Fig 2: Expression of epitope-tagged and untagged A3C derivative constructs in 293T cells. Immunoblots of 293T cells transfected with 100 ng or 400 ng of C-terminally tagged (A3C-HA), N-terminally tagged (HA-A3C), or untagged A3C variants using either an anti-HA or anti-A3C antibody for detection. Tubulin was used as a loading control

Fig 3: HIV-1 replication phenotypes following A3C disruption and variant complementation in non-permissive CEM2n cells. a Immunoblots of endogenous A3C in SupT11, H9, and CEM2n cells. Tubulin was used as a loading control. b Immunoblots of endogenous A3C in CEM2n clones following targeted disruption of A3C exon 3 by Cas9/gRNA complexes. Tubulin was used as a loading control. c A3C exon 3 sequences from parental CEM2n and an A3C-disrupted clone (CEM2n ∆A3C). d Flow cytometry plots for CEM2n ΔA3C cell pools 72 h post-transduction with GFP-reporter complementation vectors. The percentage of GFP+ cells is indicated for each population. e Immunoblots of A3C in the parental CEM2n line, CEM2n ΔA3C, and complemented CEM2n ΔA3C derivatives. Tubulin was used as a loading control. f, g Spreading infection kinetics of Vif-proficient and Vif-deficient HIV-1 (initial MOI = 0.02) in CEM2n, CEM2n ΔA3C, and the indicated complemented conditions. SupT11 cells are included as a control permissive cell type. Virus infectivity was determined by infection of CEM-GFP with culture supernatants followed 48 h later by quantification with flow cytometry. Each spreading infection experiment was performed 3 times and representative data are shown

Fig 4: A3C-Ile188 exhibits enhanced HIV-1 restriction activity in 293T cells. a Single cycle infectivity data for Vif-deficient HIV-1 viruses produced in the presence of untagged A3C-S188, A3C-I188, or catalytic mutant derivatives (E68Q). Immunoblots are shown below for viral particles (anti-A3G, anti-A3C, and anti-p24) and producer cells (anti-A3G, anti-A3C, and anti-tubulin). b Single cycle infectivity data for Vif-deficient HIV-1 viruses produced in the presence of N-terminally HA-tagged A3C-S188 or A3C-I188. Immunoblots are shown below for viral particles (anti-HA and anti-p24) and producer cells (anti-HA and anti-tubulin). All single cycle experiments were repeated at least 3 times, with representative infectivity data (mean ± SD) and immunoblots shown for one experiment