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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.

Synthetic RNA oligonucleotides composed of canonical and modified ribonucleotides are highly effective for RNA antisense therapeutics and RNA based genome engineering applications utilizing CRISPR-Cas9. Yet, synthesis of synthetic RNA using phosphoramidite chemistry is highly inefficient and expensive relative to DNA oligonucleotides, especially for relatively long RNA oligonucleotides. Thus, new biotechnologies are needed to significantly reduce costs, while increasing synthesis rates and yields of synthetic RNA. Here, we engineer human DNA polymerase theta (Polθ) variants and demonstrate their ability to synthesize long (95-200 nt) RNA oligonucleotides with canonical ribonucleotides and ribonucleotide analogs commonly used for stabilizing RNA for therapeutic and genome engineering applications. In contrast to natural promoter-dependent RNA polymerases, Polθ variants synthesize RNA by initiating from DNA or RNA primers which enables the production of highly pure RNA products. Remarkably, Polθ variants show lower capacity to misincorporate ribonucleotides compared to T7 RNA polymerase. Automation of this enzymatic RNA synthesis technology can potentially increase yields while reducing costs of synthetic RNA oligonucleotide production.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.

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., APOBEC3G (A3G) belongs to the DNA/RNA cytosine deaminase family that plays important roles in innate immunity against HIV and internal retroelements. Here the authors report the structures of two full-length A3G variants that provides insight into domain organization, multimerization, RNA binding, and viral restriction.

The emergence of precision medicine from the development of Poly (ADP-ribose) polymerase (PARP) inhibitors that preferentially kill cells defective in homologous recombination has sparked wide interest in identifying and characterizing additional DNA repair enzymes that are synthetic lethal with HR factors. DNA polymerase theta (Polθ) is a validated anti-cancer drug target that is synthetic lethal with HR factors and other DNA repair proteins and confers cellular resistance to various genotoxic cancer therapies. Since its initial characterization as a helicase-polymerase fusion protein in 2003, many exciting and unexpected activities of Polθ in microhomology-mediated end-joining (MMEJ) and translesion synthesis (TLS) have been discovered. Here, we provide a short review of Polθ‘s DNA repair activities and its potential as a drug target and highlight a recent report that reveals Polθ as a naturally occurring reverse transcriptase (RT) in mammalian cells.

Polθ reverse transcribes RNA by undergoing a significant conformational change and promotes RNA-templated DNA repair., Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Å crystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2′-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair.

APOBEC1 (APO1), a member of AID/APOBEC nucleic acid cytosine deaminase family, can edit apolipoprotein B mRNA to regulate cholesterol metabolism. This APO1 RNA editing activity requires a cellular cofactor to achieve tight regulation. However, no cofactors are required for deamination on DNA by APO1 and other AID/APOBEC members, and aberrant deamination on genomic DNA by AID/APOBEC deaminases has been linked to cancer. Here, we present the crystal structure of APO1, which reveals a typical APOBEC deaminase core structure, plus a unique well-folded C-terminal domain that is highly hydrophobic. This APO1 C-terminal hydrophobic domain (A1HD) interacts to form a stable dimer mainly through hydrophobic interactions within the dimer interface to create a four-stranded β-sheet positively charged surface. Structure-guided mutagenesis within this and other regions of APO1 clarified the importance of the A1HD in directing RNA and cofactor interactions, providing insights into the structural basis of selectivity on DNA or RNA substrates.

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with single-stranded DNA (ssDNA). Here we report a crystal structure of A3F-CD2 in complex with a 10 nucleotide (nt) ssDNA composed of poly-thymine, which reveals a novel positively-charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.

Background Each vaccine for human papillomavirus type 16 (HPV16) has been developed on the basis of a single variant, and whether these vaccines can prevent infection due to naturally occurring variants was not clear. Methods To examine this question, constructs of 39 naturally occurring single amino acid substitutions in L1 were generated for pseudovirion production, based on the analysis of 1204 HPV16 L1 protein sequences from the National Center for Biotechnology Information and Papilloma Virus Episteme. Results Thirty-one of 39 HPV16 L1 mutants produced infectious pseudovirions that exhibited similar particle-to-infectivity ratios, compared with reference pseudovirions. Twenty-one of 31 pseudovirion-producing mutants showed different susceptibilities to monoclonal antibodies, with 6 resulting in complete loss of reactivity to some of the tested monoclonal antibodies. The vaccinated sera neutralized all 31 variants. Mean neutralization titers of most variants changed by approximately 4-fold, compared with the reference pseudovirions, with the C428W and K430Q mutations displaying 9-fold and 11-fold lower susceptibilities, respectively, to neutralization by the sera than the reference pseudovirions. Conclusions These results suggest that the current HPV vaccines may not offer equal protection against all of the naturally occurring HPV16 variants discovered so far.

When a cell divides to form a new cell, it must also copy its DNA. An important step for starting DNA replication is to break or “melt” the bonds between the two strands of DNA that make up the double helix. This happens at a specific site on the DNA called the replication origin, and allows double-stranded DNA to partially unwind into two single strands. Each strand acts as a template to form a new copy of its partner strand. In many organisms, ring-shaped proteins called helicases attach directly to double-stranded DNA to melt the bonds at the replication origin. Viruses also have helicases that they can use to hijack the cell’s replication machinery and get it to copy the viral DNA. One such helicase called Large T antigen is an important part of a tumor virus called simian virus 40. Large T has a ‘hexameric’ structure, similar to the cell’s own helicases, and researchers often use it as a model for studying the replication process. However, key questions remain. How do hexameric helicases interact with the double-stranded DNA replication origin? And what are the molecular mechanisms by which these helicases melt bonds in double-stranded DNA? Gai et al. used a technique called X-ray crystallography to examine the molecular structure of the Large T helicase when it is attached to DNA. The resulting structures show that six subunits of Large T helicase assemble a ring around the replication origin, each making unique bonds with the DNA. The helicase ring squeezes the DNA, partially breaking the bonds between the DNA strands and causing local melting of the DNA. With the understanding of how the Large T hexamer helicase interacts with its DNA replication origin and how its assembly initiates DNA melting, the future challenge is to explain the process by which the partially melted origin opens up fully for replication to begin.

RNA editing is an important form of regulating gene expression and activity. APOBEC1 cytosine deaminase was initially characterized as pairing with a cofactor, A1CF, to form an active RNA editing complex that specifically targets APOB RNA in regulating lipid metabolism. Recent studies revealed that APOBEC1 may be involved in editing other potential RNA targets in a tissue-specific manner, and another protein, RBM47, appears to instead be the main cofactor of APOBEC1 for editing APOB RNA. In this report, by expressing APOBEC1 with either A1CF or RBM47 from human or mouse in an HEK293T cell line with no intrinsic APOBEC1/A1CF/RBM47 expression, we have compared direct RNA editing activity on several known cellular target RNAs. By using a sensitive cell-based fluorescence assay that enables comparative quantification of RNA editing through subcellular localization changes of eGFP, the two APOBEC1 cofactors, A1CF and RBM47, showed clear differences for editing activity on APOB and several other tested RNAs, and clear differences were observed when mouse versus human genes were tested. In addition, we have determined the minimal domain requirement of RBM47 needed for activity. These results provide useful functional characterization of RBM47 and direct biochemical evidence for the differential editing selectivity on a number of RNA targets.

APOBEC3H (A3H) is the most polymorphic member of the APOBEC3 family. Seven haplotypes (hap I-VII) and four mRNA splicing variants (SV) of A3H have been identified. The various haplotypes differ in anti-HIV activity, which is attributed to differences in protein stability, subcellular distribution, and/or RNA binding and virion packaging. Here, we report the first comparative biochemical studies of all the A3H variants using highly purified proteins. We show that all haplotypes were stably expressed and could be purified to homogeneity by Escherichia coli expression. Surprisingly, four out of the seven haplotypes showed high cytosine (C) deaminase activity, with hap V displaying extremely high activity that was comparable to the highly active A3A. Furthermore, all four haplotypes that were active in C deamination were also highly active on methylated C (mC), with hap II displaying almost equal deamination efficiency on both. The deamination activity of these A3H variants correlates well with their reported anti-HIV activity for the different haplotypes, suggesting that deaminase activity may be an important factor in determining their respective anti-HIV activities. Moreover, mC deamination of A3H displayed a strong preference for the sequence motif of T-mCpG-C/G, which may suggest a potential role in genomic mC modification at the characteristic "CpG" island motif.

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis., eLife digest Living cells store their genetic code written in molecules of DNA, with two strands of DNA twisted together to form the familiar double helix. When a cell prepares to divide, it must unwind its DNA so that the individual strands can be copied. Enzymes known as DNA helicases play a vital role in this unwinding process; yet, it is not completely clear how these enzymes move along the DNA. Schlierf et al. have now developed a new approach to see how an individual DNA helicase called G40P unwinds the DNA double helix. The experiments used a molecular ruler to measure the DNA unwinding and showed that the helicase opened the double helix one letter of genetic code at a time. Also, specific sequence of letters within the DNA molecules could slow down and stop G40P or even cause it to move backwards. DNA helicases work closely with other proteins inside cells to perform their task. DNA primases, for example, are enzymes that create the starting points for making new strands of DNA. Schlierf et al. found that the primase DnaG could also prevent G40P from moving backwards on the DNA, a new and unexpected function of DnaG. These findings contribute to an ongoing debate among researchers with partially contradictory models for how DNA helicases unwind the DNA double helix. Although originally from a virus, G40P is similar to a helicase enzyme found in bacteria. Therefore, a better understanding of this helicase may lead to new ways to stop bacteria copying their DNA, which might one day become new antibiotics to treat bacterial infections.

It is unclear whether both CD1 and CD2 of A3B can deaminate C and mC. We showed that only CD2 is active, and has weak mC deaminating activity. We successfully engineered A3BCD2 to gain over 100-fold higher mC deamination activity., 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.

BACKGROUND HIV-1 gp120/gp41 is heavily modified by n-linked carbohydrates that play important roles either in correct folding or in shielding vulnerable viral protein surfaces from antibody recognition. METHODS In our previous work, 25 potential N-linked glycosylation sites (PNGS) of a CRF07_BC isolate of HIV-1 were individually mutated, and the resulting effects on infectivity and antibody-mediated neutralization were evaluated. To further understand the functional role of these PNGS, we generated double and multiple mutants from selected individual PNGS mutants. The effects were then evaluated by examining infectivity and sensitivity to antibody-mediated neutralization by neutralizing monoclonal antibodies (nMAbs) and serum antibodies from HIV-1 positive donors. RESULTS Infectivity results showed that, among the 12 combined PNGS mutants, only 197M.1 (N197D/N301Q) lost infectivity completely, whereas all others (except for 197M.6) showed reduced viral infectivity. In terms of neutralization sensitivity to known nMAbs, we found that adding N463Q mutation to all the gp120 mutants containing N197D significantly increased neutralization sensitivity to VRC01 and VRC03, suggesting N197 and N463 have a strong synergistic effect in regulating the neutralizing sensitivity of HIV-1 to the anti-CD4bs nMAbs VRC01/VRC03. Structural analysis based on the available structures of gp120 alone and in complex with CD4 and various nMAbs elucidates a molecular rationale for this experimental observation. CONCLUSIONS The data indicate that N463 plays an important role in regulating the CD4bs MAbs VRC01/VRC03 sensitivity in the genetic background of N197D mutation of gp120, which should provide valuable information for a better understanding of the interplay between HIV-1 and VRC01/03.

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 β5 strand of the canonical five-stranded β-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.

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.

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.

Mini-chromosome maintenance (MCM) proteins are the replicative helicase necessary for DNA replication in both eukarya and archaea. Most of archaea only have one MCM gene. Here, we report a 1.8-Å crystal structure of the N-terminal MCM from the archaeon Thermoplasma acidophilum (tapMCM). In the structure, the MCM N-terminus forms a right-handed filament that contains six subunits in each turn, with a diameter of 25Å of the central channel opening. The inner surface is highly positively charged, indicating DNA binding. This filament structure with six subunits per turn may also suggests a potential role for an open-ring structure for hexameric MCM and dynamic conformational changes in initiation and elongation stages of DNA replication.

The mono-site mutations of the absolutely conserved residues, (464)LGR(466), in the α-helix 5 (h5) of HPV16 L1 completely disrupted the pentamer formation. The implication of this finding is the potential usage of a h5-like peptide as the reagent to interfere with the pentamer formation and stability as an anti-HPV reagent.

SummaryThe DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout.

SUMMARY SIX1 interacts with EYA to form a bipartite transcription factor essential for development. Loss of function of this complex causes branchio-oto-renal syndrome (BOR), while re-expression of SIX1 or EYA promotes metastasis. Here we describe the 2.0 Å structure of SIX1 bound to EYA2, which suggests a novel DNA binding mechanism for SIX1 and provides a rationale for the effect of BOR syndrome mutations. The structure also reveals that SIX1 uses predominantly a single helix to interact with EYA. Substitution of a single amino acid in this helix is sufficient to disrupt the SIX1–EYA interaction, SIX1-mediated epithelial-mesenchymal transition and metastasis in mouse models. Given that SIX1 and EYA are co-overexpressed in many tumor types, our data indicate that targeting the SIX1–EYA complex may be a potent approach to inhibit tumor progression in multiple cancer types.

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.18129.001, eLife digest When a cell divides to form a new cell, it must also copy its DNA. An important step for starting DNA replication is to break or “melt” the bonds between the two strands of DNA that make up the double helix. This happens at a specific site on the DNA called the replication origin, and allows double-stranded DNA to partially unwind into two single strands. Each strand acts as a template to form a new copy of its partner strand. In many organisms, ring-shaped proteins called helicases attach directly to double-stranded DNA to melt the bonds at the replication origin. Viruses also have helicases that they can use to hijack the cell’s replication machinery and get it to copy the viral DNA. One such helicase called Large T antigen is an important part of a tumor virus called simian virus 40. Large T has a ‘hexameric’ structure, similar to the cell’s own helicases, and researchers often use it as a model for studying the replication process. However, key questions remain. How do hexameric helicases interact with the double-stranded DNA replication origin? And what are the molecular mechanisms by which these helicases melt bonds in double-stranded DNA? Gai et al. used a technique called X-ray crystallography to examine the molecular structure of the Large T helicase when it is attached to DNA. The resulting structures show that six subunits of Large T helicase assemble a ring around the replication origin, each making unique bonds with the DNA. The helicase ring squeezes the DNA, partially breaking the bonds between the DNA strands and causing local melting of the DNA. With the understanding of how the Large T hexamer helicase interacts with its DNA replication origin and how its assembly initiates DNA melting, the future challenge is to explain the process by which the partially melted origin opens up fully for replication to begin. DOI: http://dx.doi.org/10.7554/eLife.18129.002

DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays primosome activity. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple primosome that is active in vitro. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo.

APOBEC3G (Apo3G) is a single-stranded DNA-dependent deoxycytidine deaminase, which, in the absence of the human immunodeficiency virus (HIV) viral infectivity factor, is encapsulated into HIV virions. Subsequently, Apo3G triggers viral inactivation by processively deaminating C--U, with 3'--5' polarity, on nascent minus-strand cDNA. Apo3G has a catalytically inactive N-terminal CD1 domain and an active C-terminal CD2 domain. Apo3G exists as monomers, dimers, tetramers, and higher order oligomers whose distributions depend on DNA substrate and salt. Here we use multiangle light scattering and atomic force microscopy to identify oligomerization states of Apo3G. A double mutant (F126A/W127A), designed to disrupt dimerization at the predicted CD1-CD1 dimer interface, predominantly converts Apo3G to a monomer that binds single-stranded DNA, Alu RNA, and catalyzes processive C--U deaminations with 3'--5' deamination polarity, similar to native Apo3G. The CD1 domain is essential for both processivity and polarity. We propose a structure-based model to explain the scanning and catalytic behavior of Apo3G.

The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, recently emerged as an important risk factor and drug target for human autoimmunity. Here we solved the structure of the catalytic domain of LYP, which revealed noticeable differences with previously published structures. The active center with a semi-closed conformation binds a phosphate ion, which may represent an intermediate conformation after dephosphorylation of the substrate but before release of the phosphate product. The structure also revealed an unusual disulfide bond formed between the catalytic Cys and one of the two Cys residues nearby, which is not observed in previously determined structures. Our structural and mutagenesis data suggest that the disulfide bond may play a role in protecting the enzyme from irreversible oxidation. Surprisingly, we found that the two noncatalytic Cys around the active center exert an opposite yin-yang regulation on the catalytic Cys activity. These detailed structural and functional characterizations have provided new insights into autoregulatory mechanisms of LYP function.

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC(50) values from 30 nM to 1.6 micrOM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents.

APOBEC3G (Apo3G), a cellular protein that has the ability to inhibit HIV, offers a new hope for fighting against HIV-1 infection. Recent advances in Apo3G structural and functional studies provide an opportunity for structurebased drug design and development to unleash the potent anti-HIV activity of Apo3G for AIDS prevention and therapy. Although the availability of antiretroviral regimens in the USA has significantly improved the life expectancy of patients presently receiving treatment, viral strains are emerging that are resistant to at least one or more of the six major classes of HIV drugs, which include inhibitors of viral entry, fusion, integrases, reverse transcriptase (nucleoside/nucleotide analogs and non-nucleoside inhibitors) and proteases [1]. Also troubling is the prevalent number of new infections that involve the transmission of drugresistant viral strains [1]. As HIV patients live longer, the emergence of drug-resistant HIV viral strains has become a more common occurrence. There is an urgent need for novel classes of HIV drugs that can inactivate the highly mutagenic HIV virus with high efficiency and less toxicity.

The E6 oncoproteins of the cancer-associated human papillomaviruses (high-risk HPV types) characteristically have a PDZ-binding motif at their extreme carboxy-termini. However, they interact with only some of the PDZ domain-containing proteins in the human proteome and, despite many of these proteins having multiple PDZ domains, they interact specifically through only one of those domains. Previous work has shown that the exact sequence of the C-terminal PDZ-binding motif of E6 affects substrate selection, and recently we have shown that an E6 residue peripheral to the binding motif also contributes to the specificity of binding.Here we show that substrate specificity of the HPV E6 PDZ binding is modulated both by the amino acid residues upstream of the binding domain and by the non-canonical residues within it. Using this data we have begun to construct a scheme of substrate preferences for E6 proteins from different HPV types.