Incorporation of APOBEC3G into HTLV-1 virions. (A) Overexpressed APOBEC3G was incorporated into HTLV-1 virions. HEK293T cells were cotransfected with K30, pNL43-Luc (WT), or pNL43/Δvif-Luc (ΔVif) with or without an expression vector for HA-APOBEC3G. Western blotting was performed to detect HA-APOBEC3G in HEK293T cells and produced virions with anti-HA mAb. APOBEC3G was expressed in producer cells and efficiently incorporated into produced virions (lane 2). Expression of APOBEC3G and its incorporation into HIV-1 virions were reduced by expression of Vif as described previously (lane 4). Western blotting with anti-p19 and anti-p24 mAbs showed that similar amounts of virions were produced from each transfection (bottom panel). (B) Incorporation of APOBEC3G was confirmed in HTLV-1 virions purified by sucrose density equilibrium gradient analysis. HTLV-1 K30 virions were purified by sucrose density equilibrium gradient analysis. Gradient fractions were collected and used for analyzing incorporation of APOBEC3G into virions. APOBEC3G were detected and colocalized with HTLV-1 Gag (p19) proteins (lanes 4, 5). (C) APOBEC3G, its mutants, and muAPOBEC3G were incorporated into HTLV-1 virions. Expression vectors for HA-APOBEC3G, its mutants, or HA-muAPOBEC3G were cotransfected with K30 into HEK293T cells and APOBEC3G was detected with anti-HA mAb. HA-APOBEC3G, its mutants, and HA-muAPOBEC3G were all incorporated into virions. A3G and muA3G indicate human and murine APOBEC3G, respectively. E67Q, E259Q, and E67Q/E259Q were inactive mutants of human APOBEC3G that have a point mutation in N-terminal active site, C-terminal active site, and both, respectively, as described previously . (D) Endogenous APOBEC3G was also incorporated into HTLV-1 virions. Western blotting with anti-APOBEC3G Ab revealed expression of endogenous APOBEC3G in MT-2 cells (lane 1, upper panel) and its incorporation into produced virions (lane 1, lower panel). No cytoplasmic proteins were detected with anti-β-tubulin mAb in MT-2 virions (lane 2, lower panel).