human primary CD4⁺ T cells from two different donors was determined by western blots using antibodies against CPSF6. Controls included gRNA against CXCR4 and a non-targeting gRNA. Expression of GAPDH was the control for equal loading. (B) Human primary CD4⁺ T cells depleted for CPSF6 expression were infected with HIV-1_NL4-3-GFP with the indicated concentrations of PF74, GS-CA1, or DMSO as a vehicle control. Infected GFP-positive cells were determined at 72 h post-infection. Experiments were performed in triplicates, and the mean infections ±SD are shown. Results for two different donors are shown.

Figure 2

GS-CA1 prevents the binding of CPSF6 and Nup153 to the HIV-1 core Human HEK293T cells were transfected with plasmids expressing CPSF6-FLAG (A) or Nup153-HA (B), and at 24 h post-transfection lysed cell extracts (INPUT) and stabilized wild-type HIV-1 capsid tubes were mixed with PF74 or GS-CA1 for 1 h before collection of the HIV-1 capsid tubes pellets by centrifugation. Pellets were washed two times and analyzed (BOUND). The input and bound fractions were analyzed by western blotting (a representative Western blot is shown) using anti-FLAG (A), anti-HA (B), or anti-p24 capsid antibodies. Experiments were repeated at least three times and a representative figure is shown. The amount of capsid relative to the total for three independent experiments is shown. Mean bound fractions relative to input ±SD are shown. ∗ indicates p value < 0.005, ∗∗ indicates p value < 0.001, ∗∗∗ indicates p value < 0.0005, as determined by using the unpaired t test.

Figure 3

GS-CA1 stabilizes the HIV-1 core during infection (A) Human A549 cells were infected with HIV-1-GFP (pseudotyped with VSV-G) at MOI = 2 with PF74, GS-CA1, or DMSO as a vehicle control. After incubation for 16 h 37°C cells were harvested and processed using the fate of the capsid assay, as described in methods. INPUT, SOLUBLE, and PELLET fractions of lysed cells were analyzed by western blots using antibodies against the HIV-1 p24 capsid protein. Experiments were performed 3 times, and the mean pelletable capsids ±SD are shown. (B) Human A549 cells were infected as described for (A) and infected GFP-positive cells were determined at 48 h post-infection. Infections were performed in triplicates, and the mean infections ±SD are shown. (C) Human A549 cells were infected and analyzed as described for (A) with PF74, GS-6207, or DMSO as a vehicle control. Experiments were performed 3 times, and the mean pelletable capsids ±SD are shown. (D) Human A549 cells were infected and analyzed as described for (A) and the PELLET fraction was also analyzed using antibodies against CPSF6. Experiments were performed 3 times, and the mean pelletable capsids ±SD are shown. ∗∗ indicates p value < 0.001, ∗∗∗ indicates p value < 0.0005, ∗∗∗∗ indicates p value < 0.0001, NS indicates not significant as determined by using the unpaired t test.

Figure 4

GS-CA1 does not inhibit the entry of the viral core into the nucleus (A and B) Human A549 cells were infected with wild-type HIV-1-GFP (pseudotyped with VSV-G) at MOI = 2 with PF74, GS-CA1, or DMSO as a vehicle control. (A) Capsids from the nuclear and cytosolic fractions of cells infected at 37°C for 8 h cells were determined using western blots with anti-p24 antibodies. Nopp140 and tubulin, determined using western blots with appropriate antibodies, were markers for nuclear and cytosolic content, respectively. Nuclear and cytosolic fractions of p24 were determined by western blots from three independent experiments (a representative Western blot is shown). The mean nuclear fractions relative to the cytosolic fractions ±SD are shown. ∗∗∗∗ indicates p value < 0.0001 as determined by using the unpaired t test. (B) Infected GFP-positive cells were determined at 48 h post-infection. Infections were performed in triplicates, and the mean infections ±SD are shown.

Figure 5

PF74, but not GS-CA1, disaggregates preformed CPSF6 complexes in nuclear speckles by HIV-1 infection (A) HeLa cells were infected with wild-type HIV-1-Luc virus pseudotype with VSV-G in the presence of PF74, GS-CA1, or DMSO as a vehicle control. Cells were incubated for 18 h at 37°C. Mean nuclear speckles ±SD are shown. (B) HeLa cells were infected with wild-type HIV-1-Luc virus, and 18 h post-infection were treated with PF74, GS-CA1, or DMSO as a vehicle control for 15 or 180 min. Mean nuclear speckles ±SD are shown. (C) Left panels: HeLa cells were infected with wild-type HIV-1-Luc virus in the presence of PF74, GS-CA1, PF74 and GS-CA1, or DMSO as a vehicle control. Cells were incubated for 18 at 37°C. Right panels: HeLa cells were infected with wild-type HIV-1-Luc virus, and at 18 h post-infection PF74, GS-CA1, PF74 and GS-CA1, or DMSO as a vehicle control were added for 120 min (A–C). After drug treatment, cells were immunostained using specific antibodies directed against CPSF6 (green) and SC35 (red). Nuclei were counterstained with Hoeschst (DNA). Mean nuclear speckles ±SD are shown. Scale bars, 5 μM.

Figure 6

GS-CA1 changes the conformation of the capsid Cell extracts of Human Jurkat cells (INPUT) were mixed for 1 h at room temperature with wild-type or mutant N74D HIV-1 capsid tubes in the presence of PF74, GS-CA1, or DMSO as a vehicle control. Stabilized HIV-1 capsid tubes were collected by centrifugation and washed two times (BOUND). The input and bound fractions were analyzed by western blotting using anti-CPSF6 and anti-p24 antibodies for three experiments (a representative Western blot is shown). Mean bound fractions relative to input ±SD are shown. ∗∗∗∗ indicates p value < 0.0001, NS indicates not significant as determined by using the unpaired t test.