Variantes genéticas y actividad enzimática de la citidin desaminasa: relación con la toxicidad a la capecitabina y recomendación de ajuste de las dosis / Genetic variants and enzyme activity in citidin deaminase: Relationship with capecitabine toxicity and recommendation for dose adjustment

Objetivo: la capecitabina es un fármaco antineoplásico utilizado en el tratamiento del cáncer de mama y de colon que puede dar lugar a una toxicidad grave, llegando a ser mortal en algunos pacientes. La variabilidad interindividual de esta toxicidad es debida en gran medida a las variaciones genéticas en los genes diana y las enzimas de metabolismo de este fármaco, como la timidilato sintasa y la dihidropirimidina deshidrogenasa. La enzima citidin desaminasa (CDA), imprescindible en la activación de la capecitabina, también presenta diversas variantes asociadas con un mayor riesgo de toxicidad al tratamiento, aunque su papel como biomarcador aún no está claramente definido. Por ello, nuestro objetivo principal es estudiar la asociación entre la presencia de las variantes genéticas en el gen CDA, su actividad enzimática y el desarrollo de la toxicidad grave en los pacientes tratados con capecitabina, cuya dosis inicial se haya ajustado con base en el perfil genético del gen de la dihidropirimidina deshidrogenasa (DPYD). Método: estudio de cohortes observacional multicéntrico prospectivo, centrado en el análisis de la asociación genotipo-fenotipo de la enzima CDA. Tras la fase experimental, se desarrollará un algoritmo que permita determinar el ajuste necesario de las dosis para disminuir el riesgo de toxicidad del tratamiento en función del genotipo CDA, elaborando una guía clínica para la dosificación de la capecitabina en función de las variantes genéticas en DPYD y CDA. Con base en esta guía, se creará una herramienta bioinformática que genere el informe farmacoterapéutico de manera automática, facilitando la implementación del consejo farmacogenético en la práctica clínica. Esta herramienta proporcionará un gran respaldo en la toma de decisiones farmacoterapéuticas basadas en el perfil genético del paciente, incorporando la medicina de precisión en la rutina clínica. ... (AU)

Objective: Capecitabine, an antineoplastic drug used in the treatment of breast and colon cancer, can cause severe, even fatal toxicity in some patients. The interindividual variability of this toxicity is largely due to genetic variations in target genes and enzymes of metabolism of this drug, such as thymidylate synthase and dihydropyrimidine dehydrogenase. The enzyme cytidine deaminase (CDA), involved in the activation of capecitabine, also has several variants associated with an increased risk of toxicity to treatment, although its role as a biomarker is not yet clearly defined.Therefore, our main objective is to study the association between the presence of genetic variants in CDA gen, CDA enzymatic activity and the development of severe toxicity in patients treated with capecitabine whose initial dose was adjusted based on the genetic profile of the dihydropyrimidine dehydrogenase gen (DPYD). Method: Prospective multicenter observational cohort study, focused on the analysis of the genotype-phenotype association of the CDA enzyme.After the experimental phase, an algorithm will be developed to determine the dose adjustment needed to reduce the risk of treatment toxicity according to CDA genotype, developing a clinical guide for capecitabine dosing according to genetic variants in DPYD and CDA. Based on this guide, a Bioinformatics Tool will be created to generate the pharmacotherapeutic report automatically, facilitating the implementation of pharmacogenetic advice in clinical practice. This tool will be a great support in making pharmacotherapeutic decisions based on the patient's genetic profile, incorporating precision medicine into clinical routine. Once the usefulness of this tool has been validated, it will be offered free of charge to facilitate the implementation of pharmacogenetics in hospital centers and equitably benefit all patients on capecitabine treatment. (AU)

Deubiquitinase ubiquitin-specific protease 3 (USP3) inhibits HIV-1 replication via promoting APOBEC3G (A3G) expression in both enzyme activity-dependent and -independent manners.

BACKGROUND: Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated. METHODS: Immunoblotting, real-time polymerase chain reaction, in vivo / in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4 + T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data). RESULTS: The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression ( r  = 0.5110) and CD4 + T-cell counts ( r  = 0.5083) in HIV-1-infected patients. CONCLUSIONS: USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

Compound IMB-Z inhibits hepatitis B virus replication through increasing APOBEC3G expression and incorporation into viral nucleocapsids.

OBJECTIVES: As a host restriction factor, apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) has been shown to suppress the replication of several viruses including hepatitis B virus (HBV). Recently, we reported that IMB-Z, a N-phenylbenzamide derivative, could inhibit Enterovirus 71 replication, and A3G mediated its antiviral activity. Whether IMB-Z exhibits an inhibitory effect on HBV replication has not been investigated. MATERIAL AND METHODS: HBV DNA, pregenomic RNA (pgRNA), core protein, and capsid levels were determined by a qPCR assay or Southern blot, Northern blot, Western blot, and particle gel assay, respectively. Mutation analysis of HBV DNAs was conducted by a differential DNA denaturation PCR assay. A3G encapsidation into HBV nucleocapsids was examined by Western blot analysis after ultracentrifugation and a co-immunoprecipitation (IP) assay between HBV core and A3G proteins. RESULTS: In the present study, we found that IMB-Z could considerably inhibit HBV replication in HepAD38 cells. Interestingly, IMB-Z did not alter the HBV pgRNA production but could reduce the level of core protein, viral nucleocapsids, and core-associated DNA, as well as cccDNA intracellular amplification. Similar to the action of IMB-Z's inhibition of Enterovirus 71 replication, we found that IMB-Z's inhibition of HBV replication was associated with increased level of A3G. Mechanistically, we demonstrated that the inhibitory effect of IMB-Z is independent of the cytidine deaminase activity of A3G and is exerted by increasing its incorporation into viral nucleocapsids. CONCLUSIONS: Our results indicate that IMB-Z inhibits HBV through pharmacological induction A3G expression and incorporation into HBV nucleocapsids.

DNA methylome and single-cell transcriptome analyses reveal CDA as a potential druggable target for ALK inhibitor-resistant lung cancer therapy.

Acquired resistance to inhibitors of anaplastic lymphoma kinase (ALK) is a major clinical challenge for ALK fusion-positive non-small-cell lung cancer (NSCLC). In the absence of secondary ALK mutations, epigenetic reprogramming is one of the main mechanisms of drug resistance, as it leads to phenotype switching that occurs during the epithelial-to-mesenchymal transition (EMT). Although drug-induced epigenetic reprogramming is believed to alter the sensitivity of cancer cells to anticancer treatments, there is still much to learn about overcoming drug resistance. In this study, we used an in vitro model of ceritinib-resistant NSCLC and employed genome-wide DNA methylation analysis in combination with single-cell (sc) RNA-seq to identify cytidine deaminase (CDA), a pyrimidine salvage pathway enzyme, as a candidate drug target. CDA was hypomethylated and upregulated in ceritinib-resistant cells. CDA-overexpressing cells were rarely but definitively detected in the naïve cell population by scRNA-seq, and their abundance was increased in the acquired-resistance population. Knockdown of CDA had antiproliferative effects on resistant cells and reversed the EMT phenotype. Treatment with epigenome-related nucleosides such as 5-formyl-2'-deoxycytidine selectively ablated CDA-overexpressing resistant cells via accumulation of DNA damage. Collectively, our data suggest that targeting CDA metabolism using epigenome-related nucleosides represents a potential new therapeutic strategy for overcoming ALK inhibitor resistance in NSCLC.

APOBEC3C Tandem Domain Proteins Create Super Restriction Factors against HIV-1.

Humans encode proteins, called restriction factors, that inhibit replication of viruses such as HIV-1. The members of one family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; shortened here to A3), act by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A to A3H These genes have either one or two cytidine deaminase domains, and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a "super restriction factor" that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein resulted in an even greater increase in the antiviral activity-recapitulating a similar evolution seen in A3F and A3G (double domain A3s that use only a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity but instead inhibit formation of reverse transcription products, which correlates with their ability to form large higher-order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein Vif.IMPORTANCE As a part of the innate immune system, humans encode proteins that inhibit viruses such as HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain and, furthermore, that the proteins thus generated are relatively resistant to the viral antagonist Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens such as HIV.

Analysis of APOBEC-induced mutations in yeast strains with low levels of replicative DNA polymerases.

Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length.

Arsenic programmes cellular genomic-immunity through miR-2909 RNomics.

It is widely recognized that human cells are equipped with innate antiviral-RNA armour involving the production of type I interferons and APOBEC3G (apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G) gene-product. Although arsenic has been shown to have paradoxical effect on one arm of this armour involving APOBEC3G, the exact molecular mechanism of its action in this regard is far from clear. The present study, addressed to explore as to how arsenic programmes this innate antiviral-RNA cellular-sensing pathway, clearly revealed that arsenic programmes this innate cellular antiviral genomic response through its inherent capacity to initiate cellular miR-2909 RNomics pathway, involving not only the modulation of APOBEC3G gene but also KLF4 (Kruppel-like factor 4) dependent regulation of gene coding for IKBKε (Inhibitor of nuclear factor kappa-B kinase subunit epsilon) which in turn modulates RIG-I (retinoic acid-inducible gene 1) pathway responsible for the production of IFNß (interferon beta) through restriction of CYLD (Cylindromatosis) deubiqutinating activity. This restricted inhibitory enzyme activity of CYLD upon NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) also ensures sustained expression of miR-2909. Our results for the first time show that cellular miR-2909 RNomics may constitute an innate genomic armour to promote as well as restrict retroviral infection.

Identification and antiviral activity of common polymorphisms in the APOBEC3 locus in human populations.

There are seven members of the APOBEC3 family in humans (APOBEC3A through APOBEC3H) that have antiviral activity against retroviruses and/or retroelements. To determine whether variants in APOBEC3 genes in human populations have altered antiviral activity, we identified and functionally tested novel single nucleotide variants (SNVs) in APOBEC3 genes present in the 1000 Genome Project dataset. We found that common variants minor allele frequency (> 1%) of APOBEC3A, C, F, and G do not affect protein function. However, we found that two common novel polymorphisms in APOBEC3D decrease antiviral activity against HIV-1, and one polymorphism decreases activity against Alu retrotransposons. We characterized the diversity of APOBEC3 genes in three human populations and find significant evidence that APOBEC3D has evolved under purifying selection in recent human history. These data suggest that the activity of APOBEC3D has been maintained in human populations for a cellular function in host defense.

The APP intracellular domain (AICD) potentiates ER stress-induced apoptosis.

Here we employed human SHEP neuroblastoma cells either stably or inducibly expressing the amyloid precursor protein (APP) intracellular domain (AICD) to investigate its ability to modulate stress-induced cell death. Analysis of effector caspase activation revealed that AICD overexpression was specifically associated with an increased sensitivity to apoptosis induced by the 2 endoplasmic reticulum (ER) stressors thapsigargin and tunicamycin, but not by staurosporine (STS). Basal and ER stress-induced expression of Bip/Grp78 and C/EBP-homologous protein/GADD153 were not altered by AICD implying that AICD potentiated cell death downstream or independent of the conserved unfolded protein response (UPR). Interestingly, quantitative polymerase chain reaction analysis and reporter gene assays revealed that AICD significantly downregulated messenger RNA levels of the Alzheimer's disease susceptibility gene ApoJ/clusterin, indicating transcriptional repression. Knockdown of ApoJ/clusterin mimicked the effect of AICD on ER stress-induced apoptosis, but had no discernible effect on staurosporine-induced cell death. Our data suggest that altered levels of AICD may abolish the prosurvival function of ApoJ/clusterin and increase the susceptibility of neurons to ER stress-mediated cell death, a pathway that may contribute to the pathogenesis of Alzheimer's disease.

APOBEC3A is a specific inhibitor of the early phases of HIV-1 infection in myeloid cells.

Myeloid cells play numerous roles in HIV-1 pathogenesis serving as a vehicle for viral spread and as a viral reservoir. Yet, cells of this lineage generally resist HIV-1 infection when compared to cells of other lineages, a phenomenon particularly acute during the early phases of infection. Here, we explore the role of APOBEC3A on these steps. APOBEC3A is a member of the APOBEC3 family that is highly expressed in myeloid cells, but so far lacks a known antiviral effect against retroviruses. Using ectopic expression of APOBEC3A in established cell lines and specific silencing in primary macrophages and dendritic cells, we demonstrate that the pool of APOBEC3A in target cells inhibits the early phases of HIV-1 infection and the spread of replication-competent R5-tropic HIV-1, specifically in cells of myeloid origins. In these cells, APOBEC3A affects the amount of vDNA synthesized over the course of infection. The susceptibility to the antiviral effect of APOBEC3A is conserved among primate lentiviruses, although the viral protein Vpx coded by members of the SIV(SM)/HIV-2 lineage provides partial protection from APOBEC3A during infection. Our results indicate that APOBEC3A is a previously unrecognized antiviral factor that targets primate lentiviruses specifically in myeloid cells and that acts during the early phases of infection directly in target cells. The findings presented here open up new venues on the role of APOBEC3A during HIV infection and pathogenesis, on the role of the cellular context in the regulation of the antiviral activities of members of the APOBEC3 family and more generally on the natural functions of APOBEC3A.

Recapture of hepatic apolipoprotein B mRNA editing may be a promising strategy to relieve nephrotic dyslipidemia.

A high total plasma cholesterol concentration is the most common abnormality found in patients with kidney disease, which may be associated with the increased hepatic synthesis of apoB containing lipoproteins. ApoB mRNA editing plays an important physiological role in mammalian lipid metabolism by modifying the distribution of apoB-100 and apoB-48. However, it is regretful that apoB mRNA editing cannot be found in human liver because of the absence of apobec-1 expression. In this context, we hypothesize that the recapture of hepatic apoB mRNA editing may be a promising strategy to relieve nephrotic dyslipidemia. The data presented below focus on those which support this hypothesis with regards to evidence in vitro and in vivo. (1) Human wild-type apoB mRNA can be edited only when both apobec-1 and ACF proteins are presented simultaneously in vitro. (2) Adenoviral vectors can produce short-term expression of exogenous apobec-1 in the livers and lower plasma apoB-100 and LDL levels transiently. (3) Apobec-1 transgenic animals exhibit massive hepatic editing of apoB mRNA and fundamental decreased plasma levels of apoB-100 and LDL, but are exposed to high risk of liver dysplasia and hepatocellular carcinomas. In summary, taking into account the therapeutic security, we put forward that apobec-1 recombinant adenoviral vectors can be used for the recapture of hepatic apoB mRNA editing with a transient low-level manner and may achieve satisfactory lipid-lowing effect in nephropathic animals.

Cellular pharmacology of the anti-hepatitis B virus agent beta-L-2',3'-didehydro-2',3'-dideoxy-N4-hydroxycytidine: relevance for activation in HepG2 cells.

Beta-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED(50)] = 0.03 microM; 50% cytotoxic dose [CD(50)] = 2,500 microM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[(3)H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5'-mono-, 5'-di-, and 5'-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ss-d-N(4)-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5'-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 microM l-[(3)H]Hyd4C for 24 h, the 5'-triphosphate accumulated to 19.4 +/- 2.7 pmol/10(6) cells. The predominant peak belonged to 5-diphosphate, with 43.5 +/- 4.3 pmol/10(6) cells. The intracellular half-life of the 5'-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5'-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5'-diphosphate during a drug-free incubation. The high 5'-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.

High level expression of the anti-retroviral protein APOBEC3G is induced by influenza A virus but does not confer antiviral activity.

Human APOBEC3G is an antiretroviral protein that was described to act via deamination of retroviral cDNA. However, it was suggested that APOBEC proteins might act with antiviral activity by yet other mechanisms and may also possess RNA deamination activity. As a consequence there is an ongoing debate whether APOBEC proteins might also act with antiviral activity on other RNA viruses. Influenza A viruses are single-stranded RNA viruses, capable of inducing a variety of antiviral gene products. In searching for novel antiviral genes against these pathogens, we detected a strong induction of APOBEC3G but not APOBEC3F gene transcription in infected cells. This upregulation appeared to be induced by the accumulation of viral RNA species within the infected cell and occurred in an NF-kappaB dependent, but MAP kinase independent manner. It further turned out that APOBEC expression is part of a general IFNbeta response to infection. However, although strongly induced, APOBEC3G does not negatively affect influenza A virus propagation.

Restriction of retroviral replication by APOBEC3G/F and TRIM5alpha.

Pathogenic viral infections have exerted selection pressure on their hosts to evolve cellular antiviral inhibitors referred to as restriction factors. Examples of such molecules are APOBEC3G, APOBEC3F and TRIM5alpha. APOBEC3G and APOBEC3F are cytidine deaminases that are able to strongly inhibit retroviral replication by at least two mechanisms. They are counteracted by the lentiviral Vif protein. TRIM5alpha binds to sensitive, incoming retroviruses via its C-terminal PRY/SPRY domain and rapidly recruits them to the proteasome before significant viral DNA synthesis can occur. Both of these proteins robustly block retroviral replication in a species-specific way. It remains an open but important question as to whether innate restriction factors such as these can be harnessed to inhibit HIV-1 replication in humans.

APOBEC3G-UBA2 fusion as a potential strategy for stable expression of APOBEC3G and inhibition of HIV-1 replication.

BACKGROUND: Although APOBEC3G protein is a potent and innate anti-HIV-1 cellular factor, HIV-1 Vif counteracts the effect of APOBEC3G by promoting its degradation through proteasome-mediated proteolysis. Thus, any means that could prevent APOBEC3G degradation could potentially enhance its anti-viral effect. The UBA2 domain has been identified as an intrinsic stabilization signal that protects protein from proteasomal degradation. In this pilot study, we tested whether APOBEC3G, when it is fused with UBA2, can resist Vif-mediated proteasomal degradation and further inhibit HIV-1 infection. RESULTS: APOBEC3G-UBA2 fusion protein is indeed more resistant to Vif-mediated degradation than APOBEC3G. The ability of UBA2 domain to stabilize APOBEC3G was diminished when polyubiquitin was over-expressed and the APOBEC3G-UBA2 fusion protein was found to bind less polyubiquitin than APOBEC3G, suggesting that UBA2 stabilizes APOBEC3G by preventing ubiquitin chain elongation and proteasome-mediated proteolysis. Consistently, treatment of cells with a proteasome inhibitor MG132 alleviated protein degradation of APOBEC3G and APOBEC3G-UBA2 fusion proteins. Analysis of the effect of APOBEC3G-UBA2 fusion protein on viral infectivity indicated that infection of virus packaged from HEK293 cells expressing APOBEC3G-UBA2 fusion protein is significantly lower than those packaged from HEK293 cells over-producing APOBEC3G or APOBEC3G-UBA2 mutant fusion proteins. CONCLUSION: Fusion of UBA2 to APOBEC3G can make it more difficult to be degraded by proteasome. Thus, UBA2 could potentially be used to antagonize Vif-mediated APOBEC3G degradation by preventing polyubiquitination. The stabilized APOBEC3G-UBA2 fusion protein gives stronger inhibitory effect on viral infectivity than APOBEC3G without UBA2.

Human APOBEC3G can restrict retroviral infection in avian cells and acts independently of both UNG and SMUG1.

APOBEC3 proteins are mammal-specific cytidine deaminases that can restrict retroviral infection. The exact mechanism of the restriction remains unresolved, but one model envisions that uracilated retroviral cDNA, generated by cytidine deamination, is the target of cellular glycosylases. While restriction is unaffected by UNG deficiency, it has been suggested that the SMUG1 glycosylase might provide a backup. We found that retroviral restriction can be achieved by introducing human APOBEC3G into chicken cells (consistent with the components necessary for APOBEC3-mediated restriction predating mammalian evolution) and used this assay to show that APOBEC3G-mediated restriction can occur in cells deficient in both UNG and SMUG1.

Inhibition of alpharetrovirus replication by a range of human APOBEC3 proteins.

The mammalian APOBEC3 family of cytidine deaminases includes members that can act as potent inhibitors of retroviral infectivity and retrotransposon mobility. Here, we have examined whether the alpharetrovirus Rous sarcoma virus (RSV) is susceptible to inhibition by a range of human APOBEC3 proteins. We report that RSV is highly susceptible to inhibition by human APOBEC3G, APOBEC3F, and APOBEC3B and moderately susceptible to inhibition by human APOBEC3C and APOBEC3A. For all five proteins, inhibition of RSV infectivity was associated with selective virion incorporation and with C-to-T editing of the proviral DNA minus strand. In the case of APOBEC3G, editing appeared to be critical for effective inhibition. These data represent the first report of inhibition of retroviral infectivity and induction of proviral DNA editing by human APOBEC3A and reveal that alpharetroviruses, which do not normally encounter APOBEC3 proteins in their avian hosts, are susceptible to inhibition by all human APOBEC3 proteins tested. These data further suggest that the resistance of mammalian retroviruses to inhibition by the APOBEC3 proteins expressed in their normal host species is likely to have evolved subsequent to the appearance of this family of mammalian antiretroviral proteins some 35 million years ago; i.e., the base state of a naïve retrovirus is susceptibility to inhibition.

Enzymatically active APOBEC3G is required for efficient inhibition of human immunodeficiency virus type 1.

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3G's antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be dissociated from its deaminase activity. To directly compare the antiviral activities of wild-type (wt) and deaminase-defective APO3G, we used two approaches: (i) we titrated wt and deaminase-defective APO3G in transient-transfection studies to achieve similar levels of virus-associated APO3G and (ii) we constructed stable cell lines and selected clones expressing comparable amounts of wt and deaminase-defective APO3G. Viruses produced under these conditions were tested for viral infectivity. The results from the two approaches were consistent and suggested that the antiviral activity of deaminase-defective APO3G was significantly lower than that of wt APO3G. We conclude that efficient inhibition of vif-defective human immunodeficiency virus type 1 requires catalytically active APO3G.

Human T cell leukemia virus type I is resistant to the antiviral effects of APOBEC3.

The objective of this study was to investigate whether the anti-retroviral cellular cytidine deaminase, APOBEC3, inhibits the infectivity of human T cell leukemia virus type I (HTLV-I). Sufficient quantities of cell-free HTLV-I virion for infection were obtained by cotransfecting cells with HTLV-I and human or murine APOBEC3 expression vectors along with a plasmid expressing Tax. HTLV-I viruses containing these deaminases were still capable of infecting 293T and MOLT-4 cells. No G-to-A mutations, which are characteristic of cytidine deaminases, were observed in the HTLV-I genome. These results suggest that the enzymatic activity of APOBEC3 may not contribute substantially to antiviral responses to HTLV-I.