APOBEC3G targets human T-cell leukemia virus type 1

Abstract

Background: Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cellular protein with a broad antiviral activity. It inhibits infectivitiy of a wide variety of retroviruses by deaminating deoxycytidine (dC) into deoxyuridine (dU) in newly synthesized minus strand DNA, resulting in G-to-A hypermutation of the viral plus strand DNA. To clarify the mechanism of its function, we have examined the antiviral activity of APOBEC3G on human T-cell leukemia virus type 1 (HTLV-1), the first identified human retrovirus.

Results: In this study, we have demonstrated that overexpressed as well as endogenous APOBEC3G were incorporated into HTLV-1 virions and that APOBEC3G inhibited the infection of HTLV-1. Interestingly, several inactive mutants of APOBEC3G also inhibited HTLV-1 and no G-to-A hypermutation was induced by APOBEC3G in HTLV-1 genome. Furthermore, we introduced the human immunodeficiency virus type 1 (HIV-1) vif gene into HTLV-1 producing cell line, MT-2, to antagonize APOBEC3G by reducing its intracellular expression and virion incorporation, which resulted in upregulation of the infectivity of produced viruses.

Conclusion: APOBEC3G is incorporated into HTLV-1 virions and inhibits the infection of HTLV-1 without exerting its cytidine deaminase activity. These results suggest that APOBEC3G might act on HTLV-1 through different mechanisms from that on HIV-1 and contribute to the unique features of HTLV-1 infection and transmission.

Figure 1

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 [7]. (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).

Figure 2

Inhibition of HTLV-1 infection by APOBEC3G. APOBEC3G as well as its mutants inhibited the infectivity of HTLV-1. Infectivity of HTLV-1 was measured as described in Materials and Methods. HTLV-1 proviral DNA load in target SupT1 cells was suppressed by APOBEC3G and its mutants to the level of that of heat-inactivted virus. Six independent experiments gave similar results and the data was presented as the mean of these values. Values are presented as infectivity ratio relative to K30 virus without expression of APOBEC3G.

Figure 3

No G-to-A hypermutation in HTLV-1 genome was induced by APOBEC3G. Mutations in HTLV-1 and HIV-1 ΔVif viruses were detected by sequencing p12 and Env regions, respectively. G-to-A hypermutation was induced by APOBEC3G in HIV-1 ΔVif DNA, but not in HTLV-1 DNA. We detected very few G-to-A mutations in HTLV-1 K30 genome with expression of APOBEC3G (C), but not without expression of APOBEC3G (D), whereas G-to-A hypermutation was induced in HIV-1ΔVif DNA by APOBEC3G (A). We also detected a very few G-to-A mutations in MT-2/Mock virus DNA (E) as well as MT-2/Vif virus DNA (F). G-to-A mutations are shown in red, while other mutations are denoted in black. The numbers before the sequence indicate the number of each clone, while those in parentheses indicate the total number of clones sequenced. WT indicates no mutations in this region.

Figure 4

HIV-1 Vif reduced the incorporation of APOBEC3G into HTLV-1 virions, resulting in the upregulation of the infectivity. (A) Expression of APOBEC3G in MT-2 cells and its incorporation into produced virions were reduced by HIV-1 Vif. Expression level of APOBEC3G was reduced in MT-2/Vif cells (lane 2, middle panel) as compared to MT-2/Mock cells (lane 1, middle panel). Incorporation of APOBEC3G into produced virions was also reduced in virions produced from MT-2/Vif cells (lane 2, bottom panel). Expression of Vif protein in MT-2/Vif cells was detected with anti-Vif mAb (top panel). (B) HIV Vif upregulated the infectivity of HTLV-1 produced from MT-2 cells. Infectivity of HTLV-1 virus produced from MT-2 cells was determined as described in Materials and Methods. Infectivity of viruses produced from MT-2/Vif cells was more than 4 times higher than that from MT-2/Mock cells. Four independent experiments gave similar results and the data was presented as the mean of these values. Values are presented as infectivity ratio relative to viruses from MT-2/Mock cells.