Cytidine Deaminase Activity Research Articles

Activation-induced cytidine deaminase: structure–function relationship as based on the study of mutants

Activation-induced cytidine deaminase (AID; gene symbol AICDA) is the key molecule required to induce immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM) of the variable regions of Ig genes. Its deficiency causes a form of hyper-IgM (HIGM) syndrome. The study of natural AID mutants associated with HIGM as well as engineered mutants led to the characterization of the active domains of the protein. AID, through its cytidine deaminase activity, induces a targeted DNA lesion as an early step required for both CSR and SHM. Besides its cytidine deaminase activity, AID plays a further essential role in CSR, likely by recruiting CSR-specific cofactors by its C-terminus. A similar binding of SHM-specific cofactors to the N-terminal part is suggested by the functional characteristics of N(ter) AID artificial mutants. These data require confirmation in vivo. Finally, AID acts as a homo-, di-, or multimeric complex. Together, these data strongly suggest that AID, a master molecule for antibody diversification, exerts its activity on CSR not only as a cytidine deaminase enzyme but also as a docking protein, recruiting specific cofactors to a multimeric complex.

PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells

During humoral immune responses, two distinct genetic modification events diversify the Ig genes in germinal center (GC) B cells: somatic hypermutation and class switch recombination (CSR). Both processes require the activity of activation-induced cytidine deaminase (AID), an enzyme expressed specifically in GC B cells. However, the mechanisms that regulate AID activity are largely unknown. Here we report that protein kinase A (PKA) phosphorylates AID and regulates its activity in GC B cells. AID physically interacts with the PKA holoenzyme in the cytoplasm and is phosphorylated by the PKA catalytic subunit at specific residues. AID phosphorylation is required for CSR, because substitution of the two phosphorylation targets impairs its ability to rescue CSR in AID-deficient B cells. Pharmacologic inhibition of PKA prevents isotype class switching in a murine B-cell lymphoma cell line; conversely, B cells from mice where PKA activity is made constitutive by conditional deletion of the PKA regulatory subunit gene display enhanced CSR. These findings implicate PKA in the regulation of AID function and suggest that the control of T cell-dependent immune responses may be modulated, via AID, by signals that activate PKA.

Presence and Properties of Cytidine Deaminase Enzyme in Trichomonas vaginalis

The investigation indicated the presence and the properties of cytidine deaminase(EC.3.5.4.5) activity in extract of the parasite Trichomonas vaginalis grown in Diamond’s TYM medium. The optimum conditions for the enzyme activity was determined and enzyme activity was obtained in a reaction mixture of (400)M of Tris-HCl buffer at pH (7.2) containing (7) mM of cytidine as a substrate and an amount of the enzyme ranging from (200-250)g of the enzyme extract, and an incubation period of (10) min. at (37)C. Under the optimum conditions, the specific activity was found to be (17.5) mM of cytidine deaminated per min/ mg protein in the supernatant of Trichomonas vaginalis extracts. the investigated Results of the properties of the cytidine deaminase activity showed that cytidine monophosphatet(CMP)might function as alternative substrate for enzyme activity.

Development of a Novel Assay Using Bioluminescent Reporter Bacteria To Determine the Sensitivity of AML Cells to Cytosine Arabinoside.

The nucleoside analogue cytosine arabinoside(Ara-C) has been a cornerstone of remission induction therapy for patients with AML for three decades. Even today, approximately 70% of adults fail to achieve durable complete remission with regimens including Ara-C. This project involves the development of a simple, rapid, economical test to determine Ara-C sensitivity of patient cells to enable clinicians to consider the efficacy of this drug prior to patient treatment.Bacteria expressing lux genes have been successfully used to study real time pharmacodynamics of antimicrobial agents. We have extended this elegant reporter gene technology to determine cellular penetration of Ara-C, into AML cells. AraC has no effect on E. coli since it does not express deoxycytidine kinase (dCK) and has the ability to deaminate AraC into AraU through the activity of cytidine deaminase (cdd). To render E. coli sensitive to AraC, a cdd-deficient strain was used as the host to express the human dCK gene carried on pTrc99-A plasmid under the control of the lac promoter. The expression of dCK, induced by the addition of IPTG, made E. coli sensitive to AraC. E. coli was then made self-bioluminescent by introducing the pAL2 plasmid carrying the lux operon (luxABCDE)AML cell lines were incubated with therapeutic levels of Ara-C (100 μM),washed, and re-suspended in fresh media containing bioluminescent modified E. coli and 0.1% saponin, to lyse the AML cells. Light levels and optical density were monitored using a microtitre plate reader (Fluorstar Optima, BMG Labtec).Cell lines minus drug were used so as to generate control lysates and supernantants.It was possible to detect significant increases in light levels from the lysate and supernatant of an AML cell-line known to be Ara-C sensitive (KG-1a). The lysate from a cell-line (THP-1) known to deaminate Ara-C through the over production of cytidine deaminase showed no increase in light production in the presence of Ara-C. In addition no difference in light levels was observed from the supernatant of these resistant cells. The control supernatant from both cell lines showed no change in light output. These findings indicate that this assay could be used to evaluate the uptake and metabolism of Ara-C by leukaemia cells. With refinement, the assay could be used to specify at which point in Ara-C metabolism AML cells acquire resistance. Furthermore, this technology could be modified to examine the sensitivity of AML cells to other established chemotherapeutic agents such as anthracyclines thus giving a genuinely helpful chemosensitivity profile to clinicians involved in patient care and customising treatment for individual patients.

The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation

Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.

HIV-1 Vif: HIVs Weapon Against the Cellular Defense Factor APOBEC3G

The human immunodeficiency virus type 1, HIV-1, has long been known to possess the viral infectivity factor, Vif, which supports productive viral replication in non-permissive cells, such as peripheral blood lymphocytes (PBL). In the last few years, Vif function has been elucidated by the finding that it inactivates a cellular anti-viral factor named APOBEC3G. Tremendous progress has been made since the initial observation, reflected in a large number of publications. APOBEC3G represents a novel innate defense mechanism against retroviral infection. It is expressed in non-permissive cells and possesses cytidine deaminase activity. APOBEC3G is encapsidated into viral particles and is transported into the infected cell, where it facilitates the deamination of the cytosine residues in the first strand cDNA intermediate during early steps of HIV infection. Vif counteracts APOBEC3G by direct binding, which mediates its degradation by the ubiquitin-dependent proteasomal pathway. In this review, we will summarize the current knowledge about the structure and function of both proteins, their interaction with each other and the mechanism of Vif-mediated APOBEC3G inactivation. In addition, we will discuss possible interference strategies as potential new drugs against HIV infection.

Pharmacology of 5-Aza-2′-deoxycytidine (decitabine)

The preclinical pharmacology of 5-aza-2'-deoxycytidine (decitabine, 5AZA-CdR) is reviewed. 5AZA-CdR, an analogue of deoxycytidine, is a prodrug that requires metabolic activation by deoxycytidine kinase. The active inhibitor in the cell is its triphosphate form (5AZA-dCTP), which incorporates very readily into DNA to produce an inhibition of DNA methyltransferase. The mechanism responsible for the antileukemic action of 5AZA-CdR is related to its reversal of epigenetic silencing by aberrant DNA methylation of genes that suppress leukemiogenesis. 5AZA-CdR is an S-phase-specific agent. At concentrations in the range of micromolars this analogue can induce terminal differentiation and loss of clonogenicity of human leukemic cells. Drug resistance to 5AZA-CdR occurs primarily by reduction in deoxycytidine kinase activity or increase in the activity of cytidine deaminase, the enzyme that inactivates this analogue. 5AZA-CdR is a very potent antileukemic agent in animal models, more effective than the related antileukemic drug, cytosine arabinoside. In humans, 5AZA-CdR has a short half-life of 15 to 25 minutes due to rapid inactivation by liver cytidine deaminase. The major toxicity produced by 5AZA-CdR is myelosuppression. Preliminary clinical studies in patients with hematologic malignancies indicate that 5AZA-CdR is an active chemotherapeutic agent. The optimal dose-schedule for this interesting epigenetic agent with a novel mechanism of action remains to be determined. Translation of the pharmacology of 5AZA-CdR into therapeutic regimens based on scientific rationale can be used to obtain this objective.

Prolonged fixed dose rate infusion of gemcitabine with autologous haemopoietic support in advanced pancreatic adenocarcinoma

This study aimed to define the maximum-tolerated dose (MTD) of fixed dose rate (FDR) of gemcitabine (2′-2′-difluorodeoxycitidine) infusion with circulating haemopoietic progenitor support and to evaluate the activity of the treatment. Secondary end points were pharmacokinetic of gemcitabine and difluorodeoxyuridina (dFdU) measured at first course and the activity andexpression profile of cytidine deaminase (CdA) on circulating mononuclear cells. Patients with advanced pancreatic carcinoma received escalating dose of gemcitabine 10 mg m−2 min−1 every 2 weeks with circulating haemopoietic progenitor support. First dose level was 3000 mg m−2 and the doses were increased by 500 mg m−2 until MTD. In all, 23 patients were enrolled. Toxicities were mild or moderate; the only patient treated at 7000 mg m−2 died because of toxicity; therefore; the MTD was established at 6500 mg m−2. The overall response rate was 22.2%. The AUC of gemcitabine showed a dose-dependent increase, while the AUC of dFdU reached a plateau at 4500 mg m−2. A significant relationship was found between the AUC of dFdU and CdA expression and activity (P<0.05). Moreover, progression rate and survival were significantly related to CdA expression and activity levels. The activity of high-dose gemcitabine is not superior to that reported with less intensive FDR schedules. The predictive role of CdA expression and activity on outcome deserves further investigation.

Phase I clinical and pharmacokinetic (PK) study of gemcitabine (GEM) given by hepatic arterial infusion (HAI)

3697 Background: GEM HAI may be more effective and less toxic than iv GEM in patients (pts) with liver metastases and because of the high activity of cytidine deaminase in the liver hepatic extract...

Impact of cytidine deaminase genetic polymorphisms on gemcitabine kinetics and toxicity in Japanese cancer patients

2009 Background: Gemcitabine is an anti-cancer drug effective against solid tumors. It is rapidly metabolized to its inactive metabolite 2’,2’-difluorodeoxyuridine (dFdU) by cytidine deaminase (CDA), for which several genetic polymorphisms are known. We investigated the impact of genetic polymorphisms of CDA on gemcitabine pharmacokinetics and toxicities. Methods: Two hundred fifty-two Japanese cancer patients who had not previously received gemcitabine were given a 30-min i.v. infusion of the drug at either 800 or 1000 mg/m2, and blood samples were collected before the infusion, at 3 min before the end of the infusion, and 15, 30, 60, 90, 120, and 240 min after the end of the infusion. Plasma levels of gemcitabine and its metabolite were measured by HPLC. Plasma CDA activity was also measured using gemcitabine as a substrate. Polymorphisms of the CDA gene were detected by a dideoxy sequencing method using genomic DNA obtained from peripheral blood leukocytes. Results: Two non-synonymous genetic polymorphisms of CDA - 79A>C (*2, K27Q) and 208G>A (*3, A70T) were found, and their allele frequencies were 0.207 and 0.037, respectively. Pharmacokinetic parameters and plasma CDA activities were significantly dependent on the *3 allele (Results of preliminary analysis are summarized in the Table. The data will be fixed by the meeting). In a patient homozygous for *3, very severe toxicities including grade 4 neutropenia, grade 4 thrombocytopenia, and grade 3 rash were observed. The *2 allele had no marked effects on the pharmacokinetics or toxicities of gemcitabine. Conclusions: Carriers of the CDA *3 allele have decreased clearance of gemcitabine. Attention should therefore be given to patients with the CDA *3 allele before commencing gemcitabine treatment. No significant financial relationships to disclose.

APOBEC3G &amp; HTLV-1: Inhibition without deamination

APOBEC3G is a cellular cytidine deaminase that was recently identified as the Vif-sensitive antiviral host factor responsible for the restriction of vif-defective HIV-1 in primary human cells and certain non-permissive T cell lines. Inhibition of HIV-1 replication is thought to be the result of APOBEC3G-induced hypermutation of the viral genome that occurs early during reverse transcription. Against this backdrop is a new report from the Uchiyama laboratory that proposes deaminase-independent restriction of HTLV-1 by APOBEC3G (Sasada et al. Retrovirology 2005, 2:32). These findings combined with recent reports of deaminase-independent inhibition of Hepatitis B virus as well as HIV-1 suggest that cytidine deaminase activity and antiviral activity may be separable functional properties of APOBEC3G.

APOBEC3G targets human T-cell leukemia virus type 1

BackgroundApolipoprotein 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.ResultsIn 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.ConclusionAPOBEC3G 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.

Sensitivity to Gemcitabine and Its Metabolizing Enzymes in Neuroblastoma

We examined the activity of gemcitabine against neuroblastoma in vitro and in vivo. In addition, we investigated the cellular mechanisms of high sensitivity to the agent in neuroblastoma cells. We examined 11 neuroblastoma cell lines for sensitivity to gemcitabine and other chemotherapeutic agents used clinically for neuroblastoma. The in vivo sensitivity of neuroblastoma to gemcitabine was determined in xenograft models. Furthermore, the major metabolic enzymes of gemcitabine were assessed and compared in leukemia and carcinoma cells. Apoptosis and mitochondrial membrane potentials were also evaluated. The IC50s for gemcitabine in 11 neuroblastoma lines ranged between 3 nmol/L and 4 micromol/L. The high activity of gemcitabine against neuroblastoma was confirmed in animal models. Interestingly, enzymes in neuroblastoma cells involved in the metabolism of deoxycytidine analogue have unique characteristics among solid tumors. The median of deoxycytidine kinase activity in neuroblastoma lines was similar to that in leukemia lines, which have low IC50s for cytarabine. Cytidine deaminase (CDA) activity in neuroblastoma was hardly detectable and significantly lower than that in carcinoma. The defect of CDA activity was associated with negative expression of mRNA. Furthermore, gemcitabine-induced apoptosis was observed irrespective of the caspase-8 status of neuroblastoma cells, which indicates that apoptosis depends on the mitochondrial pathway. Neuroblastoma is highly sensitive to gemcitabine. Although the cellular mechanism involved in sensitivity to gemcitabine is multifactorial, low CDA activity may contribute high sensitivity in neuroblastoma cells. These results suggest that clinical application of gemcitabine to the treatment of neuroblastoma is warranted.

Possible antitumor activity of 1‐(3‐C‐ethynyl‐β‐d‐ribo‐pentofuranosyl)cytosine (ECyd, TAS‐106) against an established gemcitabine (dFdCyd)‐resistant human pancreatic cancer cell line

We established a variant of MIAPaCa-2 human pancreatic cancer cells that is resistant to 2',2'-difluorodeoxycytidine (gemcitabine, dFdCyd), MIAPaCa-2/dFdCyd, and elucidated the biochemical characteristics and mechanism of dFdCyd-resistance in these cells. We also evaluated 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106, RNA polymerase inhibitor), a new anticancer ribonucleoside, for antitumor activity against the resistant cells in vitro and in vivo. MIAPaCa-2/dFdCyd cells were 2541-fold more resistant to dFdCyd than parental MIAPaCa-2 cells, and the major mechanism of the dFdCyd-resistance was found to be a decrease in the intracellular pool of dFdCyd and its active metabolites, which would result in a decrease in incorporation of dFdCyd triphosphate into DNA. This finding was confirmed by the discovery of decreased deoxycytidine kinase activity, increased cytidine deaminase and ribonucleotide reductase activity, and increased 5'-nucleotidase mRNA expression in the MIAPaCa-2/dFdCyd cells. The cytotoxicity of TAS-106 as an antitumor nucleoside analog was similar in both parental and dFdCyd-resistant cells, with IC(50) values of 6.25 and 6.27 nM, respectively, and this finding was supported by similar intracellular uptake and metabolism of TAS-106 in both cell lines. We also evaluated the in vivo antitumor activity of TAS-106 against MIAPaCa-2 and dFdCyd-resistant MIAPaCa-2/dFdCyd tumors implanted into nude mice. The tumor growth inhibition rate of weekly additions of TAS-106 (7 mg/kg, iv) against parental and dFdCyd-resistant tumors was 73% and 76%, respectively, while that of dFdCyd administered twice a week (240 mg/kg, iv) was 84% and 34%, respectively. These results suggest that TAS-106 would contribute to the treatment of patients with advanced pancreatic carcinomas in whom dFdCyd-based chemotherapy has failed.

Human APOBEC3G incorporation into murine leukemia virus particles

The human APOBEC3G protein exhibits broad antiretroviral activity against a variety of retroviruses. It is packaged into viral particles and executes its antiviral function in the target cell. The packaging of APOBEC3G into different viral particles requires a mechanism that confers this promiscuity. Here, APOBEC3G incorporation into murine leukemia virus (MLV) was studied using retroviral vectors. APOBEC3G uptake did not require either its cytidine deaminase activity or the presence of a retroviral vector genome. Results from immunoprecipitation and co-localization studies of APOBEC3G with a MLV Gag–CFP (cyan fluorescent protein) fusion protein imply an interaction between both proteins. RNase A treatment did not inhibit the co-precipitation of Gag–CFP and APOBEC3G, suggesting that the interaction is RNA independent. Like human immunodeficiency virus (HIV) Gag, the MLV Gag precursor protein appears to interact with APOBEC3G, indicating that Gag contains conserved structures which are used to encapsidate APOBEC3G into different retroviral particles.

Comparative Pharmacokinetics of Racivir®, (±)-β-2′,3′-Dideoxy-5-Fluoro-3′-Thiacytidine in Rats, Rabbits, Dogs, Monkeys and HIV-Infected Humans

Racivir is a 50:50 racemic mixture of the (-)- and (+)-beta-enantiomers of 2'-deoxy-3'-thia-5-fluorocytosine (FTC), which is being developed for the treatment of HIV and hepatitis B virus (HBV). The (+)-enantiomer of FTC is approximately 10-20-fold less potent than (-)-FTC, but it selects for a different HIV mutation in human lymphocytes. Plasma concentrations from a group of 54 rats, 12 pregnant rabbits and 60 dogs enrolled in large toxicity studies using a wide variety of oral doses, were compared using non-compartment pharmacokinetic modelling versus dose, treatment duration, species and gender. The pharmacokinetics of Racivir were also compared with those of a previously published pharmacokinetic study in rhesus monkeys and with data from HIV-infected human male volunteers. The (+)-FTC, but not the (-)-enantiomer, can be deaminated to the non-toxic inactive metabolite (+)-FTU. Therefore, the plasma exposure to (+)-FTU was also determined. The order of relative plasma exposure to (+)-FTU was rhesus monkeys > humans > pregnant rabbits > dogs > rats. Allometric scaling was performed to relate systemic clearance/fraction of drug absorbed (Cl/F) and terminal phase volume of distribution (Vbeta/F) versus species body weights. No individual animal species mimicked the Cl/F values in humans. However, allometric scaling using a combination of rats, pregnant rabbits and monkeys predicted the mean human Cl/F value better than a combination of rats and rabbits only (within 0.24 and SD of mean vs 0.81 SD of the observed mean value). Similarly, human Vbeta/F values were best predicted using a combination of rat and monkey data (within 0.64 SD of mean value). Species demonstrating greater deamination to (+)-FTU tended to have greater than predicted Cl/F values. The Cmax values of dogs were the closest to humans, but were statistically different. This study highlights the importance of selecting animal species that demonstrate similar cytidine deaminase activity to humans when performing preclinical dosing studies on Racivir and other antiviral agents that are substrates for mammalian cytidine deaminases.

Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F/G anti-retroviral cytidine deaminases provides insight into their DNA target site specificities

Human APOBEC3F and APOBEC3G are double-domained deaminases that can catalyze dC→dU deamination in HIV-1 and MLV retroviral DNA replication intermediates, targeting T–C or C–C dinucleotides, respectively. HIV-1 antagonizes their action through its vif gene product, which has been shown (at least in the case of APOBEC3G) to interact with the N-terminal domain of the deaminase, triggering its degradation. Here, we compare APOBEC3F and APOBEC3G to APOBEC3C, a single-domained deaminase that can also act on both HIV-1 and MLV. We find that whereas APOBEC3C contains all the information necessary for both Vif-binding and cytidine deaminase activity in a single domain, it is the C-terminal domain of APOBEC3F and APOBEC3G that confer their target site specificity for cytidine deamination. We have exploited the fact that APOBEC3C, whilst highly homologous to the C-terminal domain of APOBEC3F, exhibits a distinct target site specificity (preferring Y–C dinucleotides) in order to identify residues in APOBEC3F that might affect its target site specificity. We find that this specificity can be altered by single amino acid substitutions at several distinct positions, suggesting that the strong dependence of APOBEC3-mediated deoxycytidine deamination on the 5′-flanking nucleotide is sensitive to relatively subtle changes in the APOBEC3 structure. The approach has allowed the isolation of APOBEC3 DNA mutators that exhibit novel target site preferences.

HIV-1 Vif Can Directly Inhibit Apolipoprotein B mRNA-editing Enzyme Catalytic Polypeptide-like 3G-mediated Cytidine Deamination by Using a Single Amino Acid Interaction and Without Protein Degradation

The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G), also known as CEM-15, is a host-cell factor involved in innate resistance to retroviral infection. HIV-1 viral infectivity factor (Vif) protein was shown to protect the virus from APOBEC3G-mediated viral cDNA hypermutation. The mechanism proposed for protection of the virus by HIV-1 Vif is mediated by APOBEC3G degradation through ubiquitination and the proteasomal pathway. Here we show that in Escherichia coli the APOBEC3G-induced cytidine deamination is inhibited by expression of Vif without depletion of deaminase. Moreover, inhibition of deaminase-mediated bacterial hypermutation is dependent on a single amino acid substitution D128K that renders APOBEC3G resistant to Vif inhibition. This single amino acid was elegantly proven by other authors to determine species-specific sensitivity. Our results show that in bacteria this single amino acid substitution controls Vif-dependent blocking of APOBEC3G that is dependent on a strong protein interaction. The C-terminal region of Vif is responsible for this strong protein-protein interaction. In conclusion, our experiments suggest a complement to the model of Vif-induced degradation of APOBEC3G by bringing to relevance that deaminase inhibition can also result from a direct interaction with Vif protein.

Complementary function of the two catalytic domains of APOBEC3G

The HIV-1 viral accessory protein Vif prevents the encapsidation of the antiviral cellular cytidine deaminases APOBEC3F and APOBEC3G by inducing their proteasomal degradation. In the absence of Vif, APOBEC3G is encapsidated and blocks virus replication by deaminating cytosines of the viral cDNA. APOBEC3G encapsidation has been recently shown to depend on the viral nucleocapsid protein; however, the role of RNA remains unclear. Using APOBEC3G deletion and point mutants, we mapped the encapsidation determinant to the Zn 2+ coordination residues of the N-terminal catalytic domain (CD1). Notably, these residues were also required for RNA binding. Mutations in the two aromatic residues of CD1 but not CD2, which are conserved in cytidine deaminase core domains and are required for RNA binding, prevented encapsidation into HIV-1, HTLV-I and MLV. The Zn 2+ coordination residues of the C-terminal catalytic domain (CD2) were not required for encapsidation but were essential for cytidine deaminase activity and the antiviral effect. These findings suggest a model in which CD1 mediates encapsidation and RNA binding while CD2 mediates cytidine deaminase activity. Interestingly, HTLV-I was relatively resistant to the antiviral effects of encapsidated APOBEC3G.

Hyper‐immunoglobulin M syndromes caused by intrinsic B‐lymphocyte defects

Hyper-immunoglobulin M (IgM) syndromes are primary immunodeficiencies characterized by normal or elevated serum IgM levels with the absence of other isotypes, pinpointing to a defect in the Ig class switch recombination (CSR). The delineation of hyper-IgM syndromes made it possible to better define the mechanisms underlying the two major events of antibody maturation in humans, CSR and introduction of somatic hypermutation (SHM) in the variable region of immunoglobulins. The description of the activation-induced cytidine deaminase (AID) deficiency, characterized by a defect in both CSR and SHM, demonstrated for the first time that this molecule acts as a master player in the antigen-induced Ig gene-modification events responsible for both CSR and SHM. However, deleterious mutations located in the C-terminus lead to a CSR defect without affecting SHM, providing evidence for a role of AID in CSR distinct from the cytidine deaminase activity, likely by binding to a specific CSR cofactor. Molecular causes of two other hyper-IgM conditions have not yet been defined. However, they may be caused by either a defect in AID targeting on S regions or a CSR-specific DNA-repair defect. The mechanism of action of AID remains somewhat debated, but the observation that uracil-DNA-glycosylase deficiency leads to a severe hyper-IgM syndrome strongly argues in favor of a DNA-editing activity of AID.