Browsing by Subject "APOBEC"

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APOBEC3 subcellular localization and genomic editing
(2012-10) Lackey, Lela Lynn
The APOBEC proteins are DNA cytosine deaminases with roles in immunity, including retroviral restriction and antibody maturation. Their activity theoretically makes them a danger to genomic DNA. The subfamily of APOBEC3 genes has expanded to included seven different genes in primates. Based on their subcellular localization, only a subset of these APOBEC3 proteins have access to genomic DNA, and may potentially deaminate genomic DNA. Although the nuclear envelope breaks down during mitosis, I demonstrate that none of the APOBEC3s gain access to genomic DNA during cell division. However, APOBEC3B and other APOBEC3 proteins have access to genomic DNA during interphase. I also show that APOBEC3B is actively imported into the nuclear compartment. In general, APOBEC3 nuclear localization and deaminase activity correlate with ability to affect cell cycle progression, implicating these APOBEC3s in deamination of genomic DNA. In support of these conclusions, I observed cell death, activation of the DNA damage response and DNA mutations after ectopic expression of APOBEC3A and APOBEC3B. Moreover, endogenous APOBEC3B is demonstrably nuclear and active in breast cancer cell lines where it causes genomic deamination and mutations. Endogenous APOBEC3B is highly expressed in more than half of human breast cancers compared to normal breast tissues. In addition, sequences from tumors with higher levels of APOBEC3B have more mutations, and these mutations match APOBEC3B's deamination signature. My thesis work further defines the subcellular localization of the APOBEC3 family and provides the first evidence that APOBEC3B is involved in a human cancer type.
Biochemical and Biophysical Assay Development for Screening and Characterization of Small Molecules and Synthesized Analogues Targeting Human Cytosine Deaminases
(2023-01) Grillo, Michael
The APOBEC3 family of enzymes converts cytosine to uracil in single-stranded DNA as a part of the innate immune defense against viruses. A large body of work has demonstrated that when dysregulated, APOBEC3s contribute to mutagenesis of somatic DNA in cancer. These mutational events lead to poor clinical outcomes such as tumor recurrence, metastasis, and therapeutic resistance. Because of this, we are interested in targeting APOBEC3s for inhibition by small molecules with the goal of improving the outcome of treatment with current cancer therapies. Chapter 1 introduces APOBEC3s as targets of interest with commentary on current and potential biochemical, biophysical, cellular, and in vivo assay technologies to evaluate potential inhibitors. Chapter 2 discusses careful in silico reconstruction of APOBEC3B followed by molecular dynamics simulations and druggability analysis identifying putative allosteric sites. Virtual screening was performed, and compounds were validated in biochemical and biophysical assays to serve as potential starting points for hit to lead optimization. Chapter 3 focuses on the development of sensitive biophysical assays and implementation in fragment screening. One fragment was validated during triage and served as a starting point for preliminary structure-activity relationship studies on two potentially divergent chemical series. Chapter 4 discusses the development of a real-time fluorescence-based activity assay for human cytidine deaminase utilizing a fluorogenic substrate. This assay was validated with known small molecule inhibitors and implemented in a fragment screen to discover novel non-nucleoside inhibitors of cytidine deaminase. The long-term goal of this work is to apply the same technology to measure APOBEC3 activity. Chapter 5 outlines several attempts at synthesizing a rationally designed covalent sulfur (VI) fluoride exchange probe targeting conserved hydroxyl-containing residues in the APOBEC3 active site. A variety of standard, as well as novel, approaches to nucleoside chemistry were explored with the goal of eventually incorporating a covalent warhead into a DNA oligonucleotide. Finally, Appendix A describes attempts at expressing and purifying A3B containing 5-fluorotryptophan as a tool for protein-observed 19F-NMR experiments.
Counteraction of APOBEC3 Proteins by Herpesvirus Ribonucleotide Reductases
(2019-08) Cheng, Adam
The APOBEC3 family of DNA cytosine deaminases plays an important role in antiviral innate immunity. In this thesis, we describe the novel function of APOBEC3B as a physiologic restriction factor against herpesviruses such as Epstein-Barr virus and herpes simplex virus type 1. We additionally define the counteraction mechanism imparted by herpesviruses using the virus-encoded ribonucleotide reductase large subunit. These viral proteins directly bind A3B to inhibit enzymatic activity, relocalize it away from replicating viral DNA, and protect the virus from A3B-mediated hypermutation for preservation of the viral genome. These results have the potential to reveal new modes of antiviral therapy and have implications in the treatment of A3B-driven cancers.
Delineating the APOBEC3 enzymes responsible for the APOBEC mutation signature in cancer
(2021-08) Jarvis, Matthew
Mutations drive the initiation and progression of cancer. The leading druggable source of mutation in cancer, cytosine deamination by a subset of the nine-membered APOBEC family of DNA deaminase enzymes, leaves a distinct mutation signature on the cancer genome. This signature is characterized as C-to-T and C-to-G mutations in a TCA/T trinucleotide context, and thus APOBEC-dependent mutations can be resolved computationally from other processes of mutation in clinical next-generation tumor sequencing datasets. While specific APOBEC3 (A3) enzymes have been implicated as the main progenitors of this mutation signature (namely, APOBEC3A, APOBEC3B, and APOBEC3H, abbreviated A3A, A3B, and A3H), the literature is full of conflicting data and it is not clear which of these enzymes contributes most prominently, and whether other A3 enzymes may also contribute to mutation in cancer. In this thesis, we aim to definitively characterize the A3 enzymes that can contribute to genomic mutation in a mammalian cell, and potentially be involved in cancer mutagenesis. To accomplish this, we utilized bioinformatic approaches to understand mutational profiles in >1000 cancer cell models, the capacity of individual A3s to generate a cellular damage response and genomic mutation in culture, and the carcinogenic action of APOBECs in multiple animal systems of cancer initiation and progression. Taken together, these analyses indicate that both A3A and A3B have the capacity to generate a mutation signature in mammalian cells, and that A3A has the ability to initiate tumor formation in vivo. These novel advancements in the APOBEC biology field could prove invaluable in the design and implementation of future therapies and diagnostics targeting the A3s in cancer. An understanding of enzyme-specific mutational capacity will improve the development of targeted therapies, which could span to small molecule inhibition of enzymatic activity, synthetic lethal strategies, or immunotherapy-based approaches to selectively kill A3-expressing tumor cells, with the ultimate goal of attenuating or exploiting this mutational process to improve poor clinical outcomes (including drug resistance and metastasis).
HIV-1 Counteraction Mechanisms Versus APOBEC3-mediated Restriction
(2018-07) Richards, Christopher
Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the etiology of Acquired Immunodeficiency Syndrome (AIDS). Almost 40 years’ worth of intensive HIV-1 research have not yet led to a cure, nor is an efficacious vaccine available. The number of deaths caused by HIV-1/AIDs is declining due to effective, though non-curative, combinations of highly active antiretroviral therapy (HAART) regimens. Given that in 2016: 1.8 million newly infected people were infected with HIV-1 in 2016, 36.7 million people globally were living with HIV-, and 1 million people died from AIDS-related (UNAIDS Fact Sheet – 2018), every avenue to discovering a cure should be sought out. HIV-1’s life cycle is characterized by eight steps. Step 1 is known as attachment, where HIV-1 binds to the receptors of a CD4+ cells. Step 2 is fusion, where the viral envelope fuses with the cellular membrane, granting the virus entry to the host cell. Step 3 is reverse transcription, where the viral RNA that is now inside the host cells is converted into viral DNA. Step 4 is integration, where the HIV-1 DNA is shuttled into the nucleus, as part of a high molecular weight pre-integration complex, by co-opting host nuclear import machinery, followed by HIV-1 integrase catalyzes the insertion of the viral DNA into the host’s genome making a provirus. Step 5 is replication, where the integrated viral DNA hijacks host machinery to make viral RNA copies as well as viral structural proteins that are used as building blocks for HIV-1 particle formation. Step 6 is assembly, where newly synthesized HIV-1 proteins and viral RNA are trafficked and used to assemble an immature (noninfectious) particle at the cell membrane. Step 7 is known as budding, where the immature particle undergoes membrane scission with the host cell releasing the particle into extracellular space. Step 8 is maturation, where HIV-1 protease is activated in the newly released particle and it begins to cleave the structural proteins of the virus, releasing intra-virion proteins that are required to make the particle infectious (mature). APOBEC3 (A3) enzymes are packaged into budding virions from a cell already infected with HIV-1 (Steps 6-7). After a virion containing A3 enzymes enters a target cell (Steps 1-2), the A3s can restrict HIV-1 via deaminase-dependent and -independent mechanisms during reverse transcription (Step 3), which is described in more detail below. However, HIV-1 encodes virion infectivity factor (Vif), which allows the virus to retain high levels of infectivity via proteasomal degradation of cellular A3 restriction factors in cells producing virus. Restrictive A3 enzymes capacity to incapacitate HIV-1 is such that no appreciable infectivity is observed in Vif-null systems, thereby suggesting that modulation of the A3-Vif axis in the host’s favor could be a potentially curative antiretroviral approach. In this thesis, three separate projects combine to advance our understanding of the A3-mediated restriction mechanism and the Vif-mediated counteraction mechanism. Chapter 2 uses human APOBEC3F (A3F) to adapt HIV-1 and create a genetic and structural map of the Vif interaction surface. Chapter 3 compares the HIV-1 restriction activity of splice variants human APOBEC3H (A3H) and reports differential antiviral activities and a novel viral protease-dependent counteraction mechanism. Chapter 4 explores potential antiviral strategies using synthetic peptides derived from Vif. Collectively, these studies increase our overall understanding of how HIV-1 counteracts A3 restriction factors. Ultimately, this work informs the next generation of approaches directed at discovering ways to modulate these interactions in potentially curative ways.
HIV-1 Vif requires core binding factor Beta to degrade the APOBEC3 restriction factors and facilitate viral replication
(2012-12) Hultquist, Judd F.
While there are a number of antiretroviral drugs for the treatment of Human Immunodeficiency Virus (HIV), they are all expensive, invasive, susceptible to resistance, and are not curative. One potential future drug target is the interaction between the human antiviral APOBEC3 proteins and the HIV counterdefense protein, Vif. Vif binds to and neutralizes the DNA-mutating APOBEC3 proteins by recruitment of an E3 ubiquitin ligase complex that targets them for degradation. Design of small molecule therapeutics to disrupt this interaction and free the antiviral APOBEC3 proteins has been hampered by an incomplete understanding of the Vif E3 ubiquitin ligase complex and conflicting reports as to which of the seven different APOBEC3 proteins contribute to HIV restriction in vivo. To determine which APOBEC3 proteins contribute to HIV restriction, we performed a comprehensive analysis of both human and rhesus macaque APOBEC3 families in T cells. Based on six criteria (expression, virion incorporation, HIV restriction, viral genome mutation, neutralization by Vif, and conservation), we found that four APOBEC3 proteins have the potential to restrict HIV replication. To better understand the Vif E3 ligase complex responsible for neutralizing these proteins, we performed extensive purification experiments with HIV Vif and discovered that Vif interacts with the cellular transcription factor Core Binding Factor Beta (CBFB). We discovered that CBFB not only allows for reconstitution of the Vif E3 ligase complex in vitro, but also stabilizes Vif in vivo, subsequently facilitating ligase assembly and allowing for APOBEC3 degradation. This functional interaction is highly conserved, being required to enhance the steady-state levels of Vif proteins from all tested HIV subtypes and required for the degradation of all restrictive human and rhesus APOBEC3 proteins by their respective lentiviral Vif proteins. Mutagenesis screening revealed that CBFB interacts with Vif and its normal RUNX transcription partners on genetically separable interfaces, indicating this essential virus-host interaction may serve as a viable drug target with minimal off-target effects. Disruption of this newly identified and highly conserved CBFB-Vif interaction would release the entire multitude of restrictive APOBEC3 proteins and significantly inhibit HIV infection, making this interaction a promising new target for small molecule therapeutics.
Improved Base Editing Technologies With Novel Editors and Assays
(2018-10) St. Martin, Amber
Genome engineering is a rapidly evolving area of study. One driver of the breakneck speed with which the field is moving forward is the application of CRISPR/Cas9. Since its introduction in 2013, CRISPR/Cas9 has completely changed the ease and utility of genome engineering and has revolutionized the field. The use of CRISPR/Cas9 to directly edit genes, increase or decrease gene expression, or even image genomic loci is widely accepted and extensively used in models ranging from bacteria to mammals. The recent development of second-generation CRISPR editing tools has opened even more doors into how the human genome can be manipulated. Past methods to introduce a single-base substitution into genomes involved creating a double-strand break and taking advantage of the cellular repair pathway homologous recombination to incorporate a donor plasmid into the genomic sequence. These methods are inefficient and can result in introduction of the donor template into numerous unrelated loci throughout the genome, unwanted insertions or deletions, or chromosomal translocations. Base editing unlocks a method to introduce single-base substitutions without the need for a donor template or the creation of a double-strand break. By fusing rat APOBEC1, a natural cytosine deaminase, to Cas9 nickase and uracil DNA glycosylase inhibitor, the Liu lab was the first to create an RNA-guided base editor that can change target cytosines to thymines. In just a year and a half, significant improvements have been made on this front, including making the original base editor more efficient and specific and even introducing editors with the power to mutate adenosines to guanosines. Despite the advancements constantly being made to this technology, there is still room for improvement. The current base editors such as BE3 can edit unintended cytosines in the target sequence at high rates. In addition, the combination of the nickase activity of Cas9 and the abasic site created after deamination of the target cytosine can create a pseudo double-strand break resulting in creation of unwanted insertions or deletions. Finally, targeting at endogenous loci continues to hover between 30% and 80%, depending on the method used and model genome being targeted. Targeting rates could benefit from growth, especially as this technology is being considered for therapeutic applications. A bottleneck in the process of developing new and improved base editing technology is the time and effort that is required to quantify editing efficiencies. Most studies use next-generation sequencing to quantify editing rates. The preparation of samples and quality control required can take up to six weeks. If using a core at a larger university or research institution there is additional time spent waiting in a queue to use a sequencing instrument. The field is in need of a rapid method to quantify base editing in real time that is transferable to multiple cellular systems. Here I report two bicistronic, fluorescence-based systems for the quantification of base editing activity. By changing a 5’-TT-3’ dinucleotide motif to a 5’-TC-3’ dinucleotide motif in eGFP or mCherry, I simultaneously ablate fluorescence and create an APOBEC-preferred mutational hotspot. When the cytosine is reverted back to a thymine, fluorescence is restored. This tight off-to-on system allows for real-time quantification of base editing activity through fluorescence microscopy or flow-cytometry. After creating a novel base editing reporter system, I hypothesized that I could use the newly designed assay to create more efficient and specific base editors. Using members of the human APOBEC3 family of enzymes, I created a suite of novel base editors. These base editors have advantages over rat APOBEC1-based editors in that the structure of many APOBEC3s are known, allowing for easier structure-guided evolution to improve their editing activity. As a proof of concept, I used this knowledge to evolve APOBEC3H haplotype II into a more efficient base editor by making only a few amino acid substitution mutations. In addition, we were able to create base editors using APOBEC3A and the catalytic domain of APOBEC3B that surpass BE3 in editing efficiency. Taken together, these data contribute positively to the genome engineering field and open new doors for continuing development of this technology.
Overlapping functions of APOBEC enzymes in antiviral immunity and cancer
(2017-07) Starrett, Gabriel
APOBEC enzymes are a family of innate antiviral enzymes that form an important barrier against DNA-based pathogens. Encoding and expressing these DNA mutating enzymes, however, is an inherently risky endeavor for the stability of the host genome if not regulated appropriately. These risks have been demonstrated in numerous cancers where APOBEC3B is overexpressed and the APOBEC-associated mutation signature is enriched. Emphasizing the importance of this observation, elevated expression of APOBEC3B and presence of APOBEC-associated mutations has now been consistently linked to aggressive phenotypes and worse outcome in cancer patients. Here I present data demonstrating overlapping functions of APOBEC3 enzymes in antiviral immunity and cancer. In both of these models, APOBEC3 enzymes contribute potentially deleterious and beneficial mutations potentially impacting the survival of tumors and viruses. Additionally, the functions of these enzymes can be modulated by heritable germline mutations in the APOBEC3 locus and viral infections. DNA viruses can also act as valuable molecular probes into the regulation of APOBEC3 enzymes in tumors leading to the development of better therapies.
Regulation of APOBEC3B and the Restriction of HIV-1 in Myeloid Cells
(2018-07) Molan, Amy
The APOBEC3 (A3) DNA cytosine deaminase family comprises a fundamental arm of the innate immune response and is best known for retrovirus restriction. Several A3 enzymes restrict HIV-1 and related retroviruses by deaminating viral cDNA cytosines to uracils compromising viral genomes. Human APOBEC3B (A3B) and APOBEC3G (A3G) show strong virus restriction activities in a variety of experimental systems. A3B and A3G are also subject to tight post-translational regulation evidenced by cell-specific HIV-1 restriction activity of A3B and HIV-1 Vif-mediated degradation of A3G. After observing several potential acetylations on A3B in a mass spectrometry screen, we asked whether lysines and/or lysine post-translational modifications are required for these A3B activities. A lysine-free derivative of human A3B was constructed and shown to be indistinguishable from the wild-type enzyme in DNA cytosine deamination, HIV-1 restriction, and nuclear localization activities. However, lysine loss did render the protein resistant to degradation by SIV Vif. Taken together, we conclude that lysine side chains and modifications thereof are unlikely to be central to A3B function or regulation in human cells. HIV-1 replication in CD4-positive T lymphocytes requires counteraction of multiple different innate antiviral mechanisms. Many studies have combined to demonstrate roles for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H in HIV-1 restriction and mutation in CD4-positive T lymphocytes, whereas the APOBEC enzymes (if any) involved in HIV-1 restriction in macrophages has yet to be delineated. We show that multiple APOBEC3 genes are expressed in myeloid cell lines including THP-1. Vif-deficient HIV-1 produced from THP-1 is less infectious than Vif-proficient virus indicating the presence of at least one functional APOBEC3 enzyme. Proviral DNA resulting from such infections shows strong G-to-A mutation biases in the dinucleotide motif preferred by APOBEC3G. Moreover, Vif mutant viruses selectively sensitive to APOBEC3G show Vif-null virus-like infectivity levels and similarly strong APOBEC3G-biased G-to-A mutation spectra. These studies combine to indicate that APOBEC3G is the main HIV-1 restricting APOBEC3 family member in THP-1 cells. Overall, my thesis research provides new insights into the post-translational regulation of A3B as well as uncovering a novel restriction mechanism in myeloid lineage cells. This research provides a great backbone to build on in understanding how the HIV-1 replication cycle works in myeloid cells as well as contributing to the understanding of the post-translational regulation of A3B.
Studies on the origins of HIV-1 mutation and genetic diversity
(2013-12) Holtz, Colleen Mary
A fundamental biological property of retroviruses and RNA viruses is their ability to rapidly mutate and evolve. The ability of these viruses to generate high levels of genetic diversity during replication has clearly had a profound impact on their ability to maintain their niche in nature, and to rapidly adapt to changing environmental conditions or opportunities to expand their host range. Previous reports with HIV-1 have indicated that the cell type in which HIV-1 replicates does not have a profound impact on HIV-1 diversity. However, due to differences in dNTP pool levels and expression levels of HIV-1 DNA editing enzymes, the hypothesis that cell type does influence the diversity of HIV-1 populations was formulated. To test this, a panel of relevant cell types (i.e., CEM-GFP, U373-MAGI, 293T, and SupT1) was analyzed for their ability to influence HIV-1 mutant rate and spectra. No differences were observed in overall mutation rate, but intriguingly, cell type differences impacted HIV-1 mutation spectra. These observations represent the first description of significant differences in HIV-1 mutation spectra observed in different cell types in the absence of changes in the viral mutation rate and, imply that such differences could have a profound impact on HIV-1 pathogenesis, immune evasion, and drug resistance. The most common mutation type that arises during HIV-1 replication is transition mutations, particularly G-to-A mutations. Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) proteins create G-to-A mutations at specific cytosine dinucleotides. In order to better define the locations of APOBEC3G (A3G)-mediated G-to-A mutations, we tested the hypothesis that sequence context and DNA secondary structure influence the creation of A3G-mediated G-to-A mutations. Single-stranded DNA secondary structure as well as the bases directly 3'and 5' of the cytosine dinucleotide were found to be critically important for A3G recognition. These observations provide the first demonstration that A3G cytosine deamination hotspots are defined by both sequence context and the single-stranded DNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on the generation of HIV-1 diversity, ultimately influencing the biological properties of the progeny virus variants.

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