APOBEC3G targets specific virus species

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.     

A single amino acid determinant governs the species-specific sensitivity of APOBEC3G to Vif action

APOBEC3G (also known as CEM15) is an innate intracellular antiretroviral factor that is counteracted by the Vif protein of lentiviruses. While APOBEC3G orthologues from several species are active against a broad range of retroviruses, given Vif proteins have a narrow spectrum of activity. For instance, HIV-1 Vif efficiently blocks APOBEC3G from human but not African green monkey (AGM), whereas the reverse is observed with SIV(AGM) Vif. Here, we demonstrate that a single amino acid at position 128 of human and AGM APOBEC3G governs the virus-specific sensitivity of these proteins to Vif-mediated inhibition. Furthermore, we show that this phenotype correlates with the ability of Vif to bind APOBEC3G and interfere with its incorporation into virions. These results shed light on an important determinant of the tropism of primate lentiviruses.     

The betaretrovirus Mason-Pfizer monkey virus selectively excludes simian APOBEC3G from virion particles

The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroviruses. Therefore, any retrovirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Sch?fer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroviruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroviruses.     

APOBEC3G incorporation into human immunodeficiency virus type 1 particles

APOBEC3G is promiscuous with respect to its antiretroviral effect, requiring that it be packaged into diverse retrovirus particles. Here, we show that most virally encoded human immunodeficiency virus type 1 particle components are dispensable for APOPEC3G incorporation. However, replacement of the nucleocapsid (NC) Gag domain with a leucine zipper abolished APOBEC3G incorporation. Moreover, coprecipitation analysis showed that APOBEC3G-Gag interaction requires NC and nonspecific RNA. These observations suggest that APOBEC3G exploits an essential property of retroviruses, namely, RNA packaging, to infiltrate particles. Because it is, therefore, difficult to evolve specific sequences that confer escape from APOBEC3G, these findings may explain why lentiviruses evolved an activity that induces its destruction.     

Structure-function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities

Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.     

Differential sensitivity of murine leukemia virus to APOBEC3-mediated inhibition is governed by virion exclusion

While members of the APOBEC3 family of human intrinsic resistance factors are able to restrict the replication of Vif-deficient forms of human immunodeficiency virus type 1 (HIV-1), they are unable to block replication of wild-type HIV-1 due to the action of Vif, which induces their degradation. In contrast, HIV-1 Vif is unable to block inhibition mediated by APOBEC3 proteins expressed by several heterologous species, including mice. Here, we have asked whether the simple retrovirus murine leukemia virus (MLV) is sensitive to restriction by the cognate murine or heterologous, human APOBEC3 proteins. We demonstrate that MLV is highly sensitive to inhibition by human APOBEC3G and APOBEC3B but resistant to inhibition by murine APOBEC3 or by other human APOBEC3 proteins, including APOBEC3F. This sensitivity fully correlates with the ability of these proteins to be packaged into MLV virion particles: i.e., human APOBEC3G and APOBEC3B are packaged while murine APOBEC3 and human APOBEC3F are excluded. Moreover, this packaging in turn correlates with the differential ability of these APOBEC3 proteins to bind MLV Gag. Together, these data suggest that MLV Gag has evolved to avoid binding, and hence virion packaging, of the cognate murine APOBEC3 protein but that MLV infectivity is still restricted by certain heterologous APOBEC3 proteins that retain this ability. Moreover, these results suggest that APOBEC3 proteins may help prevent the zoonotic infection of humans by simple retroviruses and provide a mechanism for how simple retroviruses can avoid inhibition by APOBEC3 family members.     

Derepression of microRNA-mediated protein translation inhibition by apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies.     

The viral infectivity factor (Vif) of HIV-1 unveiled

The viral infectivity factor (Vif) of HIV type-1 (HIV-1) is essential for efficient viral replication, yet was, until recently, enigmatic. This resulted from the complexity and cellular specificity of its function and the correspondingly complex systems that are required for its investigation. These limitations have been overcome and Vif function has been rapidly elucidated, with implications for the development of drugs to block its activity. These studies have revealed a novel component of the innate immune system, APOBEC3G, that lethally hypermutates retroviruses, including HIV-1. For HIV-1, the competition between the virus and APOBEC3G is tipped in favor of the invader by Vif, which binds to APOBEC3G and triggers its polyubiquitination and rapid degradation, thereby preventing its entry into progeny virions.     

Identification of APOBEC3DE as another antiretroviral factor from the human APOBEC family

A tandem arrayed gene cluster encoding seven cytidine deaminase genes is present on human chromosome 22. These are APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H. Three of them, APOBEC3G, APOBEC3F, and APOBEC3B, block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. In addition, APOBEC3A and APOBEC3C block intracellular retrotransposons and simian immunodeficiency virus (SIV), respectively. In opposition to APOBEC genes, HIV-1 and SIV contain a virion infectivity factor (Vif) that targets APOBEC3F and APOBEC3G for polyubiquitylation and proteasomal degradation. Herein, we studied the antiretroviral activities of the human APOBEC3DE and APOBEC3H. We found that only APOBEC3DE had antiretroviral activity for HIV-1 or SIV and that Vif suppressed this antiviral activity. APOBEC3DE was encapsidated and capable of deaminating cytosines to uracils on viral minus-strand DNA, resulting in disruption of the viral life cycle. Other than GG-to-AG and AG-to-AA mutations, it had a novel target site specificity, resulting in introduction of GC-to-AC mutations on viral plus-strand DNA. Such mutations have been detected previously in HIV-1 clinical isolates. In addition, APOBEC3DE was expressed much more extensively than APOBEC3F in various human tissues and it formed heteromultimers with APOBEC3F or APOBEC3G in the cell. From these studies, we concluded that APOBEC3DE is a new contributor to the intracellular defense network, resulting in suppression of retroviral invasion.     

Differential requirement for conserved tryptophans in human immunodeficiency virus type 1 Vif for the selective suppression of APOBEC3G and APOBEC3F

APOBEC3G (A3G) and related cytidine deaminases, such as APOBEC3F (A3F), are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus type 1 (HIV-1) requires suppression of multiple cytidine deaminases by Vif. Whether HIV-1 Vif recognizes various APOBEC3 proteins through a common mechanism is unclear. The domains in Vif that mediate APOBEC3 recognitions are also poorly defined. The N-terminal region of HIV-1 Vif is unusually rich in Trp residues, which are highly conserved. In the present study, we examined the role of these Trp residues in the suppression of APOBEC3 proteins by HIV-1 Vif. We found that most of the highly conserved Trp residues were required for efficient suppression of both A3G and A3F, but some of these residues were selectively required for the suppression of A3F but not A3G. Mutant Vif molecules in which Ala was substituted for Trp79 and, to a lesser extent, for Trp11 remained competent for A3G interaction and its suppression; however, they were defective for A3F interaction and therefore could not efficiently suppress the antiviral activity of A3F. Interestingly, while the HIV-1 Vif-mediated degradation of A3G was not affected by the different C-terminal tag peptides, that of A3F was significantly influenced by its C-terminal tags. These data indicate that the mechanisms by which HIV-1 Vif recognizes its target molecules, A3G and A3F, are not identical. The fact that several highly conserved residues in Vif are required for the suppression of A3F but not that of A3G suggests a critical role for A3F in the restriction of HIV-1 in vivo.     

The DNA deaminase activity of human APOBEC3G is required for Ty1, MusD, and human immunodeficiency virus type 1 restriction

Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, human immunodeficiency virus type 1 (HIV-1), were used to evaluate the relative contributions of deaminase-dependent and -independent mechanisms. Although human APOBEC3G can restrict the replication of all three of these retroelements, APOBEC3G lacking the catalytic glutamate (E259Q) was clearly defective. This phenotype was particularly clear in experiments with low levels of APOBEC3G expression. In contrast, purposeful overexpression of APOBEC3G-E259Q was able to cause modest to severe reductions in the replication of Ty1, MusD, and HIV-1(ΔVif). The importance of these observations was highlighted by data showing that CEM-SS T-cell lines expressing near-physiologic levels of APOBEC3G-E259Q failed to inhibit the replication of HIV-1(ΔVif), whereas similar levels of wild-type APOBEC3G fully suppressed virus infectivity. Despite the requirement for DNA deamination, uracil DNA glycosylase did not modulate APOBEC3G-dependent restriction of Ty1 or HIV-1(ΔVif), further supporting prior studies indicating that the major uracil excision repair system of cells is not involved. In conclusion, the absolute requirement for the catalytic glutamate of APOBEC3G in Ty1, MusD, and HIV-1 restriction strongly indicates that DNA cytosine deamination is an essential part of the mechanism.