Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain.

APOBEC3G (A3G) belongs to the AID/APOBEC cytidine deaminase family and is essential for antiviral immunity. It contains two zinc-coordinated cytidine-deaminase (CD) domains. The N-terminal CD1 domain is non-catalytic but has a strong affinity for nucleic acids, whereas the C-terminal CD2 domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains in DNA binding and editing is not fully understood. Here, our studies on rhesus macaque A3G (rA3G) show that the DNA editing function in linear and hairpin loop DNA is greatly enhanced by AA or GA dinucleotide motifs present downstream (in the 3'-direction) but not upstream (in the 5'-direction) of the target-C editing sites. The effective distance between AA/GA and the target-C sites depends on the local DNA secondary structure. We present two co-crystal structures of rA3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic CD1 domain in capturing AA/GA DNA and explaining our biochemical observations. Our structural and biochemical findings elucidate the molecular mechanism underlying the cooperative function between the non-catalytic and the catalytic domains of A3G, which is critical for its antiviral role and its contribution to genome mutations in cancer.

Structural basis for polyuridine tract recognition by SARS-CoV-2 Nsp15.

SARS-CoV-2 non-structural protein 15 (Nsp15) is critical for productive viral replication and evasion of host immunity. The uridine-specific endoribonuclease activity of Nsp15 mediates the cleavage of the polyuridine [poly(U)] tract of the negative-strand coronavirus genome to minimize the formation of dsRNA that activates the host antiviral interferon signaling. However, the molecular basis for the recognition and cleavage of the poly(U) tract by Nsp15 is incompletely understood. Here, we present cryogenic electron microscopy (cryoEM) structures of SARS-CoV-2 Nsp15 bound to viral replication intermediate dsRNA containing poly(U) tract at 2.7-3.3 Å resolution. The structures reveal one copy of dsRNA binds to the sidewall of an Nsp15 homohexamer, spanning three subunits in two distinct binding states. The target uracil is dislodged from the base-pairing of the dsRNA by amino acid residues W332 and M330 of Nsp15, and the dislodged base is entrapped at the endonuclease active site center. Up to 20 A/U base pairs are anchored on the Nsp15 hexamer, which explains the basis for a substantially shortened poly(U) sequence in the negative strand coronavirus genome compared to the long poly(A) tail in its positive strand. Our results provide mechanistic insights into the unique immune evasion strategy employed by coronavirus Nsp15.

Alkyne as a Latent Warhead to Covalently Target SARS-CoV-2 Main Protease.

There is an urgent need for improved therapy to better control the ongoing COVID-19 pandemic. The main protease Mpro plays a pivotal role in SARS-CoV-2 replications, thereby representing an attractive target for antiviral development. We seek to identify novel electrophilic warheads for efficient, covalent inhibition of Mpro. By comparing the efficacy of a panel of warheads installed on a common scaffold against Mpro, we discovered that the terminal alkyne could covalently modify Mpro as a latent warhead. Our biochemical and X-ray structural analyses revealed the irreversible formation of the vinyl-sulfide linkage between the alkyne and the catalytic cysteine of Mpro. Clickable probes based on the alkyne inhibitors were developed to measure target engagement, drug residence time, and off-target effects. The best alkyne-containing inhibitors potently inhibited SARS-CoV-2 infection in cell infection models. Our findings highlight great potentials of alkyne as a latent warhead to target cystine proteases in viruses and beyond.

Structural basis for HIV-1 antagonism of host APOBEC3G via Cullin E3 ligase.

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-ß 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.

Structural basis of sequence-specific RNA recognition by the antiviral factor APOBEC3G.

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.

Understanding the structural basis of HIV-1 restriction by the full length double-domain APOBEC3G.

APOBEC3G, a member of the double-domain cytidine deaminase (CD) APOBEC, binds RNA to package into virions and restrict HIV-1 through deamination-dependent or deamination-independent inhibition. Mainly due to lack of a full-length double-domain APOBEC structure, it is unknown how CD1/CD2 domains connect and how dimerization/multimerization is linked to RNA binding and virion packaging for HIV-1 restriction. We report rhesus macaque A3G structures that show different inter-domain packing through a short linker and refolding of CD2. The A3G dimer structure has a hydrophobic dimer-interface matching with that of the previously reported CD1 structure. A3G dimerization generates a surface with intensified positive electrostatic potentials (PEP) for RNA binding and dimer stabilization. Unexpectedly, mutating the PEP surface and the hydrophobic interface of A3G does not abolish virion packaging and HIV-1 restriction. The data support a model in which only one RNA-binding mode is critical for virion packaging and restriction of HIV-1 by A3G.

Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H.

APOBEC3H (A3H) is a member of the APOBEC3 subfamily of DNA cytosine deaminases that are important for innate immune defense and have been implicated in cancer biogenesis. To understand the structural basis for A3H biochemical function, we determined a high-resolution structure of human A3H and performed extensive biochemical analysis. The 2.49 Å crystal structure reveals a uniquely long C-terminal helix 6 (h6), a disrupted ß5 strand of the canonical five-stranded ß-sheet core, and a long loop 1 around the Zn-active center. Mutation of a loop 7 residue, W115, disrupted the RNA-mediated dimerization of A3H yielding an RNA-free monomeric form that still possessed nucleic acid binding and deaminase activity. A3H expressed in HEK293T cells showed RNA dependent HMW complex formation and RNase A-dependent deaminase activity. A3H has a highly positively charged surface surrounding the Zn-active center, and multiple positively charged residues within this charged surface play an important role in the RNA-mediated HMW formation and deaminase inhibition. Furthermore, these positively charged residues affect subcellular localization of A3H between the nucleus and cytosol. Finally, we have identified multiple residues of loop 1 and 7 that contribute to the overall deaminase activity and the methylcytosine selectivity.

Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation.

The catalytic activity of human cytidine deaminase APOBEC3B (A3B) has been correlated with kataegic mutational patterns within multiple cancer types. The molecular basis of how the N-terminal non-catalytic CD1 regulates the catalytic activity and consequently, biological function of A3B remains relatively unknown. Here, we report the crystal structure of a soluble human A3B-CD1 variant and delineate several structural elements of CD1 involved in molecular assembly, nucleic acid interactions and catalytic regulation of A3B. We show that (i) A3B expressed in human cells exists in hypoactive high-molecular-weight (HMW) complexes, which can be activated without apparent dissociation into low-molecular-weight (LMW) species after RNase A treatment. (ii) Multiple surface hydrophobic residues of CD1 mediate the HMW complex assembly and affect the catalytic activity, including one tryptophan residue W127 that likely acts through regulating nucleic acid binding. (iii) One of the highly positively charged surfaces on CD1 is involved in RNA-dependent attenuation of A3B catalysis. (iv) Surface hydrophobic residues of CD1 are involved in heterogeneous nuclear ribonucleoproteins (hnRNPs) binding to A3B. The structural and biochemical insights described here suggest that unique structural features on CD1 regulate the molecular assembly and catalytic activity of A3B through distinct mechanisms.

Family-Wide Comparative Analysis of Cytidine and Methylcytidine Deamination by Eleven Human APOBEC Proteins.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins are a family of cytidine deaminases involved in various important biological processes such as antibody diversification/maturation, restriction of viral infection, and generation of somatic mutations. Catalytically active APOBEC proteins execute their biological functions mostly through deaminating cytosine (C) to uracil on single-stranded DNA/RNA. Activation-induced cytidine deaminase, one of the APOBEC members, was reported to deaminate methylated cytosine (mC) on DNA, and this mC deamination was proposed to be involved in the demethylation of mC for epigenetic regulation. The mC deamination activity is later demonstrated for APOBEC3A (A3A) and more recently for APOBEC3B and APOBEC3H (A3H). Despite extensive studies on APOBEC proteins, questions regarding whether the rest of APOBEC members have any mC deaminase activity and what are the relative deaminase activities for each APOBEC member remain unclear. Here, we performed a family-wide analysis of deaminase activities on C and mC by using purified recombinant proteins for 11 known human APOBEC proteins under similar conditions. Our comprehensive analyses revealed that each APOBEC has unique deaminase activity and selectivity for mC. A3A and A3H showed distinctively high deaminase activities on C and mC with relatively high selectivity for mC, whereas six other APOBEC members showed relatively low deaminase activity and selectivity for mC. Our mutational analysis showed that loop-1 of A3A is responsible for its high deaminase activity and selectivity for mC. These findings extend our understanding of APOBEC family proteins that have important roles in diverse biological functions and in genetic mutations.

Crystal structures of APOBEC3G N-domain alone and its complex with DNA.

APOBEC3G (A3G) is a potent restriction factor of HIV-1. The N-terminal domain of A3G (A3G-CD1) is responsible for oligomerization and nucleic acid binding, both of which are essential for anti-HIV activity. As a countermeasure, HIV-1 viral infectivity factor (Vif) binds A3G-CD1 to mediate A3G degradation. The structural basis for the functions of A3G-CD1 remains elusive. Here, we report the crystal structures of a primate A3G-CD1 (rA3G-CD1) alone and in complex with single-stranded DNA (ssDNA). rA3G-CD1 shares a conserved core structure with the previously determined catalytic APOBECs, but displays unique features for surface charge, dimerization and nucleic acid binding. Its co-crystal structure with ssDNA reveals how the conformations of loops and residues surrounding the Zn-coordinated centre (Zn-centre) change upon DNA binding. The dimerization interface of rA3G-CD1 is important for oligomerization, nucleic acid binding and Vif-mediated degradation. These findings elucidate the molecular basis of antiviral mechanism and HIV-Vif targeting of A3G.

DNA cytosine and methylcytosine deamination by APOBEC3B: enhancing methylcytosine deamination by engineering APOBEC3B.

APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) is a family of enzymes that deaminates cytosine (C) to uracil (U) on nucleic acid. APOBEC3B (A3B) functions in innate immunity against intrinsic and invading retroelements and viruses. A3B can also induce genomic DNA mutations to cause cancer. A3B contains two cytosine deaminase domains (CD1, CD2), and there are conflicting reports about whether both domains are active. Here we demonstrate that only CD2 of A3B (A3BCD2) has C deamination activity. We also reveal that both A3B and A3BCD2 can deaminate methylcytosine (mC). Guided by structural and functional analysis, we successfully engineered A3BCD2 to gain over two orders of magnitude higher activity for mC deamination. Important determinants that contribute to the activity and selectivity for mC deamination have been identified, which reveals that multiple elements, rather than single ones, contribute to the mC deamination activity and selectivity in A3BCD2 and possibly other APOBECs.

Evidence that YycJ is a novel 5'-3' double-stranded DNA exonuclease acting in Bacillus anthracis mismatch repair.

The most important system for correcting replication errors that survive the built in editing system of DNA polymerase is the mismatch repair (MMR) system. We have identified a novel mutator strain yycJ in Bacillus anthracis. Mutations in the yycJ gene result in a spontaneous mutator phenotype with a mutational frequency and specificity comparable to that of MMR-deficient strains such as those with mutations in mutL or mutS. YycJ was annotated as a metallo-ß-lactamase (MßL) super family member with unknown activity. In this study we carried out a biochemical characterization of YycJ and demonstrated that a recombinant YycJ protein possesses a 5'-3' exonuclease activity at the 5' termini and at nicks of double-stranded DNA. This activity requires a divalent metal cofactor Mn2+ and is stimulated by 5'-phosphate ends of duplex DNA. The mutagenesis of conserved amino acid residues revealed that in addition to the five MßL family conserved motifs, YycJ appears to have its specific motifs that can be used to distinguish YycJ from other closely related MßL family members. A phylogenetic survey showed that putative YycJ homologs are present in several bacterial phyla as well as in members of the Methanomicrobiales and Thermoplasmales from Archaea. We propose that YycJ represents a new group of MßL fold exonucleases, which is likely to act in the recognition of MMR entry point and subsequent removal of the mismatched base in certain MutH-less bacterial species.

The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

The role of Bacillus anthracis RecD2 helicase in DNA mismatch repair.

DNA mismatch repair (MMR) systems can be classified as either MutH-dependent or MutH-independent. In bacteria, extensive studies have been conducted with the MutH-dependent MMR in Escherichia coli and its close relatives. The picture of MutH-independent MMR in other bacteria is less clear, as MMR components other than MutS and MutL have not been identified in the majority of bacteria. Bacillus anthracis is one of the MutH-less Gram(+) bacteria in the phylum of Firmicutes. We used papillation as a tool to search for B. anthracis new mutator strains and identified a spontaneous mutator that carries a minitransposon insertion in the BAS4289 locus. The mutational frequency and specificity exhibited in this mutant were comparable to that of MMR-deficient strains with knockouts of mutL or mutS. It retained a similar UV sensitivity profile as that of the wild type. BAS4289 encodes a putative DNA helicase RecD2 that shares 30% sequence identity with Deinococcus radiodurans RecD2, a well characterized superfamily 1B helicase whose homologs are widely present in Firmicutes complete genomes. We demonstrated that the N-terminal region of RecD2, a unique sequence extension used to distinguish RecD2 from RecD1, was important for B. anthracis RecD2, as mutations in the N-terminal conserved motifs affected its DNA repair function. This is the first report of a RecD2 helicase being associated with MMR. RecD2 and our recently described YycJ protein are likely to be two additional components in the B. anthracis MutH-independent MMR system.

Papillation in Bacillus anthracis colonies: a tool for finding new mutators.

Colonies of Bacillus anthracis Sterne allow the growth of papillation after 6 days of incubation at 30°C on Luria-Bertani medium. The papillae are due to mutations that allow the cells to overcome the barriers to continued growth. Cells isolated from papillae display two distinct gross phenotypes (group A and group B). We determined that group A mutants have mutations in the nprR gene including frameshifts, deletions, duplications and base substitutions. We used papillation as a tool for finding new mutators as the mutators generate elevated levels of papillation. We discovered that disruption of yycJ or recJ leads to a spontaneous mutator phenotype. We defined the nprR/papillation system as a new mutational analysis system for B. anthracis. The mutational specificity of the new mutator yycJ is similar to that of mismatch repair-deficient strains (MMR⁻) such as those with mutations in mutL or mutS. Deficiency in recJ results in a unique specificity, generating only tandem duplications.

Selective advantage of resistant strains at trace levels of antibiotics: a simple and ultrasensitive color test for detection of antibiotics and genotoxic agents.

Many studies have examined the evolution of bacterial mutants that are resistant to specific antibiotics, and many of these focus on concentrations at and above the MIC. Here we ask for the minimum concentration at which existing resistant mutants can outgrow sensitive wild-type strains in competition experiments at antibiotic levels significantly below the MIC, and we define a minimum selective concentration (MSC) in Escherichia coli for two antibiotics, which is near 1/5 of the MIC for ciprofloxacin and 1/20 of the MIC for tetracycline. Because of the prevalence of resistant mutants already in the human microbiome, allowable levels of antibiotics to which we are exposed should be below the MSC. Since this concentration often corresponds to low or trace levels of antibiotics, it is helpful to have simple tests to detect such trace levels. We describe a simple ultrasensitive test for detecting the presence of antibiotics and genotoxic agents. The test is based on the use of chromogenic proteins as color markers and the use of single and multiple mutants of Escherichia coli that have greatly increased sensitivity to either a wide range of antibiotics or specific antibiotics, antibiotic families, and genotoxic agents. This test can detect ciprofloxacin at 1/75 of the MIC.

Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli.

We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of "codrugs" that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's "intrinsic resistome," which provides innate resistance to antibiotics.

Deletion of dnaN1 generates a mutator phenotype in Bacillus anthracis.

The dnaN gene in eubacteria is an essential gene that encodes the beta subunit of replicative DNA polymerase. Nearly all eubacterial genomes sequenced to date predict a single copy of the dnaN gene in a well-conserved neighboring gene context. However, 19 genomes out of 348 scanned, including Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus weihenstephanensis, predict more than one dnaN gene. In most cases, these genomes appear to maintain a copy of the dnaN homolog in its usual neighboring gene context (designated as dnaN1) in addition to a second copy (designated as dnaN2) in an entirely different gene context. We used B. anthracis as our model system to investigate the role of these DnaNs. We constructed a single knockout mutant of dnaN1 and of dnaN2; however, we could not make a viable double knockout mutant of dnaN1 and dnaN2. The dnaN1 knockout mutant displays a markedly reduced colony size. It also displays a significantly increased mutation rate, which is similar to that of a mismatch repair deficient strain and to a strain deficient both in dnaN1 and mismatch repair. The dnaN2 knockout mutant, however, has a similar growth rate and a comparable mutation rate to that of the wild type. This is the first study demonstrating the existence of two functional DnaN homologs in the B. anthracis genome, with DnaN1 appearing to be more crucial than DnaN2. Our results also suggest the direct involvement of DnaN1 in the DNA mismatch repair process, which is consistent with previous findings.