Letteratura scientifica selezionata sul tema "APOBEC family"

ABSTRACT 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.

Abstract The hallmark activity of APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) family of cytidine deaminases, including activation-induced deaminase (AID) and APOBEC3 genes, has been detected in somatic mutation signatures by ultra-deep sequencing of the genomes of many cancers, including chronic lymphocytic leukemia (CLL). The acquisition of these mutations is hypothesized to lead to the progression towards aggressive disease in cancer. To examine this in CLL, we tested if increased APOBEC family member gene expression in CLL cells, as measured by microarray and quantitative real time PCR, correlated with worse patient outcome. Higher levels of AID, APOBEC3B, APOBEC3F and APOBEC3H in CLL cells correlated with worse patient outcome, whereas APOBEC3G did not. Interestingly, higher levels of a truncated splice variant of APOBEC3F tended to correlate with better patient outcome. The expression of truncated APOBEC3F may possibly interfere with APOBEC family member mutation activity. To test mutation activity, CLL cells were activated by transfer into NOD-scid IL2Rγnull mice, a xenograft model of human CLL, and hallmark mutation signatures in the expressed immunoglobulin variable region (IGHV) of CLL cells were analyzed by targeted ultra-deep sequencing. Induced IGHV mutation hallmarks consistent with AID were found. These data support the hypothesis that expression and mutation activity of APOBEC family members, such as AID, in CLL cells could lead to adverse patient consequences.

Abstract The APOBEC cytidine deaminase enzyme family is linked to mutational signatures identified in cancer. While previous work has provided insights into the role of APOBEC3A and APOBEC3B in mutational processes in cancer, understanding of the mutational signatures induced by other APOBEC family members is limited. In this issue of Cancer Research, Liu and colleagues investigated the role of APOBEC3G (A3G) in bladder cancer. The authors revealed that transgenic expression of A3G in a murine bladder cancer model promotes tumorigenesis and induces a unique mutational signature distinct from previously identified APOBEC signatures. Expression of this A3G-related mutational signature correlated with significantly worse survival in patients with urothelial bladder carcinoma, and A3G expression was identified in 21 different cancer types. These findings suggest that different APOBEC3 enzymes induce unique mutation signatures and play distinct roles in cancer evolution. More complete understanding of the function of each APOBEC3 enzyme will improve anticancer therapy. See related article by Liu et al., p. 506

Abstract A mutational signature consistent with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) activity has been identified in somatic mutations found in large-scale surveys of ultra-deep sequencing data from many human cancers including chronic lymphocytic leukemia (CLL). APOBEC is a cytidine deaminase family made up of eleven genes, including AID (activation-induced cytidine deaminase) and APOBEC3B, both of which have been implicated in somatic mutation in various cancers, including CLL. These observations have led to the hypothesis that APOBEC cytidine deaminases may be driving somatic mutations leading to the development of more aggressive cancers. Therefore, we examined APOBEC gene family member RNA expression levels in CLL to test for correlations with expression levels and patient outcome. We further examined if CLL cells generated de novo APOBEC family member mutational patterns in the immunoglobulin variable region gene (IGHV) after implantation in a mouse xenograft model of CLL. CLL peripheral blood mononuclear cells (PBMCs) and associated clinical data were collected from patients after informed consent as approved by the Institutional Review Board at the North Shore-Long Island Jewish Health System and in accordance with the Helsinki Declaration. CLL samples were chosen based on availability with no pre-established inclusion/exclusion criteria. CLL RNA expression levels were examined by microarray or quantitative real-time PCR (qPCR). For microarray studies, CLL B cells were purified prior to RNA isolation and acquisition of microarray expression data using Illumina Human WG6 and HT12 bead chips, followed by quantile normalization using GenomeStudio software (Illumina). For qPCR, RNA expression from CLL PBMCs was measured relative to glyceraldehyde 3-phosphate dehydrogenase gene expression by Taqman assay with Roche UPL probes and LightCycler 480. To examine de novo mutations in CLL, the IGHV region was ultra-deep sequenced (Roche 454 FLX system) from human CLL cells recovered from the NOD/Shi-scid,γcnull (NSG) xenograft mouse model of CLL as approved by the Institutional Animal Care and Use Committee at the North Shore-Long Island Jewish Health System. CLL patient (N = 65) RNA expression by microarray showed very low levels of APOBEC1, 2, 3A, 3B, 3D, 4, and AID, modest levels of APOBEC3C and 3H, and high levels of APOBEC3F and 3G. Higher AID expression levels significantly correlated (P <0.05) with shorter time to first treatment (TFT), which was anticipated based on previous reports. Interestingly APOBEC3B and APOBEC3F expression differences showed possible trends correlating with worse patient outcome. Therefore, we tested select APOBEC gene family members by qPCR. For qPCR, we utilized the CLL patient cohort (N= 83) previously found to indicate that AID expression was a risk factor for worse patient outcome in a multivariate analysis (Patten et al. 2012 Blood 120:4802). RNA expression by qPCR followed the same pattern as the microarray data: AID and APOBEC3B had very low levels, APOBEC3H had modest levels, and APOBEC3F and 3G had high levels. Similar to AID, patients could be grouped based on the presence or absence of detectable APOBEC3B, with its presence showing a significant correlation (P <0.05) with worse TFT and overall survival. Higher levels of APOBEC3F and 3H showed a trend towards a correlation with shorter TFT, while differences in APOBEC3G expression had no significant correlation with patient outcome. Thus, not only did we confirm the correlation of AID expression with worse patient outcome, but we also found APOBEC3B and potentially APOBEC3F and 3H correlate with worse patient outcome. To test if CLL cells can acquire de novo mutations indicative of APOBEC gene family member activity, human CLL cells were transferred into NSG mice. After CLL cells proliferated for 4-14 weeks in this xenograft model, the IGHV region was amplified, ultra-deep sequenced, and analyzed for specific mutational characteristics of various APOBEC gene family members. The results of these ongoing analyses will be presented. In conclusion, the expression levels of the APOBEC gene family members AID, APOBEC3B, and potentially APOBEC3F and 3H, correlate with worse patient outcome. These data are consistent with the hypothesis that APOBEC gene family member activity may promote new mutations at sites outside the IG gene loci leading to the evolution of aggressive CLL. Disclosures Barrientos: Pharmacyclics, Celgene, and Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead, Pharmacyclics, and AbbVie: Research Funding.

ABSTRACT The activation-induced deaminase/apolipoprotein B-editing catalytic subunit 1 (AID/APOBEC) family comprises four groups of proteins. Both AID, a lymphoid-specific DNA deaminase that triggers antibody diversification, and APOBEC2 (function unknown) are found in all vertebrates examined. In contrast, APOBEC1, an RNA-editing enzyme in gastrointestinal cells, and APOBEC3 are restricted to mammals. The function of most APOBEC3s, of which there are seven in human but one in mouse, is unknown, although several human APOBEC3s act as host restriction factors that deaminate human immunodeficiency virus type 1 replication intermediates. A more primitive function of APOBEC3s in protecting against the transposition of endogenous retroelements has, however, been proposed. Here, we focus on mouse APOBEC2 (a muscle-specific protein for which we find no evidence of a deaminating activity on cytidine whether as a free nucleotide or in DNA) and mouse APOBEC3 (a DNA deaminase which we find widely expressed but most abundant in lymphoid tissue). Gene-targeting experiments reveal that both APOBEC2 (despite being an ancestral member of the family with no obvious redundancy in muscle) and APOBEC3 (despite its proposed role in restricting endogenous retrotransposition) are inessential for mouse development, survival, or fertility.

Abstract Background: Mutations drive the initiation and progression of cancer. A leading druggable source of mutation in cancer is enzymatic deamination of single-stranded DNA cytosines by cellular APOBEC3 enzymes. Cytosine-to-uracil deamination can result in a variety of different mutational outcomes including DNA breakage and chromosomal aberrations as well as single base substitution mutations. The latter are comprised of C-to-T and C-to-G mutations in TCA or TCT trinucleotides and attributable to the intrinsic preference of several APOBEC3 family members for binding to these motifs. This mutation pattern, commonly called the “APOBEC signature”, is evident in approximately one-quarter of primary breast tumors and one-third of metastatic breast tumors. Although multiple APOBEC3 enzymes have been implicated as a source of this signature in breast cancer (namely, APOBEC3A, APOBEC3B, and APOBEC3H), the literature is full of conflicting views and it is not clear which of these enzymes contributes most significantly to the mutational landscape of breast cancer. Methods: The near-haploid human cell line, HAP1, was engineered to express the HSV-1 TK gene as a mutation reporter. Candidate APOBEC3 enzymes were expressed individually and confirmed by immunoblotting and activity assays. DNA breakage was measured directly by COMET assays and DNA damage responses indirectly by phosphorylated gamma-H2AX staining. Mutation frequencies were quantified by assaying rates of drug resistance, and mutation patterns were analyzed by sequencing locally in TK and globally across whole genomes. Results: APOBEC3A and APOBEC3B both caused significant increases in chromosomal DNA breakage and DNA damage responses. These enzymes also elevated drug resistance mutation frequencies. In contrast, expression of active APOBEC3H or catalytic mutant derivatives of APOBEC3A and APOBEC3B failed to trigger increases beyond normal spontaneous levels. Interestingly, APOBEC3A and APOBEC3B both inflicted mutation signatures that were indistinguishable locally in TK and globally across whole genomes. The vast majority of these APOBEC signature mutations were dispersed (non-kataegic) and not associated with obvious mesoscale chromosomal features such as single-stranded loop regions of stem-loop structures. Computational comparisons of the broader pentanucleotide APOBEC3A and APOBEC3B mutation signatures and those extracted from 794 primary breast tumor genomes (ICGC cohort) revealed an APOBEC3A-biased subset, an APOBEC3B-biased subset, and a larger group of tumors best explained by combinatorial action of both of these enzymes. Conclusions: Our results indicate that APOBEC3A and APOBEC3B contribute combinatorially in most instances to the observed APOBEC mutation signature in breast cancer. These results provide a framework for developing diagnostic and therapeutic approaches for APOBEC-positive breast cancer. Citation Format: Reuben S Harris, Matthew C Jarvis, Michael A Carpenter, Margaret R Brown, Prokopios P Argyris, William Brown, Douglas Yee. Apobec mutation signature in breast cancer explained by combinatorial action of apobec3a and apobec3b [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-01.

Abstract The APOBEC family of cytidine deaminases include AID (activity induced deaminase) and 10 related APOBEC enzymes (A1,A2,A3A,A3B,A3C,A3D,A3F,A3G,A3H and A4). AID is well studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes. APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Recently, a causal role for the AID/APOBECs in inducing somatic mutations in myeloma has been proposed and we have previously published that APOBEC signature mutations as a frequent event in Myeloma. We have also observed that APOBEC-mediated mutations may account for mutations associated with progression of smoldering myeloma to MM. We further investigated the role of APOBEC in genomic changes in MM and observed that APOBEC expression and activity is elevated in myeloma cell lines as well as patient samples. Using knockdown and over expression approaches, we showed that depletion of APOBECs in myeloma cell lines reduces genomic instability. Following APOBEC3G knock down we observed decreased DNA damage (by g-H2AX), decrease in acquisition of new copy number events over time, and reduced mutational load, strongly suggesting that inhibiting APOBECs could be a potential approach to reduce genome evolution in MM. We next investigated the effect of APOBEC inhibition on myeloma cell proliferation. We observed that Sh-RNA-based APOBEC knock down in MM1S and H929 MM cell lines, led to significant inhibition of MM cell proliferation, and induction of apoptotic cell death. Associated with APOBEC knockdown, we also observed increased levels of Cyclin-dependent kinase inhibitor 1B (p27Kip1) at both RNA and protein level. By immunoprecipitation we found that APOBEC3G interacts and inhibits the RNA binding protein DEAD-END 1 (DND1), thereby preventing it from inhibiting miR-221-mediated targeting of p27 transcripts. Knockdown of DND1, or over-expression of miR-221 in APOBEC-depleted cells rescued the cell proliferation defects with concomitant decrease in p27 levels. These results show that APOBCs bind to and sequester DND1, leading to miR-221-mediated depletion of p27. In the absence of APOBEC, DND1 prevents the degradation of p27 mRNA, leading to elevated p27 levels and inhibition of cell cycle, suggesting a role for APOBECs in regulating MM cell proliferation that might be independent of its RNA/DNA mutator function. Taken together, these results indicate a significant functional role for APOBECs both in genome evolution as well as cell growth in myeloma and may constitute an important therapeutic target. Disclosures Munshi: OncoPep: Other: Board of director.

Abstract The AID/APOBEC family of cytidine deaminase proteins includes AID (activity induced deaminase), and 10 related APOBEC enzymes (A1, A2, A3A, A3B, A3C, A3D, A3F, A3G, A3H and A4). AID has been well-studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes whereas APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Dysregulated activity of APOBECs causes C >T transitions or C>G, C>A transversions in DNA. We have recently shown APOBEC signature mutation pattern in multiple myeloma (MM) genomes (Bolli et al Nat. Comm. 2014), and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in myeloma. A role for the AID/APOBECs in generation of somatic mutations has also been proposed in a variety of other cancers based on identification of APOBEC signature mutations In order to understand which APOBECs are dysregulated in myeloma, we performed RNA sequencing analysis of primary myeloma cells from 409 newly-diagnosed MM patients and myeloma cell lines. Our analysis showed elevated expression of several APOBEC family members; mainly A3A, A3B, A3C, and A3G. We then optimized a plasmid-based functional assay and found high cytidine deaminase activity in extracts from a number of myeloma cell lines and patient derived CD138+ cells compared to CD138+ cells from healthy donors, suggesting that APOBECs are dysregulated in myeloma. We then investigated the impact of elevated APOBEC expression/function on overall genome maintenance and acquisition of genomic changes (such as amplifications, deletions) overtime. We used shRNA-mediated knockdown of specific APOBEC proteins in myeloma cell lines and investigated the acquisition of genomic changes in control and knockdown cells during their growth in culture, using SNP (Single Nucleotide Polymorphism) arrays and WGS (whole genome sequencing) platforms. Our results with both approaches showed significant reduction in the accumulation of copy number changes (both amplifications and deletions) and overall mutation load after APOBEC knockdown. Evaluation with both the SNP and WGS showed that when control and APOBEC knockdown cells were cultured for three weeks, the acquisition of new copy number and mutational changes throughout genome were reduced by ~50%. We next investigated the relationship between APOBEC expression/activity in MM and other DNA repair pathways. Using an in vitro HR activity assay, we measured HR activity in extracts from control and APOBEC knockdown cells. Depletion of APOBEC proteins resulted in 50-80% reduction in in vitro HR activity of the extracts. We also evaluated correlation between HR activity and gene expression using RNA-seq data from myeloma cells derived from 100 patients at diagnosis and identified the genes whose expression correlated with HR activity. Elevated expression of APOBECs 3D, 3G and 3F significantly correlated with high HR activity (R=0.3; P≤0.02), suggesting their relevance to HR. Analyzing genomic copy number information for each patient we have also observed significant correlation between higher expression of A3G and increased genomic instability in this dataset (P=0.0045). In summary, our study shows that dysregulated APOBECs induce mutations and genomic instability, and inhibiting APOBEC activity could reduce the rate of accumulation of ongoing genomic changes. This data sheds light on biology of the disease as well as clonal evolution. Disclosures Munshi: Amgen: Consultancy; Oncopep: Patents & Royalties; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Pfizer: Consultancy.

Hepatitis B virus (HBV) is a DNA virus that causes liver disease and replicates by reverse transcription of an RNA template. Previous studies have reported that HBV genomes bearing G→A hypermutation are present at low frequency in human serum. These mutations are most likely due to the activity of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) cytosine deaminases, cellular proteins known to confer innate immunity against retroviruses by generating lethal hypermutations in viral genomes. This study assessed APOBEC3G, APOBEC3C and APOBEC3H, three members of this protein family present in human liver, for their ability to edit HBV genomes. Transfection of human HepG2 hepatoma cells with a plasmid encoding the APOBEC3C protein resulted in abundant G→A mutations in the majority of newly formed HBV genomes. By contrast, transfection of APOBEC3G- and APOBEC3H-encoding plasmids only marginally increased hypermutation rates above the level caused by the cytosine deaminases naturally present in HepG2 cells. APOBEC3G- and APOBEC3H-mediated hypermutation, however, was clearly revealed by transfection of chicken LMH hepatoma cells, which lack endogenous cytosine deaminases. These results indicate that APOBEC3G, APOBEC3C and APOBEC3H have the ability to edit HBV DNA and that each protein is likely to contribute to various degrees to the generation of modified genomes in human liver cells.

Cancer is considered a group of diseases characterized by uncontrolled growth and spread of abnormal cells and is propelled by somatic mutations. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) family of enzymes are endogenous sources of somatic mutations found in multiple human cancers. While these enzymes normally act as an intrinsic immune defence against viruses, they can also catalyse ‘off-target’ cytidine deamination in genomic single-stranded DNA intermediates. The deamination of cytosine forms uracil, which is promutagenic in DNA. Key factors to trigger the APOBEC ‘off-target’ activity are overexpression in a non-normal cell type, nuclear localization and replication stress. The resulting uracil-induced mutations contribute to genomic variation, which may result in neutral, beneficial or harmful consequences for the cancer. This review summarizes the functional and biochemical basis of the APOBEC3 enzyme activity and highlights their relationship with the most well-studied cancers in this particular context such as breast, lung, bladder, and human papillomavirus-associated cancers. We focus on APOBEC3A, APOBEC3B and APOBEC3H haplotype I because they are the leading candidates as sources of somatic mutations in these and other cancers. Also, we discuss the prognostic value of the APOBEC3 expression in drug resistance and response to therapies.

The thesis is focus on RNA editing mediated by two AID/APOBEC family members. The aim of my work was the investigation of possible novel factors that regulate hAPOBEC1 expression or cofactors which help the deaminase to exert its activity. First, I characterised cellular models for their proliferation and clonogenic activities as well as cell cycle distribution evaluating a combinatorial effect of hAPOBEC1 and RBM47 which lead to a decrease in cell growth. I investigated the role of RNA editing beyond the lipid transport by high-throughput sequencing which provided me information regarding new deamination events, RNA stability, and also a differential gene expression in presence or absence of the editosome components. By Differential expression analysis, I got a list of genes that are differentially expressed in clones with hAPOBEC1 and RBM47 which need to be analysed for their biological meaning. From the mRNA-seq I got a consistent list of putative edited sites even though some of them were validated with no success. Moreover, I applied a genetic library screen to activate a high number of genes in cells expressing RBM47 to evaluate an eventual up-regulation of APOBEC1 and find factors which trigger its expression. The cells in which editing happened have been selected thanks to a specific fluorescent reporter containing ApoB target. The results have still to be analysed. The second aim of my project was to study APOBEC3A regulation, by chemical and genetic screenings, through the development of a specific sensitive reporter system to detect APOBEC3A-mediated RNA editing. In this work I presented the design of an artificial fluorescent reporter containing a target of APOBEC3A like SDHB or DDOST properly built to produce a stop codon in the middle of the target and optimised for the levels of editing. I checked its specificity for APOBEC3A and not for other APOBEC proteins like APOBEC1 and APOBEC3B. This let me also detected a novel putative editing site mediated by APOBEC3A by Sanger sequencing. Moreover, I designed another fluorescent reporter system able to evaluate APOBEC3A RNA editing by fluorescent microscopy. I created stable cell lines expressing all the lentiviral reporter plasmids to further investigate induction of endogenous APOBEC3A and its regulation. In a future perspective the dual fluorescent reporter could be a useful tool to identify novel RNA editing targets upon the application of an activation library screen.

APOBEC family deaminases are capable of causing mutations by deaminating cytosine in DNA to uracil. This is exploited in diversification of the repertoire of antibodies by somatic hypermutation, and also in restricting the spread of retroviruses. APOBEC family induced mutations are not distributed entirely at random throughout the genes they deaminate; rather each APOBEC family member has its own preferred local sequence that will be preferentially targeted. Work presented in this thesis elucidates these preferred motifs for a number of different deaminases and investigates the structural basis of their specificity using viral and bacterial genetic assays. To determine the local sequence specificities of APOBEC proteins active in E. coli, a novel selection system was devised based on the conditional-lethal sacB gene. By varying the activity and orientation of promoters it was possible to target mutations to a chromosomally integrated sacB gene under certain conditions. Selecting for viable mutants generated mutation spectra for the AID, APOBEC1 and APOBEC3G deaminases. This enabled their preferred sequence motifs to be identified and correlated with particular mutation patterns found in vivo.

APOBEC3s (A3s) are a family of human cytidine deaminases that play important roles in both innate immunity and cancer. A3s protect host cells against retroviruses and retrotransposons by deaminating cytosine to uracil on foreign pathogenic genomes. However, when mis-regulated, A3s can cause heterogeneities in host genome and thus promote cancer and the development of therapeutic resistance. The family consists of seven members with either one (A3A, A3C and A3H) or two zinc-binding domains (A3B, A3D, A3D and A3G). Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA-bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using a combination of computational molecular modeling and parallel molecular dynamics (pMD) simulations followed by experimental verifications, I investigated the roles of active site residues and surrounding loops in determining the substrate specificity and RNA versus DNA binding among A3s. Starting with A3B, I revealed the structural basis and gatekeeper residue for DNA binding. I also identified a unique auto-inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity compared to the highly similar A3A. Besides, I investigated the structural mechanism of substrate specificity and ssDNA binding conformation in A3s. I found an interdependence between substrate conformation and specificity. Specifically, the linear DNA conformation helps accommodate CC dinucleotide motif while the U-shaped conformation prefers TC. I also identified the molecular mechanisms of substrate sequence specificity at -1’ and -2’ positions. Characterization of substrate binding to A3A revealed that intra-DNA interactions may be responsible for the specificity in A3A. Finally, I investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive analysis of A3s in this thesis shed light into the structural mechanism of substrate specificity and broaden the understanding of molecular interactions underlying the biological function of these enzymes. These results have implications for designing specific A3 inhibitors as well as base editing systems for gene therapy.

Avec la découverte de l’édition des acides nucléiques, le dogme selon lequel l’information génétique est transmise de manière fidèle jusqu’aux protéines a été remis en question. Mise en évidence sur les ARN, l’édition est définie comme la modification de certains transcrits résultant dans la production d'une protéine différente de la séquence codée par le gène. Sur l’ADN, ce processus serait un mécanisme de protection, empêchant l’invasion du génome cellulaire par des gènes exogènes. Chez l’homme, la conversion C-U est catalysée par des cytidines désaminases dont font partie APOBEC3. L’objectif de ce travail a consisté à étudier les enzymes de la famille APOBEC3 impliquées dans les phénomènes de restriction virale observés sur le génome viral du VIH-1, en particulier dans la région de l'ORF Vpr. Des transitions C-T et G-A conduisant à l’inactivation de vpr, corrélées à la variation de l'expression des apobec3, suggèrent que les protéines de la famille APOBEC3 pourraient être en partie responsables de la présence de virus défectifs, et ainsi être impliquées dans la chronicité de l’infection observée pour les cellules H9/LAI et pour au moins un patient non progresseur à long terme. Des tests de désamination in vitro ont permis de montrer une activité d’APOBEC3G au niveau de 2 résidus C. Nous n’avons pas pu mettre en évidence d’activité de désamination sur ce même substrat pour les autres APOBEC3 testées. Ceci suggère que la protéine APOBEC3G pourrait être responsable des modifications observées sur le génome du VIH-1. Des expériences préliminaires à la recherche des partenaires cellulaires potentiels, suggèrent l’existence d’un complexe composé d’au moins 5 protéines
RNA editing is a post-transcriptional process that changes the informational capacity within the RNA. This process modifies transcripts in many organisms and thus contributes to expanding the number of gene products without the generation of new genes. Base changes on DNA by C deaminases can be considered as a protection mechanism preventing the invasion of the cell by exogenous genes. In human, A-I and C-U conversion have been described. The C-to-U changes are catalyzed by APOBEC cytidine deaminases, with the APOBEC3 family involved in DNA modifications. The aim of this work is to study the APOBEC3 proteins that are involved in viral restriction phenomenon observed particularly in HIV-1 infections. One of the targets for deamination, the vpr orf, was chosen as model. The correlation between C-T and G-A transitions inactivating vpr with the variation of apobec3 expression, led us to postulate that APOBEC3 family proteins could be partially responsible of the presence of defective viruses. In that way, the activity of restriction deaminases may be involved in chronic infection observed in the H9/LAI cells and, in some cases, on long-term non-progressor patients. In vitro deamination assays showed that two C residues in vpr can be modified in Us by APOBEC3G, but not by other APOBEC3 deaminases, suggesting that APOBEC3G is responsible of the changes observed on the HIV-1 genome. I also look for potential cellular partners for APOBEC3G using a TAP-tag approach. Preliminary experiments indicate a complex composed of at least five proteins

PhD (Microbiology)
Department of Microbiology
Introduction Human Immunodeficiency Virus (HIV-1) continues to be a global public health concern, even though Antiretroviral drugs (ARV), especially Highly Active Antiretroviral Therapy (HAART) has significantly reduced morbidity and mortality due to AIDS globally in developed and developing countries. However, there is still a great need to explore every avenue for new therapeutic interventions due to the limitations and side effects of HAART. Potential major breakthroughs for future therapeutic development were the discoveries more than 10 years ago of the role of HIV-1 co-receptors and anti-viral activities of host restriction factors such as APOBEC3G protein, which is a member of the DNA cytosine deaminase family. Entry of HIV in to the host cell is through the attachment of the viral envelope glycoprotein to the CD4 receptor, and subsequent interaction, mainly with either CCR5 or CXCR4 co-receptors. Inhibitors, such as Maraviroc, which binds to CCR5 inhibiting entry of CCR5 utilizing viruses (R5 viruses), is currently reserved for salvage therapy in many countries including South Africa. In the course of HIV infection, CXCR4 utilizing viruses (X4 viruses) may emerge and outgrow R5 viruses, and potentially limit the effectiveness of Maraviroc. Several host cell APOBEC3 genes (A3D, A3F, A3G and A3H) have been shown to restrict HIV, and the HIV viral infectivity factor (Vif) protein serves to antagonize the action of APOBEC3 proteins, promoting viral replication. The CCR5 co-receptor and the HIV Env V3 loop have also been documented as playing roles in HIV-1 disease progression. The interplay between host and viral genes still needs widespread attention, given that disease outcomes of HIV depend on many factors, including host cell genetics. Since the discovery of these genes and their role in HIV replication, many studies have been conducted that show their association with viral polymorphism. The polymorphisms found in host cell genes can have significant effects on viral replication, transmission and fitness and can also contribute to the overall diversity in HIV-1 populations. It is hypothesized that there are significant polymorphisms in HIV-1 and cellular genes that may differ among different populations. Population-based studies have tried to establish a relationship between host factors such as APOBEC3 and CCR5 polymorphism and the rate of disease progression, but most studies have focused on Caucasian populations. In vi contrast, little information is available for the effects of variation in these genes in African populations such as South Africa, where the HIV epidemic has expanded at an alarming rate. Although several population studies have focused on African Americans, these do not give us a complete picture of the potential variation in Africans, though the studies can be a good guide on which to base additional studies. A more comprehensive analysis involving different African populations will likely provide a better understanding of the mechanisms underlying host-pathogen interactions, especially in view of the fact that African Americans are primarily infected with HIV subtype B, which is rarely seen in Africa. Methodology This study characterized the genetic variability of the APOBEC3 D, F, G and H genes as well as the HIV-1 vif, in an ethnically diverse HIV-1 infected South African cohort using Next Generation Sequencing (NGS). In addition, polymorphism in CCR5 was analyzed in conjunction with an analysis of the V3 loop sequences in HIV-1 from this cohort. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) of 192 HIV-1 infected drug-experienced individuals who presented for routine care at the HIV/AIDS Prevention Group Wellness Clinic (HAPG) in Bela-Bela, Donald Fraser Hope Clinic (DFHC) in Vhufhuli and in local clinics in the Vhembe district of Limpopo Province, South Africa. Next generation sequencing custom based (Tn5 tagmentation and amplicon based) protocols to prepare libraries for host and HIV-1 genes were developed and validated with commercially available library preparation kits. The Tn5 tagmentation methods were used for longer DNA fragments and the custom amplicon based methods were used mainly for the shorter DNA fragments. To determine the variability of the APOBEC3 and CCR5 host genes, gene-specific primers were designed to amplify complete 12.16 kb A3D, 13.31 kb A3F, 10.74 kb A3G, 6.8 kb A3H and 1.3 kb CCR5 genes targeting the regions containing the exons. Libraries for the resulting amplicons were prepared using Tn5 transposase tagmentation methods and sequenced on an NGS Illumina MiSeq platforms generating millions of reads with good read coverage for variant calling. Single nucleotide polymorphisms (SNPs) and indels were determined, verified in dbSNPs and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap. A Chi-square goodness-of-fit was used to verify if whether observed genotype frequencies were in agreement with the Hardy-Weinberg Equilibrium. Haplotypes and Linkage disequilibrium were inferred to determine SNP association. vii The HIV-1 vif and env V3 loop genes were also sequenced to determine their degree of variability of these genes and to infer co-receptor usage in the South African population. Gene-specific primers were designed to amplify the 579 bp Vif region and 440 bp containing the 105 bp V3 loop. Sequencing libraries from the resulting amplicons were prepared using either the Tn5 transposase or custom-based library preparation methods and sequenced on either an Illumina MiSeq or a MiniSeq platform generating millions of reads with good read coverage for variant calling. Phylogenetic analysis was done to determine the relatedness of the sequences. Major and minor variants were determined for HIV-1 and env V3 loop quasispecies was analysed for co-receptor usage; in an effort to draw inferences for the subsequent utility of Maraviroc as salvage therapy in South Africa. Results and Discussion Next generation library preparation; Tn5 tagmentation based and custom amplicon based protocols to sequence host and HIV genes were successfully developed and used to sequence and characterize variability in host cell APOBEC3D, F, G H, CCR5 and the HIV-1 vif gene and the V3 loop region of the env gene. The HIV-1 env V3 loop sequences generated (and quasispecies analyzed) were used to infer co-receptor usage in treatment-experienced individuals; in an effort to draw inferences for the subsequent utility of Maraviroc as salvage therapy in South Africa. Quality V3 loop sequences were obtained from 72 patients, with 5 years (range: 0-16) median duration on treatment. Subtypes A1, B and C viruses were identified at frequencies of 4% (3/72), 4% (3/72) and 92% (66/72) respectively. Fifty four percent (39/72) of patients were predicted to exclusively harbor R5 viral quasispecies; and 21% (15/72) to exclusively harbor X4 viral quasispecies. Twenty five percent of patients (18/72) were predicted to harbor a dual/mixture of R5X4 quasispecies. Of these 18 patients, about 28% (5/18) were predicted to harbor the R5+X4, a mixture with a majority R5 and minority X4 viruses, while about 72% (13/18) were predicted to harbor the R5X4+ a mixture with a majority X4 and minority R5 viruses. The proportion of all patients who harboured X4 viruses either exclusively or dual/mixture was 46% (33/72). Thirty-five percent (23/66) of the patients who were of HIV-1 subtype C were predicted to harbor X4 viruses (χ2=3.58; p=0.058), and 57% of these (13/23) were predicted to harbor X4 viruses exclusively. CD4+ cell count less than 350 cell/μl was associated with the presence of X4 viruses (χ2=4.99; p=0.008). The effectiveness of Maraviroc as a component in salvage therapy may be compromised for a significant number of chronically infected patients harboring CXCR4 utilizing viii viruses in the study cohort. Although from the current study a subset of patients harboring CCR5 utilizing viruses may benefit from Maraviroc, characterizing and identifying if variation in CCR5 are located at Maraviroc binding sites was of importance to investigate. The following variants; P35P, S75S, Y89Y, A335V and Y339F and their varying frequencies were detected in the CCR5 gene. The A335V variant was detected at a higher frequency of 17.4% (29/167). The G265S variant is reported for the first time in this study at 0.6% (1/167) frequency. The SNPs detected were in strong LD (D’= 1, R2 = 0.0) with minor deviation from the Hardy-Weinberg Equilibrium. These variants were not located at the binding motif of Maraviroc. The variants A335V and Y339F were detected at a higher frequency in this study than previously reported in South Africa. Variability in APOBEC3 host cell genes was also characterized in our study cohort. The following APOBEC3 variants compared to the GRCh37 consensus sequence were detected: R97C, R248K and T316T in A3D; R48P, A78V, A108S, S118S, R143R, I87L, Q87L, V231I, E245E, S229S, Y307C and S327S in A3F; S60S, H186R, R256H, Q275E and G363R in A3G and N15Δ, G105R, K140E, K121D, E178D in A3H. Minor allele frequency variants (MAF<5%); L221L, T238I, C224Y and C320Y in A3D; I87L, P97L and S229S in A3F; R256H, A109A, F119F and L371L in A3G, which are frequent in the European population, were also detected. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were detected. Most of the SNPs were in strong LD with minor deviation from the Hardy-Weinberg Equilibrium. Four, six, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively. In general, polymorphism in A3D, 3F, 3G and 3H were higher in our South African cohort than previously reported among other African, European and Asian populations. The APOBEC3 antagonist HIV-1 vif gene was also sequenced to determine the level of diversity in a South African population and also correlated with APOBEC3 variation. Functional Vif without frameshift mutation was observed in all samples except in 4 samples. The functional domain and motifs, such as Zn binding motifs, proline-rich domain, human casein kinase, and the N and C-terminal CBF interaction site were highly conserved. APOBEC binding motifs and the nuclear localization signal were less conserved in the South African HIV-1 Vif. APOBEC3 H variation strongly correlates with Vif variation. All the Vif sequences were subtype C, except one sample, which was identified as an A1/C recombinant. The vif gene in a South African population was under purifying selection, with the dS= 0.2581 and dN= 0.0684 and the dN/dS value = 0.265. There is a high genetic diversity in the South African vif gene, which may ix influence the neutralization and restriction of APOBEC genes. Conclusions In conclusion, the protocols developed in this study can be applied to amplify and sequence any host and HIV-1 genes of interest allowing much deeper and more sensitive profiling of host gene and HIV-1 genetic diversity. Our findings show that a highly significant number of chronically HIV-1 subtype C infected patients in Maraviroc-free treatment harbor CXCR4 utilizing viruses. The data is useful in the consideration of whether to include entry antagonists such as Maraviroc in alternative forms of treatment for patients failing second line treatment regimen in the study setting. The determination of co-receptor usage prior to initiation of therapy consisting of Maraviroc is suggested. Variation in the CCR5 coding region were observed at higher frequencies compare to other studies conducted in South African populations at different locations. This data may suggest that different populations in South Africa have different SNP frequencies. All the polymorphisms identified in the study were not located at the Maraviroc binding motif, therefore the subset of patient infected by R5 viruses may benefit from this drug. We have shown that significant APOBEC3 variation exists among an ethnically diverse population of South Africa by providing extensive data for 4 different A3 genes that are known to restrict HIV infection, but have only been sparsely studied in African populations. This study provides a baseline for future studies that would functionally characterize SNPs identified in this population, in order to understand the role of novel and/or low frequency variants observed. Ex vivo and in vivo studies will increase our understanding of how these variants might have cumulatively impacted the epidemic in Northern South Africa. This study also shows that there is a high level of HIV-1 Vif diversity in the study area. This diversity may impact the expression and packaging of Vif proteins, and the infectivity of HIV. In addition, a significant correlation was observed between HIV-1 Vif variation and APOBEC3 H haplotypes.
NRF

Risk for Alzheimer’s disease (AD) is strongly influenced by genetics. In fact, following age, the second strongest risk factor for AD is family history. To date, genetic studies have thus far demonstrated that the inheritance of AD is dichotomous. Roughly half of the cases of the rare early-onset (<60 years) familial form of AD (EO-FAD) are caused by rare mutations in APP, PSEN1, and PSEN2.These mutations usually guarantee onset and account for ~5% of AD. The major gene influencing risk for late-onset AD (LOAD) is APOE. The ε‎4 and ε‎2 variants increase and decrease risk for AD, respectively. Together, these four genes account for up to 50% of the genetic variance of AD. To find the remaining AD genes, multiple genome-wide association studies (GWAS) have resulted in the identification of eleven other AD candidate genes. In this review, I summarize the current state of our knowledge of the genetic factors influencing risk for AD and prospects for future studies.

Este artículo relata un viaje del proyecto de doctorado dirigido por la práctica del investigador, Tangohia mai te taura (“Toma esta soga”). El estudio implica investigar, dirigir y producir un documental sobre agravios históricos dentro de Te Whakatōhea y Te Whānau ā Mokomoko. Específicamente, explora el potencial de la práctica y la forma documental en relación con Mātauranga Māori (costumbres y conocimientos maoríes) y kaupapa Māori (enfoques de investigación maoríes). El estudio busca cuestionar ciertas narrativas construidas por Pākehā sobre el exotizado asesinato del misionero, el reverendo Carl Sylvius Völkner en 1885. Como consecuencia de una acusación de asesinato, mi antepasado Mokomoko fue arrestado por el crimen, encarcelado y ahorcado, mientras protestaba su inocencia. En represalia, a nuestro pueblo le confiscaron sus codiciadas tierras por parte del gobierno y se convirtieron en parias de múltiples relatos históricos. La tesis pregunta cómo un documentalista maorí de esta iwi (tribu) podría llegar al dolor y la injusticia de tal evento de maneras culturalmente sensibles, para contar la historia del impacto generacional. La investigación considera cuatro rasgos distintivos del enfoque del autor como cineasta indígena. WHAKAPAPA - GENEALOGÍA: En el pensamiento maorí, whakapapa conecta al realizador con la película, los entrevistados y la comunidad. Sin embargo, las conexiones de whakapapa conllevan la responsabilidad de navegar la realización de películas con respeto y cuidado. WHENUA y WHANAU - TIERRA Y FAMILIA: Metodológicamente mi enfoque a través de la encarnación. Paso tiempo viviendo y reconectando con mi familia extendida y las tierras en las que vivimos. Camino, pienso,escucho y siento mi camino a través de un mundo complejo, buscando activamente oportunidades para asistir a wānanga (discusiones) y apoyar las kapa haka (artes escénicas maoríes) relacionadas con nuestra tierra y nuestra familia. Mi posición es de humildad y cocreación. Soy consciente de que el rōpū (equipo) con el que trabajo será llamado al corazón confiado de mi whanau. Así, semanas antes de que comience la producción, vivimos con el mundo que el documental busca grabar. TIKANGA - ADUANAS: El proceso y las estructuras de realización de esta película siguen siendo conscientes de tikanga Māori (costumbres maoríes). Karakia y waiata (oraciones y canciones maoríes) acompañan el proceso de creación de la obra. La tripulación, en gran parte maorí, está atenta a los protocolos y sensibilidades. Estas prácticas también afirman nuestro rōpū (grupo) como familia. KOHA - RECIPROCACIÓN: A diferencia de muchos enfoques convencionales para la realización de documentales, donde las películas se “filman” de manera económica y eficiente, este proyecto se basa en el concepto de koha (reciprocidad). Se entiende que las comunidades regalan su tiempo e historias y, en respuesta, los regalos del proyecto regresan. Como artista, hago esfuerzos conscientes para apoyar a la iwi, repatriar conocimientos y artefactos que ubico en mi investigación, ser un miembro activo dentro de la ciudad y apoyar iniciativas comunitarias. Como cineasta, soy miembro de una generación que se ha ido alejando gradualmente de la historia y encarnado el dolor de mi whanau. Vengo a buscar mi pasado en un esfuerzo por comprender y contribuir con algo útil que apoye las aspiraciones y la capacidad de mi pueblo para lograr valor, curación y reparación histórica.

Smoking is the most common and important risk factor for bladder cancer. The reason lying behind the fact is that – the smoke toxins accelerate other DNA damaging events and attention being focused on a family of enzymes called “APOBEC.