Catalytic activity of APOBEC3F is required for efficient restriction of Vif-deficient human immunodeficiency virus

Abstract

APOBEC3 proteins are DNA cytosine deaminases that restrict the replication of human immunodeficiency virus deficient in the counterdefense protein Vif. Here, we address the capacity of APOBEC3F to restrict via deaminase-dependent and -independent mechanisms by monitoring spreading infections in diverse T cell lines. Our data indicate that only a deaminase-proficient protein is capable of long-term restriction of Vif-deficient HIV in T cells, analogous to prior reports for APOBEC3G. This indicates that the principal mechanism of APOBEC3F restriction is deaminase-dependent.

Keywords: APOBEC3F; APOBEC3G; Deaminase; HIV restriction; Vif.

Fig. 1. APOBEC3 expression levels in the cell lines used in this study

(A) A3F levels found in SupT11 cells stably transfected with untagged A3F (code letter B followed by a number to indicate a distinct, clonal line), A3F E251Q (code letter D) or a vector control (code letter A) in comparison with naturally nonpermissive lines CEM2n and H9. (B) A3G levels found in SupT11 cells stably transfected with untagged A3G (code letter C), A3G E259Q (code letter E) or a vector control (code letter A) in comparison with naturally nonpermissive lines CEM2n and H9.

Fig. 2. Encapsidation of A3F catalytic mutants E251Q and W277A

Plasmids expressing A3F-V5 or either of two catalytic mutant derivatives were cotransfected with Vif-deficient HIV_IIIB. Virus-containing supernatants and producer cells were harvested approximately two days post-transfection, and expression and encapsidation were analyzed by western blot.

Fig. 3. Deaminase activity is required for restriction of Vif-deficient HIV by both A3F and A3G

Representative spreading infection curves infected at MOI 0.05 from one of three independent experiments demonstrating the inability of catalytic mutants of A3F or A3G to efficiently restrict Vif-deficient HIV. (A–B) Wildtype or Vif-deficient HIV_IIIB on four independent cell lines expressing wildtype A3F. (C–D) On four independent cell lines expressing A3F E251Q. (E–F) On four independent cell lines expressing wildtype A3G. (G–H) On four independent cell lines expressing A3G E259Q. Cell lines in all panels are named in as Fig. 1. Variation in the time to peak and in the height of peaks reflects the fact that each cell line used is an independent subclone. Note also that the peak of infectious virion production is typically followed shortly thereafter by a rapid drop in detected infectious virus. This reflects viral cytopathic effects in permissive cultures where HIV successfully spreads, which results in the death of the permissive producer cell cultures shortly after peak infectious virion production. In the absence of surviving producer cells, no infectious virion production is detected.

Fig. 4. Mutation of the putative A3F-interacting region of Vif but not the putative A3G-interacting region of Vif yields viruses selectively susceptible to the predicted APOBEC3 protein

(A) Growth of wildtype, Vif-deficient and the indicated Vif mutant derivatives of HIV_IIIB in the presence of A3F. (B) Growth of wildtype, Vif-deficient and the indicated Vif mutants in the presence of A3G. (C) Growth of wildtype, Vif-deficient and the indicated Vif mutants in naturally nonpermissive CEM2n cells. Results are representative of 2–3 independent experiments. The symbols associated with viruses showing no growth are boxed to facilitate comparison.