Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s.

The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy.

Insights into the Structures and Multimeric Status of APOBEC Proteins Involved in Viral Restriction and Other Cellular Functions.

Apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) proteins belong to a family of deaminase proteins that can catalyze the deamination of cytosine to uracil on single-stranded DNA or/and RNA. APOBEC proteins are involved in diverse biological functions, including adaptive and innate immunity, which are critical for restricting viral infection and endogenous retroelements. Dysregulation of their functions can cause undesired genomic mutations and RNA modification, leading to various associated diseases, such as hyper-IgM syndrome and cancer. This review focuses on the structural and biochemical data on the multimerization status of individual APOBECs and the associated functional implications. Many APOBECs form various multimeric complexes, and multimerization is an important way to regulate functions for some of these proteins at several levels, such as deaminase activity, protein stability, subcellular localization, protein storage and activation, virion packaging, and antiviral activity. The multimerization of some APOBECs is more complicated than others, due to the associated complex RNA binding modes.

Elucidation of the Complicated Scenario of Primate APOBEC3 Gene Evolution.

APOBEC3 proteins play pivotal roles in defenses against retroviruses, including HIV-1, as well as retrotransposons. Presumably due to the evolutionary arms race between the hosts and retroelements, APOBEC3 genes have rapidly evolved in primate lineages through sequence diversification, gene amplification and loss, and gene fusion. Consequently, modern primates possess a unique set or "repertoire" of APOBEC3 genes. The APOBEC3 gene repertoire of humans has been well investigated. There are three types of catalytic domains (Z domain; A3Z1, A3Z2, and A3Z3), 11 Z domains, and 7 independent genes, including 4 genes encoding double Z domains. However, the APOBEC3 gene repertoires of nonhuman primates remain largely unclear. Here, we characterize APOBEC3 gene repertoires among primates and investigated the evolutionary scenario of primate APOBEC3 genes using phylogenetic and comparative genomics approaches. In the 21 primate species investigated, we identified 145 APOBEC3 genes, including 69 double-domain type APOBEC3 genes. We further estimated the ages of the respective APOBEC3 genes and revealed that APOBEC3B, APOBEC3D, and APOBEC3F are the youngest in humans and were generated in the common ancestor of Catarrhini. Notably, invasion of the LINE1 retrotransposon peaked during the same period as the generation of these youngest APOBEC3 genes, implying that LINE1 invasion was one of the driving forces of the generation of these genes. Moreover, we found evidence suggesting that sequence diversification by gene conversions among APOBEC3 paralogs occurred in multiple primate lineages. Together, our analyses reveal the hidden diversity and the complicated evolutionary scenario of APOBEC3 genes in primates.IMPORTANCE In terms of virus-host interactions and coevolution, the APOBEC3 gene family is one of the most important subjects in the field of retrovirology. APOBEC3 genes are composed of a repertoire of subclasses based on sequence similarity, and a paper by LaRue et al. provides the standard guideline for the nomenclature and genomic architecture of APOBEC3 genes. However, it has been more than 10 years since this publication, and new information, including RefSeq, which we used in this study, is accumulating. Based on accumulating knowledge, APOBEC3 genes, particularly those of primates, should be refined and reannotated. This study updates knowledge of primate APOBEC3 genes and their genomic architectures. We further inferred the evolutionary scenario of primate APOBEC3 genes and the potential driving forces of APOBEC3 gene evolution. This study will be a landmark for the elucidation of the multiple aspects of APOBEC3 family genes in the future.

Potential APOBEC-mediated RNA editing of the genomes of SARS-CoV-2 and other coronaviruses and its impact on their longer term evolution.

Members of the APOBEC family of cytidine deaminases show antiviral activities in mammalian cells through lethal editing in the genomes of small DNA viruses, herpesviruses and retroviruses, and potentially those of RNA viruses such as coronaviruses. Consistent with the latter, APOBEC-like directional C→U transitions of genomic plus-strand RNA are greatly overrepresented in SARS-CoV-2 genome sequences of variants emerging during the COVID-19 pandemic. A C→U mutational process may leave evolutionary imprints on coronavirus genomes, including extensive homoplasy from editing and reversion at targeted sites and the occurrence of driven amino acid sequence changes in viral proteins. If sustained over longer periods, this process may account for the previously reported marked global depletion of C and excess of U bases in human seasonal coronavirus genomes. This review synthesizes the current knowledge on APOBEC evolution and function and the evidence of their role in APOBEC-mediated genome editing of SARS-CoV-2 and other coronaviruses.

The interesting relationship between APOBEC3 deoxycytidine deaminases and cancer: a long road ahead.

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.

Structural Insights into APOBEC3-Mediated Lentiviral Restriction.

Mammals have developed clever adaptive and innate immune defense mechanisms to protect against invading bacterial and viral pathogens. Human innate immunity is continuously evolving to expand the repertoire of restriction factors and one such family of intrinsic restriction factors is the APOBEC3 (A3) family of cytidine deaminases. The coordinated expression of seven members of the A3 family of cytidine deaminases provides intrinsic immunity against numerous foreign infectious agents and protects the host from exogenous retroviruses and endogenous retroelements. Four members of the A3 proteins-A3G, A3F, A3H, and A3D-restrict HIV-1 in the absence of virion infectivity factor (Vif); their incorporation into progeny virions is a prerequisite for cytidine deaminase-dependent and -independent activities that inhibit viral replication in the host target cell. HIV-1 encodes Vif, an accessory protein that antagonizes A3 proteins by targeting them for polyubiquitination and subsequent proteasomal degradation in the virus producing cells. In this review, we summarize our current understanding of the role of human A3 proteins as barriers against HIV-1 infection, how Vif overcomes their antiviral activity, and highlight recent structural and functional insights into A3-mediated restriction of lentiviruses.

Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors.

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer.

APOBEC3s: DNA-editing human cytidine deaminases.

Nucleic acid editing enzymes are essential components of the human immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins. Among these enzymes are cytidine deaminases of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) super family, each with unique target sequence specificity and subcellular localization. We focus on the DNA-editing APOBEC3 enzymes that have recently attracted attention because of their involvement in cancer and potential in gene-editing applications. We review and compare the crystal structures of APOBEC3 (A3) domains, binding interactions with DNA, substrate specificity, and activity. Recent crystal structures of A3A and A3G bound to ssDNA have provided insights into substrate binding and specificity determinants of these enzymes. Still many unknowns remain regarding potential cooperativity, nucleic acid interactions, and systematic quantification of substrate preference of many APOBEC3s, which are needed to better characterize the biological functions and consequences of misregulation of these gene editors.

The preferred nucleotide contexts of the AID/APOBEC cytidine deaminases have differential effects when mutating retrotransposon and virus sequences compared to host genes.

The AID / APOBEC genes are a family of cytidine deaminases that have evolved in vertebrates, and particularly mammals, to mutate RNA and DNA at distinct preferred nucleotide contexts (or "hotspots") on foreign genomes such as viruses and retrotransposons. These enzymes play a pivotal role in intrinsic immunity defense mechanisms, often deleteriously mutating invading retroviruses or retrotransposons and, in the case of AID, changing antibody sequences to drive affinity maturation. We investigate the strength of various hotspots on their known biological targets by evaluating the potential impact of mutations on the DNA coding sequences of these targets, and compare these results to hypothetical hotspots that did not evolve. We find that the existing AID / APOBEC hotspots have a large impact on retrotransposons and non-mammalian viruses while having a much smaller effect on vital mammalian genes, suggesting co-evolution with AID / APOBECs may have had an impact on the genomes of the viruses we analyzed. We determine that GC content appears to be a significant, but not sole, factor in resistance to deaminase activity. We discuss possible mechanisms AID and APOBEC viral targets have adopted to escape the impacts of deamination activity, including changing the GC content of the genome.

RNA binding to APOBEC deaminases; Not simply a substrate for C to U editing.

Apolipoprotein B mRNA Editing Catalytic Polypeptide-like 1 or APOBEC1 was discovered in 1993 as the zinc-dependent cytidine deaminase responsible for the production of an in frame stop codon in apoB mRNA through modification of cytidine at nucleotide position 6666 to uridine. At the time of this discovery there was much speculation concerning the mechanism of base modification RNA editing which has been rekindled by the discovery of multiple C to U RNA editing events in the 3' UTRs of mRNAs and the finding that other members of the APOBEC family while able to bind RNA, have the biological function of being DNA mutating enzymes. Current research is addressing the mechanism for these nucleotide modification events that appear not to adhere to the mooring sequence-dependent model for APOBEC1 involving the assembly of a multi protein containing editosome. This review will summarize our current understanding of the structure and function of APOBEC proteins and examine how RNA binding to them may be a regulatory mechanism.

The APOBEC Protein Family: United by Structure, Divergent in Function.

The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins have diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss) DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated.