The On-Going Conflict Between APOBEC3 Immune Factors and HIV-1 Vif

CIHR/IRSC, NSERC/CRSN

APOBEC3D excludes APOBEC3F from HIV-1 virions by competitive binding of RNA

The human family of APOBEC3 enzymes are primarily studied as single-stranded DNA deoxycytidine deaminases that act as host restriction factors for a number of viruses and retroelements. The deamination of deoxycytidine to deoxyuridine causes inactivating mutations in target DNA and the nucleic acid binding ability may also cause deamination independent restriction. There are seven APOBEC3 enzymes in humans, named A-H, excluding E, each of which has restriction activity against a subset of viruses or retroelements. There are primarily four, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H that have been found to restrict replication of HIV-1, however their restriction activity varies and they have primarily been studied individually despite co-expression in the cells that HIV-1 infects. It is known that APOBEC3F hetero-oligomerizes with APOBEC3G and APOBEC3H and that this influences host restriction outcomes during HIV-1 infection in tissue culture. Here, we examined if APOBEC3F interacts with APOBEC3D and the functional outcomes. We found that APOBEC3D mRNA expression was similar to or higher than APOBEC3F mRNA in multiple donors, suggesting that the proteins would be co-expressed, allowing for interactions to occur. We determined that APOBEC3F and APOBEC3D interacted primarily through an RNA intermediate; however, this interaction resulted in APOBEC3D competitively excluding APOBEC3F from virions. Although HIV-1 restriction still occurred when APOBEC3F and APOBEC3D were co-expressed, it was due to primarily APOBEC3D-mediated deamination-independent restriction. The APOBEC3D-mediated exclusion of APOBEC3F from HIV-1 encapsidation could be recapitulated in vitro through RNA capture experiments in which APOBEC3D decreased or abrogated the ability of APOBEC3F to bind to HIV-1 protease or 5’UTR RNA, respectively. Overall, the data suggest that there are mechanisms at the protein level that segregate APOBEC3s into different virus particles

Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.fals

Atrial fibrillation is an independent predictor for in-hospital mortality in patients admitted with SARS-CoV-2 infection.

Background Atrial fibrillation (AF) is the most encountered arrhythmia and has been associated with worse in-hospital outcomes. Objective This study was to determine the incidence of AF in patients hospitalized with coronavirus disease 2019 (COVID-19) as well as its impact on in-hospital mortality. Methods Patients hospitalized with a positive COVID-19 polymerase chain reaction test between March 1 and April 27, 2020, were identified from the common medical record system of 13 Northwell Health hospitals. Natural language processing search algorithms were used to identify and classify AF. Patients were classified as having AF or not. AF was further classified as new-onset AF vs history of AF. Results AF occurred in 1687 of 9564 patients (17.6%). Of those, 1109 patients (65.7%) had new-onset AF. Propensity score matching of 1238 pairs of patients with AF and without AF showed higher in-hospital mortality in the AF group (54.3% vs 37.2%; P \u3c .0001). Within the AF group, propensity score matching of 500 pairs showed higher in-hospital mortality in patients with new-onset AF as compared with those with a history of AF (55.2% vs 46.8%; P = .009). The risk ratio of in-hospital mortality for new-onset AF in patients with sinus rhythm was 1.56 (95% confidence interval 1.42-1.71; P \u3c .0001). The presence of cardiac disease was not associated with a higher risk of in-hospital mortality in patients with AF (P = .1). Conclusion In patients hospitalized with COVID-19, 17.6% experienced AF. AF, particularly new-onset, was an independent predictor of in-hospital mortality

Effect of a Behavioral Nudge on Adoption of an Electronic Health Record-Agnostic Pulmonary Embolism Risk Prediction Tool: A Pilot Cluster Nonrandomized Controlled Trial

OBJECTIVE: Our objective was to determine the feasibility and preliminary efficacy of a behavioral nudge on adoption of a clinical decision support (CDS) tool. MATERIALS AND METHODS: We conducted a pilot cluster nonrandomized controlled trial in 2 Emergency Departments (EDs) at a large academic healthcare system in the New York metropolitan area. We tested 2 versions of a CDS tool for pulmonary embolism (PE) risk assessment developed on a web-based electronic health record-agnostic platform. One version included behavioral nudges incorporated into the user interface. RESULTS: A total of 1527 patient encounters were included in the trial. The CDS tool adoption rate was 31.67%. Adoption was significantly higher for the tool that included behavioral nudges (39.11% vs 20.66%; DISCUSSION: We demonstrated feasibility and preliminary efficacy of a PE risk prediction CDS tool developed using insights from behavioral science. The tool is well-positioned to be tested in a large randomized clinical trial

Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA

deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. on long ssDNA regions. This resembles the “hit and run” single base substitution events observed in yeast., we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance

Twin gradients in APOBEC3 edited HIV-1 DNA reflect the dynamics of lentiviral replication

The human immunodeficiency virus (HIV) Vif protein blocks incorporation of two host cell cytidine deaminases, APOBEC3F and 3G, into the budding virion. Not surprisingly, on a vif background nascent minus strand DNA can be extensively edited leaving multiple uracil residues. Editing occurs preferentially in the context of TC (GA on the plus strand) and CC (GG) depending on the enzyme. To explore the distribution of APOBEC3F and –3G editing across the genome, a product/substrate ratio (AA + AG)/(GA + GG) was computed for a series of 30 edited genomes present in the data bases. Two highly polarized gradients were noted each with maxima just 5′ to the central polypurine tract (cPPT) and LTR proximal polypurine tract (3′PPT). The gradients are in remarkable agreement with the time the minus strand DNA remains single stranded. In vitro analyses of APOBEC3G deamination of nascent cDNA spanning the two PPTs showed no pronounced dependence on the PPT RNA:DNA heteroduplex ruling out the competing hypothesis of a PPT orientation effect. The degree of hypermutation varied smoothly among genomes indicating that the number of APOBEC3 molecules packaged varied considerably

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein–DNA Complexes That Carry Out DNA Repair

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein–protein and protein–DNA interactions. It can be used to capture snapshots of protein–DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein–protein and protein–DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein–protein and protein–DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein–DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein–protein and protein–nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure–function relationships that govern DNA repair processes

Histone H2A and H2B Are Monoubiquitinated at AID-Targeted Loci

Background: Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined. Methodology/Principal Findings: Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt\u27s B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed V(H) and S gamma 3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci. Conclusions/Significance: Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation

Evolution of the Primate APOBEC3A Cytidine Deaminase Gene and Identification of Related Coding Regions

The APOBEC3 gene cluster encodes six cytidine deaminases (A3A-C, A3DE, A3F-H) with single stranded DNA (ssDNA) substrate specificity. For the moment A3A is the only enzyme that can initiate catabolism of both mitochondrial and nuclear DNA. Human A3A expression is initiated from two different methionine codons M1 or M13, both of which are in adequate but sub-optimal Kozak environments. In the present study, we have analyzed the genetic diversity among A3A genes across a wide range of 12 primates including New World monkeys, Old World monkeys and Hominids. Sequence variation was observed in exons 1–4 in all primates with up to 31% overall amino acid variation. Importantly for 3 hominids codon M1 was mutated to a threonine codon or valine codon, while for 5/12 primates strong Kozak M1 or M13 codons were found. Positive selection was apparent along a few branches which differed compared to positive selection in the carboxy-terminal of A3G that clusters with A3A among human cytidine deaminases. In the course of analyses, two novel non-functional A3A-related fragments were identified on chromosome 4 and 8 kb upstream of the A3 locus. This qualitative and quantitative variation among primate A3A genes suggest that subtle differences in function might ensue as more light is shed on this increasingly important enzyme