APOBEC3G targets specific virus species

Abstract

Human APOBEC3G (huAPOBEC3G), also known as CEM15, is a broad antiretroviral host factor that deaminates dC to dU in the minus strand DNA of human immunodeficiency virus type 1 (HIV-1), other lentiviruses, and murine leukemia virus (MLV), thereby creating G-to-A hypermutation in the plus strand DNA to inhibit the infectivity of these viruses. In this study, we examined the antiretroviral function of a murine homologue of APOBEC3G (muAPOBEC3G) on several retrovirus systems with different producer cells. MuAPOBEC3G did not suppress the infectivity of murine retroviral vectors produced from human or murine cells, whereas it showed antiviral activity on both wild-type and Deltavif virions of HIV-1 in human cells. In contrast, huAPOBEC3G showed broad antiviral activity on HIV-1 and murine retroviral vectors produced from human cells as well as murine cells. These data suggested that muAPOBEC3G does not possess antiretroviral activity on murine retroviruses and has a different target specificity from that of huAPOBEC3G and that huAPOBEC3G works as a broad antiviral factor not only in human cells but also in murine cells. A functional interaction study between human and murine APOBEC3G supported the former hypothesis. Furthermore, studies on the expression of APOBEC3G in producer cells and its incorporation into virions revealed that muAPOBEC3G is incorporated into HIV-1 virions but not into MLV virions. Thus, muAPOBEC3G cannot suppress the infectivity of murine retrovirus because it is not incorporated into virions. We suggest that murine retroviruses can replicate in murine target cells expressing muAPOBEC3G because they are not targets for this enzyme.

FIG. 1.

Effect of muAPOBEC3Gwt on the infectivity of HIV-1 virions. We transfected pNL43-Luc (wild type) or pNL43/Δvif-Luc (Δvif) with pDON-based vectors (increasing amounts of huAPOBEC3G or muAPOBEC3Gwt, with the empty parental vector making up the balance) into HEK293T cells. Viruses from these cells were used to challenge M8166 cells, and productive infection was measured by luciferase activity. Values are presented as the percent infectivity relative to the wild-type virus not expressing APOBEC3G. (A) Expression of muAPOBEC3Gwt suppressed the infectivity of wild-type as well as Δvif virions to the same extent, while huAPOBEC3G showed a more potent antiviral effect on Δvif virions than wild-type virions. (B) muAPOBEC3Gwt introduced G-to-A hypermutation in the viral DNA of wild-type as well as Δvif virus. A DNA sequence analysis of the env region was performed with DNA synthesized in endogenous reverse transcription reactions (a, b, and c) or DNA amplified from target cells (d and e). G-to-A mutations are shown in red, with nucleic acid numbers corresponding to the pNL43 sequence, 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. (a) Δvif virions with huAPOBEC3G. (b and d) Δvif virions with muAPOBEC3Gwt. (c and e) Wild-type virions with muAPOBEC3Gwt.

FIG. 2.

MuAPOBEC3Gwt did not suppress the infectivity of MLV-based vectors produced from human cells. We transfected pDON-Luc and pVSV-G with pcDNA3/HA-based vectors into GP293 cells. An adjusted amount of virus as determined by reverse transcriptase values was used to challenge HEK293T cells, and productive infection was measured by luciferase activity. Values are presented as the percent infectivity relative to the value with MLV not expressing APOBEC3G.

FIG. 3.

MuAPOBEC3Gwt worked synergistically with huAPOBEC3G on the infectivity of HIV-1 virions but not on MLV virions. (A) We transfected pNL43-Luc (wild type) or pNL43/Δvif-Luc (Δvif) with a combination of expression vectors for APOBEC3G (increasing amounts of muAPOBEC3Gwt or huAPOBEC3G with a fixed amount of huAPOBEC3G or muAPOBEC3Gwt, respectively) into HEK293T cells. An infectivity assay was performed, and values are presented as described in the legend to Fig. 1. (B) We transfected pDON/Luc with a combination of expression vectors for APOBEC3G as described in A into GP293 cells. An infectivity assay was carried out, and values are presented as described in the legend to Fig. 2A. (C) A coimmunoprecipitation assay revealed the physical interaction between huAPOBEC3G and muAPOBEC3Gwt. We transfected pcDNA3/HA-based vectors with pDON/EGFP-based vectors. Cell lysates were immunoprecipitated (IP) with anti-EGFP monoclonal antibody (Ab) and subjected to immunoblotting with anti-HA monoclonal antibody. Solid arrow, huAPOBEC3G; open arrow, muAPOBEC3Gwt.

FIG. 4.

HuAPOBEC3G suppressed the infectivity of MLV-based vector produced from murine cells, but muAPOBEC3Gwt did not. (A) We transfected pDON-Luc with pcDNA3/HA-based vectors into PT67 cells. An infectivity assay was performed 2 days after transfection or 2 weeks after selection with G418 and hygromycin B, and values are presented as described in the legend to Fig. 2A. (B) The protein expression of APOBEC3G in PT67 cells was confirmed by Western blot analysis with anti-HA monoclonal antibody. Lane 1 is a positive control, and lanes 2 to 6 correspond to the lanes in panel A. Solid arrow, huAPOBEC3G; open arrow, muAPOBEC3Gwt.

FIG. 5.

MuΔexon5 and muAPOBEC3Gwt showed similar antiviral effects. (A) We transfected pNL43-Luc (wild type) or pNL43/Δvif-Luc (Δvif) with pcDNA/HA-based vectors into HEK293T cells with the indicated amount of plasmid. An infectivity assay was carried out, and values are presented as described in the legend to Fig. 1. MuΔexon5 and muAPOBEC3Gwt showed similar antiviral effects. (B) Protein expression of APOBEC3G in producer cells (upper column) and incorporation of APOBEC3G into virions (lower column) were detected by Western blot analysis with anti-HA monoclonal antibody. The expression of β-actin and the incorporation of p24 were used as internal controls for protein expression in the cell lysate and protein incorporation into virions, respectively. The expression of HIV-1 Vif protein reduced the protein expression of huAPOBEC3G in producer cells as well as the incorporation of huAPOBEC3G into HIV-1 virions, while it did not affect the protein expression or incorporation of muAPOBEC3Gwt and muΔexon5 into virions.

FIG. 6.

MuAPOBEC3G (muAPOBEC3Gwt or muΔexon5) did not show antiviral effects on MLV because it was not incorporated into MLV virions. We transfected pDON-Luc and pVSV-G with the indicated amount of pcDNA3/HA-based vectors into GP293 cells. An infectivity assay was performed, and values are presented as described in the legend to Fig. 2A. (A) HuAPOBEC3G suppressed the infectivity of MLV, but muAPOBEC3G did not. (B) Protein expression of APOBEC3G in producer cells (upper column) and the incorporation of APOBEC3G into virions (lower column) were detected by Western blot analysis with anti-HA monoclonal antibody. MuAPOBEC3G was not incorporated into MLV virions, although it was expressed in producer cells at a level comparable to huAPOBEC3G. Solid arrow, huAPOBEC3G or muΔexon5; open arrow, muAPOBEC3Gwt. (C) The expression of muAPOBEC3G was not impaired by the expression of MLV.