-
MBio Aug 2023HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4 T lymphocytes and macrophages. Previous studies have demonstrated that the apolipoprotein B...
HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4 T lymphocytes and macrophages. Previous studies have demonstrated that the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) family of proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1 restriction in CD4 T lymphocytes. Virus-encoded virion infectivity factor (Vif) counteracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4 T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades only A3 proteins during viral replication is currently unknown. Herein, we describe the development and characterization of -, -, and -to--null THP-1 cells. In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced infectivity in parental and -null THP-1 cells, and a more modest decrease in infectivity in -null cells. Remarkably, disruption of A3A-A3G protein expression completely restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate that the primary function of Vif during infectious HIV-1 production from THP-1 cells is the targeting and degradation of A3 enzymes. IMPORTANCE HIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However, it is currently unclear whether Vif has additional essential cellular targets. To address this question, we disrupted 3 to 3 genes in the THP-1 myeloid cell line using CRISPR and compared the infectivity of wild-type HIV-1 and Vif mutants with the selective A3 neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1 and the other Vif mutants is fully restored by ablating the expression of cellular A3A to A3G proteins. These results indicate that A3 proteins are the only essential target of Vif that is required for fully infectious HIV-1 production from THP-1 cells.
Topics: Humans; HIV-1; Cytidine Deaminase; vif Gene Products, Human Immunodeficiency Virus; Protein Binding; APOBEC-3G Deaminase; Cytosine Deaminase; Cell Line; HIV Infections; Myeloid Cells; Virion; APOBEC Deaminases
PubMed: 37555667
DOI: 10.1128/mbio.00782-23 -
Frontiers in Immunology 2024The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition.... (Review)
Review
The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition. Secreted novel AID/APOBEC-like deaminases (SNADs), characterized by the presence of a signal peptide are unique among all of intracellular classical AID/APOBECs, which are the central part of antibody diversity and antiviral defense. To date, there is no available knowledge on SNADs including protein characterization, biochemical characteristics and catalytic activity. We used various approaches to define the phylogeny of SNADs, their common structural features, and their potential structural variations in fish species. Our analysis provides strong evidence of the universal presence of SNAD1 proteins/transcripts in fish, in which expression commences after hatching and is highest in anatomical organs linked to the immune system. Moreover, we searched published fish data and identified previously, "uncharacterized proteins" and transcripts as SNAD1 sequences. Our review into immunological research suggests SNAD1 role in immune response to infection or immunization, and interactions with the intestinal microbiota. We also noted SNAD1 association with temperature acclimation, environmental pollution and sex-based expression differences, with females showing higher level. To validate predictions we performed expression studies of several SNAD1 gene variants in carp, which revealed distinct patterns of responses under different conditions. Dual sensitivity to environmental and pathogenic stress highlights its importance in the fish and potentially enhancing thermotolerance and immune defense. Revealing the biological roles of SNADs represents an exciting new area of research related to the role of DNA and/or RNA editing in fish biology.
Topics: Animals; APOBEC-1 Deaminase; Cytidine Deaminase; DNA; Nucleic Acids; Cytidine
PubMed: 38601149
DOI: 10.3389/fimmu.2024.1340273 -
The Journal of Biological Chemistry May 2024Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a...
Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.
PubMed: 38796062
DOI: 10.1016/j.jbc.2024.107410 -
Rheumatology (Oxford, England) May 2024
Topics: Humans; Autoimmune Diseases; Cytidine Deaminase; APOBEC Deaminases
PubMed: 38060252
DOI: 10.1093/rheumatology/kead663 -
Nature Jun 2024Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases. Most mutations begin as nucleotide mismatches or damage...
Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.
Topics: Humans; DNA Damage; DNA Mismatch Repair; Deamination; Neoplasms; Mutation; Sequence Analysis, DNA; Cytidine Deaminase; Base Pair Mismatch; Cytosine; Single Molecule Imaging; APOBEC Deaminases; DNA, Single-Stranded; DNA Replication; Proteins
PubMed: 38867045
DOI: 10.1038/s41586-024-07532-8 -
BMC Pulmonary Medicine Dec 2023Small cell transformation was one mechanism by which EGFR-mutation NSCLC acquired resistance after tyrosine kinase inhibitors (TKIs) treatment. A few reports of small...
Small cell transformation was one mechanism by which EGFR-mutation NSCLC acquired resistance after tyrosine kinase inhibitors (TKIs) treatment. A few reports of small cell transformation occurred in other oncogene-driven lung cancers. We found the first case of transformation of a RET-rearranged lung adenocarcinoma to SCLC after selpercatinib, a novel highly selective RET TKIs. Whole-exome sequencing (WES) was used to explore alteration in gene expression in tumor tissue at initial diagnosis and after transformation into small cell carcinoma. We found that transformed into SCLC tumor tissue had inactivation of RB1 and TP53, with RET fusion was still present. In addition, the APOBEC family of cytidine deaminases appeared amplification. Although RET rearrangement still existed, using another RET TKIs was ineffective, and etoposide plus platinum might be an effective rescue treatment.
Topics: Humans; Lung Neoplasms; Exome Sequencing; ErbB Receptors; Protein Kinase Inhibitors; Small Cell Lung Carcinoma; Adenocarcinoma of Lung; Mutation; Proto-Oncogene Proteins c-ret
PubMed: 38057798
DOI: 10.1186/s12890-023-02799-5 -
Leukemia May 2024[Image: see text]
[Image: see text]
Topics: Multiple Myeloma; Humans; Inflammation; APOBEC Deaminases; Biomarkers, Tumor; Prognosis
PubMed: 38461190
DOI: 10.1038/s41375-024-02210-0 -
RNA Biology Jan 2024In mammals, RNA editing events involve the conversion of adenosine (A) in inosine (I) by ADAR enzymes or the hydrolytic deamination of cytosine (C) in uracil (U) by the...
In mammals, RNA editing events involve the conversion of adenosine (A) in inosine (I) by ADAR enzymes or the hydrolytic deamination of cytosine (C) in uracil (U) by the APOBEC family of enzymes, mostly APOBEC1. RNA editing has a plethora of biological functions, and its deregulation has been associated with various human disorders. While the large-scale detection of A-to-I is quite straightforward using the Illumina RNAseq technology, the identification of C-to-U events is a non-trivial task. This difficulty arises from the rarity of such events in eukaryotic genomes and the challenge of distinguishing them from background noise. Direct RNA sequencing by Oxford Nanopore Technology (ONT) permits the direct detection of Us on sequenced RNA reads. Surprisingly, using ONT reads from wild-type (WT) and APOBEC1-knock-out (KO) murine cell lines as well as in vitro synthesized RNA without any modification, we identified a systematic error affecting the accuracy of the Cs call, thereby leading to incorrect identifications of C-to-U events. To overcome this issue in direct RNA reads, here we introduce a novel machine learning strategy based on the isolation Forest (iForest) algorithm in which C-to-U editing events are considered as sequencing anomalies. Using in vitro synthesized and human ONT reads, our model optimizes the signal-to-noise ratio improving the detection of C-to-U editing sites with high accuracy, over 90% in all samples tested. Our results suggest that iForest, known for its rapid implementation and minimal memory requirements, is a promising tool to denoise ONT reads and reliably identify RNA modifications.
Topics: Mice; Animals; Humans; RNA; RNA Editing; Base Sequence; APOBEC Deaminases; Mammals; Sequence Analysis, RNA
PubMed: 38090878
DOI: 10.1080/15476286.2023.2290843 -
BioRxiv : the Preprint Server For... Nov 2023Replication of the complex retrovirus mouse mammary tumor virus (MMTV) is antagonized by murine Apobec3 (mA3), a member of the Apobec family of cytidine deaminases. We...
Replication of the complex retrovirus mouse mammary tumor virus (MMTV) is antagonized by murine Apobec3 (mA3), a member of the Apobec family of cytidine deaminases. We have shown that MMTV-encoded Rem protein inhibits proviral mutagenesis by the Apobec enzyme, activation-induced cytidine deaminase (AID) during viral replication in BALB/c mice. To further study the role of Rem , we have infected C57BL/6 (B6) mice with a superantigen-independent lymphomagenic strain of MMTV (TBLV-WT) or a mutant strain (TBLV-SD) that is defective in Rem and its cleavage product Rem-CT. Unlike MMTV, TBLV induced T-cell tumors in µMT mice, indicating that mature B cells, which express the highest AID levels, are not required for TBLV replication. Compared to BALB/c, B6 mice were more susceptible to TBLV infection and tumorigenesis. The lack of Rem expression accelerated B6 tumorigenesis at limiting doses compared to TBLV-WT in either wild-type B6 or AID-deficient mice. However, unlike proviruses from BALB/c mice, high-throughput sequencing indicated that proviral G-to-A or C-to-T changes did not significantly differ in the presence and absence of Rem expression. stimulation showed higher levels of mA3 relative to AID in B6 compared to BALB/c splenocytes, but effects of agonists differed in the two strains. RNA-Seq revealed increased transcripts related to growth factor and cytokine signaling in TBLV-SD-induced tumors relative to those from TBLV-WT, consistent with a third Rem function. Thus, Rem-mediated effects on tumorigenesis in B6 mice are independent of Apobec-mediated proviral hypermutation.
PubMed: 37961113
DOI: 10.1101/2023.11.02.565355 -
Methods in Cell Biology 2024The APOBEC3 family of cytosine deaminases, which target single-stranded DNA and RNA of viruses and retroelements as part of the innate immune defense, generate mutations...
The APOBEC3 family of cytosine deaminases, which target single-stranded DNA and RNA of viruses and retroelements as part of the innate immune defense, generate mutations in many human cancers. Although the APOBEC3A paralog is a major endogenous source of these mutations, low APOBEC3A mRNA levels and protein abundance have hampered functional characterization. Extensive homology across APOBEC3 paralogs have further challenged the development of specific detection reagents. Here, we describe the isolation and use of monoclonal antibodies with specificity for APOBEC3A and the APOBEC3A/APOBEC3B/APOBEC3G proteins. We provide protocols and technical advice for detection and measurement of APOBEC3A protein across human cancer cell lines using standard immunoblotting and immunofluorescence protocols.
Topics: Humans; Proteins; Neoplasms; Cell Line; Mutation; Cytidine Deaminase; Minor Histocompatibility Antigens
PubMed: 38359985
DOI: 10.1016/bs.mcb.2022.10.008