Sole copy of Z2-type human cytidine deaminase APOBEC3H has inhibitory activity against retrotransposons and HIV-1.

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.

Interaction with 7SL RNA but not with HIV-1 genomic RNA or P bodies is required for APOBEC3F virion packaging.

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F), like APOBEC3G, has broad antiviral activity against diverse retroelements, including Vif-deficient human immunodeficiency virus (HIV)-1. Its antiviral functions are known to rely on its virion encapsidation and be suppressed by HIV-1 Vif, which recruits Cullin5-based E3 ubiquitin ligases. However, the factors that mediate A3F virion packaging have not yet been identified. In this study, we demonstrate that A3F specifically interacts with cellular signal recognition particle (SRP) RNA (7SL RNA), which is selectively packaged into HIV-1 virions. Efficient packaging of 7SL RNA as well as A3F was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. Reducing 7SL RNA packaging by overexpression of SRP19 protein inhibited A3F virion packaging. Although A3F has been shown to interact with P bodies and viral genomic RNA, our data indicated that P bodies and HIV-1 genomic RNA were not required for A3F packaging. Thus, in addition to its well-known function in SRPs, 7SL RNA, which is encapsidated into diverse retroviruses, also participates in the innate antiviral function of host cytidine deaminases.

Cytidine deaminase APOBEC3B interacts with heterogeneous nuclear ribonucleoprotein K and suppresses hepatitis B virus expression.

The cytidine deaminase apolipoprotein B mRNA editing catalytic subunit-3 (APOBEC3) proteins have been identified as potent inhibitors of diverse retroviruses, retrotransposons and hepatitis B virus (HBV). The mechanism of APOBEC3 proteins in the control of HBV infection, however, is less clear. Here we report that APOBEC3B (A3B) displays dual inhibitory effects on both HBsAg and HBeAg expression as well as HBV core-associated DNA synthesis. Heterogeneous nuclear ribonucleoprotein K (hnRNP K), a positive regulator of HBV expression, has been identified as a major interaction partner of A3B protein. A3B protein inhibited the binding of hnRNP K to the enhancer II of HBV (Enh II), and S gene transcription of HBV. Moreover, A3B directly suppressed HBV S gene promoter activity. Individual variation in A3B expression was observed in both normal primary hepatocytes and liver tissues. Interestingly, A3B was able to inhibit CMV and SV40 promoter-mediated gene expression. In conclusion, A3B suppresses HBV replication in hepatocytes by inhibiting hnRNP K-mediated transcription and expression of HBV genes as well as HBV core DNA synthesis. In addition, A3B protein may be a broad antiviral host factor. Thus, regulated A3B expression may contribute to non-cytolytic HBV clearance in vivo.

7SL RNA mediates virion packaging of the antiviral cytidine deaminase APOBEC3G.

Cytidine deaminase APOBEC3G (A3G) has broad antiviral activity against diverse retroviruses and/or retrotransposons, and its antiviral functions are believed to rely on its encapsidation into virions in an RNA-dependent fashion. However, the cofactors of A3G virion packaging have not yet been identified. We demonstrate here that A3G selectively interacts with certain polymerase III (Pol III)-derived RNAs, including Y3 and 7SL RNAs. Among A3G-binding Pol III-derived RNAs, 7SL RNA was preferentially packaged into human immunodeficiency virus type 1 (HIV-1) particles. Efficient packaging of 7SL RNA, as well as A3G, was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. A3G mutants that had reduced 7SL RNA binding but maintained wild-type levels of mRNA and tRNA binding were packaged poorly and had impaired antiviral activity. Reducing 7SL RNA packaging by overexpression of SRP19 proteins inhibited 7SL RNA and A3G virion packaging and impaired its antiviral function. Thus, 7SL RNA that is encapsidated into diverse retroviruses is a key cofactor of the antiviral A3G. This selective interaction of A3G with certain Pol III-derived RNAs raises the question of whether A3G and its cofactors may have as-yet-unidentified cellular functions.

Differential inhibition of long interspersed element 1 by APOBEC3 does not correlate with high-molecular-mass-complex formation or P-body association.

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.

Cell-specific regulation of APOBEC3F by interferons.

Human cytidine deaminase APOBEC3F (A3F) has broad anti-viral activity against hepatitis B virus and retroviruses including human immunodeficiency virus type 1. However, its regulation in viral natural target cells such CD4+ T lymphocytes, macrophages, and primary liver cells has not been well studied. Here we showed that A3F was up-regulated by interferon (IFN)-alpha in primary hepatocytes and multiple liver cell lines as well as macrophages. Although the IFN-alpha signaling pathway was active in T lymphoid cells and induction of other IFN stimulated genes such as PKR was detected, A3F and APOBEC3G (A3G) were not induced by IFN-alpha in these cells. Thus, additional factors other than known IFN-stimulated genes also regulated IFN-alpha-induced A3F expression distinctly. A3F and A3G expression levels in primary hepatocytes, especially after IFN-alpha stimulation, were comparable to those in CD4+ T lymphocytes in some individuals. Significant variations of A3F and A3G expression in primary hepatocytes from various subjects were observed. Individual variations in A3F and/or A3G regulation and expression might influence the clinical outcomes of hepatitis B infection.

STAT1-independent cell type-specific regulation of antiviral APOBEC3G by IFN-alpha.

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-alpha in liver cells and macrophages, but not in T lymphoid cells or epithelial 293T cells. In contrast, other IFN-alpha-stimulated genes such as dsRNA-activated protein kinase were induced in all these cells, suggesting additional cellular factors may regulate IFN-alpha-induced A3G expression. Consistent with this idea, IFN-alpha-mediated induction of A3G, but not other IFN-alpha-stimulated genes, was potently inhibited by the drug Rottlerin, through a mechanism independent of STAT1 activation. The canonical IFN-alpha-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-gamma induction of A3G in these cells. Our results indicate that A3G may participate in antiviral cellular defenses through a novel IFN-mediated signaling pathway.

Regulation of Apobec3F and human immunodeficiency virus type 1 Vif by Vif-Cul5-ElonB/C E3 ubiquitin ligase.

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, a residue critical for h-A3G recognition by HIV-1 Vif. However, HIV-1 Vif, but not SIVagm Vif, was able to bind h-A3F and induce its polyubiquitination and degradation through the Cul5-containing E3 ubiquitin ligase. Interference with Cul5-E3 ligase function by depletion of Cul5, through RNA interference or overexpression of Cul5 mutants, blocked the ability of HIV-1 Vif to suppress h-A3F. A BC-box mutant of HIV-1 Vif that failed to recruit Cul5-E3 ligase but was still able to interact with h-A3F failed to suppress h-A3F. Interestingly, interference with Cul5-E3 ligase function or overexpression of h-A3F or h-A3G also increased the stability of HIV-1 Vif, suggesting that like the substrate molecules h-A3F and h-A3G, the substrate receptor protein Vif is itself also regulated by Cul5-E3 ligase. Our results indicate that Cul5-E3 ligase appears to be a common pathway hijacked by HIV-1 Vif to defeat both h-A3F and h-A3G. Developing inhibitors to disrupt the interaction between Vif and Cul5-E3 ligase could be therapeutically useful, allowing multiple host antiviral factors to suppress HIV-1.

Determinant of HIV-1 mutational escape from cytotoxic T lymphocytes.

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.

Chimeric immune receptor T cells bypass class I requirements and recognize multiple cell types relevant in HIV-1 infection.

Transduction of T cells with a chimeric immune T cell receptor (CIR) has been proposed as a strategy to generate cellular immunity against viral pathogens such as HIV-1. In the case of the CD4-CD3-zeta chain (CD4-zeta) CIR, specificity for HIV-1 is conferred by binding of the CD4 moiety to gp120 on the surface of infected cells. However, it is unclear whether CD4-zeta-T cells may differ from naturally derived CD8(+) cytotoxic T cells (CTL) in their susceptibility to viral escape mechanisms or ability to recognize different cell types that support viral replication. We demonstrate that CIR-T cells can mediate antiviral activity against HIV-1 in cells that are resistant to class I-restricted CTL-mediated activity. Furthermore, CIR-T cells can suppress virus in multiple cell types, including monocytes, dendritic cells, and lymphocyte-dendritic cell clusters. These results provide evidence that T cells can be redirected against novel targets, and that independence from the class I pathway may have distinct advantages.