APOBEC3F determinants of HIV-1 Vif sensitivity

Abstract

HIV-1 Vif counteracts restrictive APOBEC3 proteins by targeting them for proteasomal degradation. To determine the regions mediating sensitivity to Vif, we compared human APOBEC3F, which is HIV-1 Vif sensitive, with rhesus APOBEC3F, which is HIV-1 Vif resistant. Rhesus-human APOBEC3F chimeras and amino acid substitution mutants were tested for sensitivity to HIV-1 Vif. This approach identified the α3 and α4 helices of human APOBEC3F as important determinants of the interaction with HIV-1 Vif.

FIG 1

Vif-A3F interaction model and A3F experimental constructs. (A) Model illustrating that while only a single amino acid change (e.g., E324K) will convert huA3F from HIV-1 Vif sensitive to Vif resistant (11), multiple mutations may be required to sensitize rhA3F to HIV-1 Vif-mediated degradation. (B) To-scale amino acid alignment of rhA3F, huA3F, and each rhA3F-huA3F chimera construct used in this study. Each construct is depicted as a horizontal line, with amino acid differences relative to the rhA3F sequence labeled and the residue number indicated above the top sequence. To facilitate comparison, α helices are shaded dark gray, and β strands are shaded light gray. The zinc-coordinating and catalytic residues in the C-terminal domain (CTD) are indicated by black dots.

FIG 2

Humanized rhA3F restricts single-cycle HIV-1 replication in a Vif-dependent manner. (A and B) Histograms depicting the relative (normalized to the vector control) infectivities of Vif-deficient and Vif-proficient HIV-1 clone pIIIB produced in the presence of the indicated controls or hu/rhA3F-V5 chimeric constructs (n = 3; mean plus standard deviation shown). Representative immunoblots for each series of infectivity data are shown below each histogram. The infected cells were blotted for V5 to detect A3F, Vif, and tubulin (TUB). Purified viral particles were blotted for V5 to detect A3F and p24 (Gag). A schematic of each construct is shown, with huA3F-derived residues represented in white and rhA3F residues represented in gray. (C) The ratio of Vif-proficient HIV-1 infectivity to Vif-deficient infectivity is plotted using the data from panel B. One-way analysis of variance (ANOVA) was used to compare these values, and Dunnett's method was employed for post hoc testing. Data for constructs significantly different from rhA3F (P < 0.05) are represented by black bars, and P values are shown to highlight the most important comparisons (ns, not significant).

FIG 3

HIV-1 replication kinetics in SupT11 T-cell clones stably expressing humanized rhA3F constructs. (A) A representative immunoblot showing A3F levels in the indicated clones, compared to endogenous levels of A3F in H9 cells. (B) Spreading-infection kinetics of Vif-proficient HIV-1 clone pIIIB (solid lines) in SupT11 clones stably expressing the indicated A3F expression constructs. Vif-deficient HIV-1 clone pIIIB had no detectable replication under any of the conditions, except in cells expressing low rhA3F levels or the empty vector (dashed lines and data not shown). All infections were initiated with a 1% multiplicity of infection.

FIG 4

Vif interaction surface of A3F. (A) The residues identified as being important for converting rhA3F from HIV-1 Vif resistant to HIV-1 Vif sensitive are depicted in yellow and labeled on the huA3F crystal structure (PDB accession no. 4IOU). (B) The residues indicated define a negatively charged surface (red), as seen in an electrostatic potential surface map of A3F. (C) Alignment of the regions determined to be important for conveying sensitivity to degradation by HIV-1 Vif in huA3F and rhA3F. The residues in α helices three and four that differ between huA3F and rhA3F are highlighted in yellow. Active-site residues that coordinate the zinc atom are indicated by black dots.