Fig 2: Depletion of IQGAP1 Increases Production of Infectious HIV-1 Virions(A) KO of IQGAP1 in HEK293 cells increases release of HIV-1 virions. IQGAP1 KO and control HEK293 cells were transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg). After 48 h the culture supernatants were collected, and the cells were harvested and lysed. Virions in the supernatants were isolated by pelleting through 25% sucrose cushions. Gag protein and its cleavage products in the cells (cell lysate) and in released virions (viral pellet) were detected by HIV-1-specific antibody. Shown are representative western blots from three independent experiments (n = 3).(B) Densitometric analysis of data in (A). Shown are fold changes in protein expression levels for total Gag (Pr55gag and all its cleavage products), Pr55gag, and p24CA relative to control transfected cells, all normalized to GAPDH for each experiment. The data are mean ± SEM from three independent experiments (n = 3). p values are shown when increase of p24CA from KO cells relative to control cells is significant.(C) KO of IQGAP1 in HEK293 cells does not affect expression of viral genomes. Cell lysates from (A) were assayed to measure the luciferase reporter expressed from the Nef ORF in the transfected viral genomes, with the levels in the control transfected cells set at 100. The data are mean ± SEM from three independent experiments (n = 3).(D) Re-expression of WT IQGAP1 in IQGAP1 KO cells reduces HIV-1 viral release to normal levels of control cells. IQGAP1-KO cells were co-transfected with pNL4–3.Luc.R-E-viral DNA genome (1 µg) and either an empty vector (EV) or a CRISPR-resistant IQGAP1-expressing DNA (IQGAP1XPR) Control cells were co-transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg) and an empty vector (EV). All transfection were performed at a molar ratio of 3:1 (EV or IQGAP1XPR to pNL4–3.Luc.R-E-). After 48 h culture supernatants and cells were processed as in (A). Shown are representative western blots from three independent experiments (n = 3).(E) Expression of HIV-1 proteins with VSV-G in IQGAP1-depleted cells. IQGAP1 KO or control HEK293 cells were transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg) and pVSVG (0.5 µg). At 48 h after transfection, the cells were harvested and lysed. Intracellular Gag protein and its cleavage products as well as the VSV-G env protein were detected with specific antibodies. Shown is a representative western blot out of three independent experiments (n = 3).(F) KO of IQGAP1 enhances the production of infectious HIV-1 virus. Two serial dilutions (1/10 and 1/100) of culture supernatants harvested from producer cells in (E) were used to infect HEK293A cells, and luciferase activities were measured 48 h after infection. In each infecting dilution, the luciferase activity from cells infected with virus packaged from control cells was set as 1. The data are mean ± SEM and p values are for virus yield from KO cells relative to control cells from three independent experiments (n = 3).(G) KO of IQGAP1 increases release of HIV-1 virions from infected cells. IQGAP1 KO and control HEK293 cells were infected with HIV-1-VSV for 48 h. Cell lysates (cell lysate) and culture supernatants (viral pellet) were assessed by western blots as in (A). Shown are representative western blots from three independent experiments (n = 3).(H) KD of IQGAP1 in human T cell lymphoblast-like cell line increases release of HIV-1 virions from infected cells. Stable KO Jurkat cells expressing specific IQGAP1 shRNAs (IQGAP-A and IQGAP1-B) or expressing control non-targeting (NT) shRNA were infected and processed as in (G). Shown are representative western blots from three independent experiments (n = 3).

Fig 3: IQGAP1 Depletion Enhances HIV-1 Gag Targeting to The Plasma Membrane(A) Depletion of IQGAP1 enhances Gag association to the plasma membrane. Control or IQGAP1 KO HEK293 cells were transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg). After 48 h, the cells were fixed and stained for HIV-1 Gag using HIV-1-specific antibody (red) and for cell nuclei using DAPI (blue). Random fields of view were imaged to acquire a minimum of 100 Gag-expressing cells. Shown are four representative fields of view from a single experiment out of three independent experiments (n = 3). Scale bar, 10 µm.(B) IQGAP1 KO or control cells were transfected as in (A), and membrane floatation assay was performed as in Figure 6B. Shown are representative western blots from three independent experiments (n = 3).(C) Densitometric analysis of the data in (B) was calculated as described in Figure 6C. The data are mean ± SEM from three independent experiments (n = 3). p value is for membrane-bound Gag in IQGAP1-KO cells relative to control.

Fig 4: IQGAP1 Interacts with the Nucleocapsid and p6 Domains of HIV-1 Gag(A) IQGAP1 interacts with the HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg) together with DNAs expressing GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to pNL4–3.Luc.R-E-). Cell lysates were subjected to immunoprecipitation using anti-GFP antibody. GFP, GFP-IQGAP1, and Gag were detected using western blot. Shown are representative western blots from three independent experiments (n = 3).(B) IQGAP1 interacts with unmyristylated HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (Gag WT; 1 µg) or with an HIV-1 genome encoding a Gag mutant that cannot be myristylated (Gag G2A; 1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to Gag WT or Gag G2A) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3).(C) Schematic presentation of the Rev-independent flag-tagged Gag domains proteins used for this experiment.(D) The nucleocapsid domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with DNAs encoding GFP or GFP-IQGAP1 together with 1 µg of the indicated Gag constructs from (C) at a molar ratio of 3 (GFP or GFP-IQGAP1 to HIV-1 Gag constructs) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3).(E) The p6 domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with the GagDNC construct (C) (1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to Gag?DNC) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3).

Fig 5: The C Terminus Region of HIV-1 Gag Is Required for IQGAP1 Regulation of Viral Release Independently of Gag L Domains(A) Schematic presentation of Rev-independent flag-tagged Gag domains proteins used for this experiment.(B and C) Replacement of the C terminus region of Gag with the GCN4 leucine zipper abolishes IQGAP1 regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent Gag-flag (B) or GagZ-flag (C) constructs (0.5 µg) with increasing amounts of DNA encoding GFP-IQGAP1 or GFP at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the culture supernatants were purified. CA levels in pelleted virions and intracellular GFP-IQGAP1 and Gag levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Shown are representative western blots from three independent experiments (n = 3).(D and E) Either the p6 or the nucleocapsid domains in Gag are sufficient for IQGAP1 negative regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent Gag?p6-flag (D) or GagDNC-flag (E) DNAs (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNA and processed as in (B). Shown are representative western blots from three independent experiments (n = 3).(F) Schematic presentation of Rev-independent flag-tagged Gag mutant proteins used for this research.(G–J) IQGAP1 overexpression reduces HIV-1 Gag VLPs lacking the ability to recruit the cellular ESCRT machinery. HEK293T cells were co-transfected with DNA constructs from (F) (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the supernatants were isolated. CA levels in pelleted virions and cellular GFP-IQGAP1 and Gag expression levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Cells were transfected with (G) wild-type, (H) PTAPmut, (I) LYPXnLmut, (J) and PTAP/LYPXnLmut Gag expression constructs. Shown are representative western blots from three independent experiments (n = 3).