Fig. 8
From: Determinants of FIV and HIV Vif sensitivity of feline APOBEC3 restriction factors
The impact of mutations in FcaA3s on FIV infectivity. a FIVΔvif luciferase reporter virions were produced in the presence of feline A3 expression plasmids (FcaA3Z3 wild-type, FcaA3Z3.A65I, FcaA3Z3.LI-AA or FcaA3Z3.A65I + LI-AA, 250 ng plasmid) with increasing amounts of FIV Vif (0, 40, 80 and 160 ng plasmid), pcDNA3.1 (+) was added as a control (vector). Infectivity of reporter vectors was determined by quantification of luciferase activity in 293T cells transduced with vector particles. b Cell lysates of FIV producer cells examined in a were used to detect the expression of FcaA3Z3 and FIV Vif by anti-HA and anti-V5 antibodies, respectively. Cell lysates were also analyzed for equal amounts of total proteins using anti-tubulin antibody. c The impact of FcaA3Z2Z3 and FcaA3Z2Z3-M on infectivity of FIVΔvif luciferase reporter vectors in the presence of FIV Vif. 293T cells were co-transfected FIVΔvif luciferase reporter viruses with 1000 ng FcaA3s and 100 ng FIV Vif expression plasmids. 48 h later, FIV particles were used to infect 293T cells, and infectivity was determined by quantification of luciferase activity. d Cell lysates for FIV producer cells used in c were analyzed by immunoblots to detect the expression of FcaA3Z3s and FIV Vif by anti-HA and anti-V5 antibodies, respectively. Cell lysates were also analyzed using anti-tubulin antibody. e, f Spreading replication of HIV-1 expressing FIV Vif (NL-Bal.vif FIV) is inhibited by feline A3Z2Z3-M. e Immunoblot analysis of HOS.CD4.CCR5 cells stable expressing feline A3Z2Z3 proteins. A3 proteins were detected by anti-HA antibody. Anti-tubulin served to demonstrate equal protein loading. f HOS.CD4.CCR5 cells expressing either wild-type A3Z2Z3 or A3Z2Z3-M were infected by NL-Bal.vif FIV with an MOI of 0.01 and virus replication was monitored by using the cell culture supernatant for infection of TZM-bl luciferase reporter cells. Asterisks represent statistically significant differences: ***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05; ns, p > 0.05 (Dunnett t test)