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Science Advances Jan 2023Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral...
Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral infectivity factor (Vif) breaches this host A3G immunity by hijacking a cellular E3 ubiquitin ligase complex to target A3G for ubiquitination and degradation. The molecular mechanism of A3G targeting by Vif-E3 ligase is unknown, limiting the antiviral efforts targeting this host-pathogen interaction crucial for HIV-1 infection. Here, we report the cryo-electron microscopy structures of A3G bound to HIV-1 Vif in complex with T cell transcription cofactor CBF-β and multiple components of the Cullin-5 RING E3 ubiquitin ligase. The structures reveal unexpected RNA-mediated interactions of Vif with A3G primarily through A3G's noncatalytic domain, while A3G's catalytic domain is poised for ubiquitin transfer. These structures elucidate the molecular mechanism by which HIV-1 Vif hijacks the host ubiquitin ligase to specifically target A3G to establish infection and offer structural information for the rational development of antiretroviral therapeutics.
Topics: Humans; Ubiquitin-Protein Ligases; vif Gene Products, Human Immunodeficiency Virus; HIV-1; Cullin Proteins; Cryoelectron Microscopy; HIV Infections; Ubiquitin; Protein Binding; APOBEC-3G Deaminase
PubMed: 36598981
DOI: 10.1126/sciadv.ade3168 -
Journal of Virology Jan 2023Activation-induced cytidine deaminase/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) proteins are cytosine deaminases implicated in diverse...
Activation-induced cytidine deaminase/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) proteins are cytosine deaminases implicated in diverse biological functions. APOBEC1 (A1) proteins have long been thought to regulate lipid metabolism, whereas the evolutionary significance of A1 proteins in antiviral defense remains largely obscure. Endogenous retroviruses (ERVs) document past retroviral infections and are ubiquitous within the vertebrate genomes. Here, we identify the gene repertoire, characterize the A1-mediated mutation footprints in ERVs, and interrogate the evolutionary arms race between genes and ERVs across vertebrate species. We find that genes are widely present in tetrapods, recurrently amplified and lost in certain lineages, suggesting that genes might have originated during the early evolution of tetrapods. A1-mediated mutation footprints can be detected in ERVs across tetrapods. Moreover, genes appear to have experienced episodic positive selection in many tetrapod lineages. Taken together, we propose that a long-running arms race between genes and retroviruses might have persisted throughout the evolutionary course of tetrapods. () genes have been thought to function in defense against retroviruses, whereas the evolutionary significance of A1 proteins in antiviral defense remains largely obscure. In this study, we identify the gene repertoire, characterize the A1-mediated mutation footprints in endogenous retroviruses (ERVs), and explore the evolutionary arms race between genes and ERVs across vertebrate species. We found A1 proteins originated during the early evolution of tetrapods, and detected the footprints of A1-induced hypermutations in retroviral fossils. genes appear to have experienced pervasive positive selection in tetrapods. Our study indicates a long-running arms race between genes and retroviruses taking place throughout the evolutionary course of tetrapods.
Topics: Animals; APOBEC-1 Deaminase; Endogenous Retroviruses; Evolution, Molecular; Mutation; Phylogeny; Retroviridae Infections; Vertebrates
PubMed: 36598198
DOI: 10.1128/jvi.01795-22 -
Chinese Medical Journal Nov 2022Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some...
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.
Topics: Humans; HIV-1; HIV Infections; Virus Replication; Ubiquitin-Specific Proteases; Deubiquitinating Enzymes; APOBEC-3G Deaminase; Cytidine Deaminase
PubMed: 36574218
DOI: 10.1097/CM9.0000000000002478 -
Clinical Cancer Research : An Official... May 2023Over recent years, members of the APOBEC3 family of cytosine deaminases have been implicated in increased cancer genome mutagenesis, thereby contributing to intratumor... (Review)
Review
Over recent years, members of the APOBEC3 family of cytosine deaminases have been implicated in increased cancer genome mutagenesis, thereby contributing to intratumor and intertumor genomic heterogeneity and therapy resistance in, among others, breast cancer. Understanding the available methods for clinical detection of these enzymes, the conditions required for their (dysregulated) expression, the clinical impact they have, and the clinical implications they may offer is crucial in understanding the current impact of APOBEC3-mediated mutagenesis in breast cancer. Here, we provide a comprehensive review of recent developments in the detection of APOBEC3-mediated mutagenesis and responsible APOBEC3 enzymes, summarize the pathways that control their expression, and explore the clinical ramifications and opportunities they pose. We propose that APOBEC3-mediated mutagenesis can function as a helpful predictive biomarker in several standard-of-care breast cancer treatment plans and may be a novel target for treatment.
Topics: Humans; Female; Breast Neoplasms; Mutagenesis; Cytosine Deaminase; Genome; Cytidine Deaminase; APOBEC Deaminases
PubMed: 36478188
DOI: 10.1158/1078-0432.CCR-22-2861 -
ACS Chemical Biology Dec 2022Nucleic acid structure plays a critical role in governing the selectivity of DNA- and RNA-modifying enzymes. In the case of the APOBEC3 family of cytidine deaminases,...
Nucleic acid structure plays a critical role in governing the selectivity of DNA- and RNA-modifying enzymes. In the case of the APOBEC3 family of cytidine deaminases, these enzymes catalyze the conversion of cytosine (C) to uracil (U) in single-stranded DNA, primarily in the context of innate immunity. DNA deamination can also have pathological consequences, accelerating the evolution of viral genomes or, when the host genome is targeted by either APOBEC3A (A3A) or APOBEC3B (A3B), promoting tumor evolution leading to worse patient prognosis and chemotherapeutic resistance. For A3A, nucleic acid secondary structure has emerged as a critical determinant of substrate targeting, with a predilection for DNA that can form stem loop hairpins. Here, we report the development of a specific nanomolar-level, nucleic acid-based inhibitor of A3A. Our strategy relies on embedding the nucleobase 5-methylzebularine, a mechanism-based inhibitor, into a DNA dumbbell structure, which mimics the ideal substrate secondary structure for A3A. Structure-activity relationship studies using a panel of diverse inhibitors reveal a critical role for the stem and position of the inhibitor moiety in achieving potent inhibition. Moreover, we demonstrate that DNA dumbbell inhibitors, but not nonstructured inhibitors, show specificity against A3A relative to the closely related catalytic domain of A3B. Overall, our work demonstrates the feasibility of leveraging secondary structural preferences in inhibitor design, offering a blueprint for further development of modulators of DNA-modifying enzymes and potential therapeutics to circumvent APOBEC-driven viral and tumor evolution.
Topics: Humans; Cytidine Deaminase; DNA, Single-Stranded; Structure-Activity Relationship
PubMed: 36475588
DOI: 10.1021/acschembio.2c00796 -
Nature Communications Dec 2022An essential step in restricting HIV infectivity by the antiviral factor APOBEC3G is its incorporation into progeny virions via binding to HIV RNA. However, the...
An essential step in restricting HIV infectivity by the antiviral factor APOBEC3G is its incorporation into progeny virions via binding to HIV RNA. However, the mechanism of APOBEC3G capturing viral RNA is unknown. Here, we report crystal structures of a primate APOBEC3G bound to different types of RNAs, revealing that APOBEC3G specifically recognizes unpaired 5'-AA-3' dinucleotides, and to a lesser extent, 5'-GA-3' dinucleotides. APOBEC3G binds to the common 3'A in the AA/GA motifs using an aromatic/hydrophobic pocket in the non-catalytic domain. It binds to the 5'A or 5'G in the AA/GA motifs using an aromatic/hydrophobic groove conformed between the non-catalytic and catalytic domains. APOBEC3G RNA binding property is distinct from that of the HIV nucleocapsid protein recognizing unpaired guanosines. Our findings suggest that the sequence-specific RNA recognition is critical for APOBEC3G virion packaging and restricting HIV infectivity.
Topics: Animals; APOBEC-3G Deaminase; Cytidine Deaminase; HIV-1; Antiviral Agents; Nucleoside Deaminases; Virion; RNA, Viral; HIV Infections
PubMed: 36470880
DOI: 10.1038/s41467-022-35201-9 -
Nucleic Acids Research Nov 2022The human APOBEC family of eleven cytosine deaminases use RNA and single-stranded DNA (ssDNA) as substrates to deaminate cytosine to uracil. This deamination event has...
The human APOBEC family of eleven cytosine deaminases use RNA and single-stranded DNA (ssDNA) as substrates to deaminate cytosine to uracil. This deamination event has roles in lipid metabolism by altering mRNA coding, adaptive immunity by causing evolution of antibody genes, and innate immunity through inactivation of viral genomes. These benefits come at a cost where some family members, primarily from the APOBEC3 subfamily (APOBEC3A-H, excluding E), can cause off-target deaminations of cytosine to form uracil on transiently single-stranded genomic DNA, which induces mutations that are associated with cancer evolution. Since uracil is only promutagenic, the mutations observed in cancer genomes originate only when uracil is not removed by uracil DNA glycosylase (UNG) or when the UNG-induced abasic site is erroneously repaired. However, when ssDNA is present, replication protein A (RPA) binds and protects the DNA from nucleases or recruits DNA repair proteins, such as UNG. Thus, APOBEC enzymes must compete with RPA to access their substrate. Certain APOBEC enzymes can displace RPA, bind and scan ssDNA efficiently to search for cytosines, and can become highly overexpressed in tumor cells. Depending on the DNA replication conditions and DNA structure, RPA can either be in excess or deficient. Here we discuss the interplay between these factors and how despite RPA, multiple cancer genomes have a mutation bias at cytosines indicative of APOBEC activity.
Topics: Humans; Replication Protein A; DNA, Single-Stranded; Cytidine Deaminase; Uracil-DNA Glycosidase; DNA Replication; Cytosine; DNA; Uracil; APOBEC Deaminases; Deamination
PubMed: 36444883
DOI: 10.1093/nar/gkac1121 -
Cells Nov 2022RNA editing is an epitranscriptomic modification, leading to targeted changes in RNA transcripts. It is mediated by the action of ADAR (adenosine deaminases acting on...
RNA editing is an epitranscriptomic modification, leading to targeted changes in RNA transcripts. It is mediated by the action of ADAR (adenosine deaminases acting on double-stranded (ds) RNA and APOBEC (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) deaminases and appears to play a major role in the pathogenesis of many diseases. Here, we assessed its role in experimental autoimmune encephalomyelitis (EAE), a widely used non-clinical model of autoimmune inflammatory diseases of the central nervous system (CNS), which resembles many aspects of human multiple sclerosis (MS). We have analyzed in silico data from microglia isolated at different timepoints through disease progression to identify the global editing events and validated the selected targets in murine tissue samples. To further evaluate the functional role of RNA editing, we induced EAE in transgenic animals lacking expression of APOBEC-1. We found that RNA-editing events, mediated by the APOBEC and ADAR deaminases, are significantly reduced throughout the course of disease, possibly affecting the protein expression necessary for normal neurological function. Moreover, the severity of the EAE model was significantly higher in APOBEC-1 knock-out mice, compared to wild-type controls. Our results implicate regulatory epitranscriptomic mechanisms in EAE pathogenesis that could be extrapolated to MS and other neurodegenerative disorders (NDs) with common clinical and molecular features.
Topics: Humans; Mice; Animals; RNA Editing; APOBEC-1 Deaminase; Encephalomyelitis, Autoimmune, Experimental; RNA, Double-Stranded; Mutagenesis, Site-Directed; Mice, Knockout
PubMed: 36429012
DOI: 10.3390/cells11223582 -
Nature Communications Nov 2022APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine to uridine in single-stranded DNA (ssDNA) substrates, thus playing a key role in innate...
APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine to uridine in single-stranded DNA (ssDNA) substrates, thus playing a key role in innate antiviral immunity. However, the APOBEC3 family has also been linked to many mutational signatures in cancer cells, which has led to an intense interest to develop inhibitors of A3's catalytic activity as therapeutics as well as tools to study A3's biochemistry, structure, and cellular function. Recent studies have shown that ssDNA containing 2'-deoxy-zebularine (dZ-ssDNA) is an inhibitor of A3s such as A3A, A3B, and A3G, although the atomic determinants of this activity have remained unknown. To fill this knowledge gap, we determined a 1.5 Å resolution structure of a dZ-ssDNA inhibitor bound to active A3G. The crystal structure revealed that the activated dZ-HO mimics the transition state by coordinating the active site Zn and engaging in additional stabilizing interactions, such as the one with the catalytic residue E259. Therefore, this structure allowed us to capture a snapshot of the A3's transition state and suggests that developing transition-state mimicking inhibitors may provide a new opportunity to design more targeted molecules for A3s in the future.
Topics: APOBEC-3G Deaminase; Oligonucleotides; Cytidine Deaminase; DNA, Single-Stranded; Catalytic Domain
PubMed: 36402773
DOI: 10.1038/s41467-022-34752-1 -
Journal of Global Antimicrobial... Dec 2022As 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...
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.
Topics: Humans; Antiviral Agents; APOBEC-3G Deaminase; Cytidine Deaminase; Hepatitis B; Hepatitis B virus; Nucleocapsid; Virus Replication
PubMed: 36396043
DOI: 10.1016/j.jgar.2022.11.006