Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G [A3G]) and other APOBEC3 proteins (25) are related to a family of proteins that also includes apolipoprotein B-editing catalytic subunit 1 (APOBEC1), APOBEC2, and activation-induced cytidine deaminase (AID) (23,66). These proteins have cytidine deaminase activities that modify RNA or DNA. A3G was the first APOBEC3 protein to be identified as a potent inhibitor of HIV-1 in the absence of Vif (59). A major outcome of virion packaging of A3G is the induction of C-to-U mutations in the minus-strand viral DNA during reverse transcription (22,32,42,43,63,73,77). Virion-packaged A3G and A3F can also reduce the accumulation of viral DNA (3,21,27,40,45,57,71) and the formation of proviral DNA (40,45).

The human cytidine deaminase APOBEC3G (A3G) and other APOBEC3 proteins exhibit differential inhibitory activities against diverse endogenous retroelements and retroviruses, including Vif-deficient human immunodeficiency virus type 1. The potential inhibitory activity of human APOBEC proteins against long interspersed element 1 (LINE-1) has not been fully evaluated. Here, we demonstrate inhibition of LINE-1 by multiple human APOBEC3 cytidine deaminases, including previously unreported activity for A3DE and A3G. More ancient members of APOBEC, cytidine deaminases AID and APOBEC2, had no detectable activity against LINE-1. A3A, which did not form high-molecular-mass (HMM) complexes and interacted poorly with P bodies, was the most potent inhibitor of LINE-1. A3A specifically recognizes LINE-1 RNA but not the other cellular RNAs tested. However, in the presence of LINE-1, A3A became associated with HMM complexes containing LINE-1 RNA. The ability of A3A to recognize LINE-1 RNA required its catalytic domain and was important for its LINE-1 suppression. Although the mechanism of LINE-1 restriction did not seem to involve DNA editing, A3A inhibited the accumulation of nascent LINE-1 DNA, suggesting interference with LINE-1 reverse transcription and/or integration or intracellular movement of LINE-1 ribonucleoprotein. Thus, association with P bodies or cellular HMM complexes could not predict the potency of APOBEC3 anti-LINE-1 activities. The catalytic domain of APOBEC3 proteins may be important for proper folding and target factors such as RNA or protein interaction in addition to cytidine deamination.A3G and other APOBEC3 proteins (22) belong to a family of proteins that also includes activation-induced cytidine deaminase (AID), apolipoprotein B-editing catalytic subunit 1 (APOBEC1), and APOBEC2 (1,16,19,35,39,45). These proteins have cytidine deaminase activities that can modify RNA or DNA. A3G was the first APOBEC3 protein to be identified as a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) in the absence of Vif (41). Subsequently, several other human APOBEC3 proteins, including APOBE C3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOB EC3DE (A3DE), and APOBEC3F (A3F), were identified as broad antiviral factors against HIV-1, simian immunodeficiency virus, murine leukemia virus, and various endogenous retroelements (3, 4, 11-14, 24, 26, 37, 40, 48, 49, 53) as well as hepatitis B virus (44).Like AID, which edits single-stranded immunoglobulin gene DNA, APOBEC3 proteins prefer minus-strand retroviral DNAs as targets (1,10,16,19,29,35,39,45,51). In the absence of Vif-induced A3G degradation in virus-producing cells, virion-packaged A3G induces C-to-U mutations in minus-strand viral DNA during reverse transcription (18,25,30,31,43,50,52). Both cytidine deamination-dependent and -independent antiviral functions of APOBEC3 proteins have been reported (2,8,17,20,28,33,36,44).The potential inhibitory activity of certain human APOBEC3 proteins against LINE-1 has been reported. A3A and A3B are potent inhibitors...

The human cytidine deaminase Apobec3F (h-A3F), a protein related to the previously recognized antiviral factor Apobec3G (h-A3G), has antiviral activity against human immunodeficiency virus type 1 (HIV-1) that is suppressed by the viral protein Vif. The mechanism of HIV-1 Vif-mediated suppression of h-A3F is not fully understood. Here, we demonstrate that while h-A3F, like h-A3G, was able to suppress primate lentiviruses other than HIV-1 (simian immunodeficiency virus from African green monkeys [SIVagm] and Rhesus macaques [SIVmac]), the interaction between Vif proteins and h-A3F appeared to differ from that with h-A3G. H-A3F showed no change in its species specificity against HIV-1 or SIVagm Vif when a negatively charged amino acid was replaced with a lysine at position 128,

CD8+ class I–restricted cytotoxic T lymphocytes (CTLs) usually incompletely suppress HIV-1 in vivo, and while analogous partial suppression induces antiretroviral drug-resistance mutations, epitope escape mutations are inconsistently observed. However, escape mutation depends on the net balance of selective pressure and mutational fitness costs, which are poorly understood and difficult to study in vivo. Here we used a controlled in vitro system to evaluate the ability of HIV-1 to escape from CTL clones, finding that virus replicating under selective pressure rapidly can develop phenotypic resistance associated with genotypic changes. Escape varied between clones recognizing the same Gag epitope or different Gag and RT epitopes, indicating the influence of the T cell receptor on pressure and fitness costs. Gag and RT escape mutations were monoclonal intra-epitope substitutions, indicating limitation by fitness constraints in structural proteins. In contrast, escape from Nef-specific CTL was more rapid and consistent, marked by a polyclonal mixture of epitope point mutations and upstream frameshifts. We conclude that incomplete viral suppression by CTL can result in rapid emergence of immune escape, but the likelihood is strongly determined by factors influencing the fitness costs of the particular epitope targeted and the ability of responding CTL to recognize specific epitope variants.

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins have been classified as either Z1- or Z2-type cytidine deaminases on the basis of phylogenetic analysis of their catalytic domains. Despite the identification of a number of Z1-type domain-containing cytidine deaminases, only one copy of Z2-type cytidine deaminase has been detected in each of the mammalian species evaluated thus far. Z1-type human APOBEC3 proteins are known to exhibit broad activities against diverse retroelements. However, the potential role of the only human Z2-type cytidine deaminase, APOBEC3H (A3H), in the restriction of retroelements has not yet been fully characterized. Here, we demonstrate that human A3H is a potent inhibitor of non-LTR LINE-1 transposition. Interestingly, it was also as efficient as A3G in inhibiting Alu retrotransposition, despite its poor association with Alu RNA. We have further demonstrated, for the first time, that human APOBEC3DE is also a potent inhibitor of Alu retrotransposition. Variants of A3H have divergent antiviral activities against HIV-1-Vif-deficient viruses. Unlike the anti-HIV-1 cytidine deaminases A3G and A3F, A3H is moderately regulated by interferons. These observations suggest that human Z2-type cytidine deaminase A3H variants have varying intrinsic abilities to restrict retroelements and that various APOBEC3 proteins may have evolved distinct inhibitory mechanisms against retroelements.

APOBEC3G (A3G) has broad antiviral activity against retroviruses and hepatitis B virus. However, the role of IFNs in regulating A3G during innate immunity has not been established. In this study, we show that the A3G gene is uniquely regulated by IFNs in a cell type-dependent manner. A3G was up-regulated by IFN-α in liver cells and macrophages, but not in T lymphoid cells or epithelial 293T cells. In contrast, other IFN-α-stimulated genes such as dsRNA-activated protein kinase were induced in all these cells, suggesting additional cellular factors may regulate IFN-α-induced A3G expression. Consistent with this idea, IFN-α-mediated induction of A3G, but not other IFN-α-stimulated genes, was potently inhibited by the drug Rottlerin, through a mechanism independent of STAT1 activation. The canonical IFN-α-mediated pathway of gene transcription requires both STAT1 and STAT2. Surprisingly, induction of A3G was STAT1 independent, but STAT2 dependent in liver cells. However, STAT1 signaling was functional and required for IFN-γ induction of A3G in these cells. Our results indicate that A3G may participate in antiviral cellular defenses through a novel IFN-mediated signaling pathway.

Although CD8+ CTLs are presumed to be an important mediator of protective immunity in HIV-1 infection, the factors that determine CTL antiviral efficiency are poorly understood. Two factors that have been proposed to influence CTL antiviral function are antigenic avidity and epitope specificity. In this study we evaluate these by examining the activity of HIV-1-specific CTL against acutely infected cells. The ability of CTL to kill infected cells is variable and depends more on epitope specificity than functional avidity within the range for the tested clones (50% of maximal killing, 50 pg/ml to 100 ng/ml); killing efficiency is similar for different clones recognizing the same epitope, despite their variation in avidity. When CTL clones are tested for their ability to suppress viral replication, similar results are observed. Inhibition is more dependent on epitope specificity than functional avidity among the tested clones (50% of maximal killing, 20 pg/ml to 20 ng/ml). Thus, CTL specificity can be an overriding factor in the ability of CTL to interact with HIV-1-infected cells, indicating that factors determining the process of epitope presentation on infected cells have a key influence on CTL efficiency. These results suggest that CTL specificity may have a pivotal role in the immunopathogenesis of infection, and that simple quantitative measures of CTL may be insufficient indicators of the CTL response to HIV-1.

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