Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.

[Poxvirus-encoded DNA replication proteins: potential targets for antivirals]. / Le complexe de réplication des poxvirus : cible potentielle de molécules antivirales.

In the spring of 2022, an epidemic due to human monkeypox virus (MPXV) of unprecedented magnitude spread across all continents. Although this event was surprising in its suddenness, the resurgence of a virus from the Poxviridae family is not surprising in a world population that has been largely naïve to these viruses since the eradication of the smallpox virus in 1980 and the concomitant cessation of vaccination. Since then, a vaccine and two antiviral compounds have been developed to combat a possible return of smallpox. However, the use of these treatments during the 2022 MPXV epidemic showed certain limitations, indicating the importance of continuing to develop the therapeutic arsenal against these viruses. For several decades, efforts to understand the molecular mechanisms involved in the synthesis of the DNA genome of these viruses have been ongoing. Although many questions remain unanswered up to now, the three-dimensional structures of essential proteins, and in particular of the DNA polymerase holoenzyme in complex with DNA, make it possible to consider the development of a model for poxvirus DNA replication. In addition, these structures are valuable tools for the development of new antivirals targeting viral genome synthesis. This review will first present the molecules approved for the treatment of poxvirus infections, followed by a review of our knowledge of the replication machinery of these viruses. Finally, we will describe how these proteins could be the target of new antiviral compounds.

The Vaccinia Virus DNA Helicase Structure from Combined Single-Particle Cryo-Electron Microscopy and AlphaFold2 Prediction.

Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323-785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.

Solution Structure of the C-terminal Domain of A20, the Missing Brick for the Characterization of the Interface between Vaccinia Virus DNA Polymerase and its Processivity Factor.

Poxviruses are enveloped viruses with a linear, double-stranded DNA genome. Viral DNA synthesis is achieved by a functional DNA polymerase holoenzyme composed of three essential proteins. For vaccinia virus (VACV) these are E9, the catalytic subunit, a family B DNA polymerase, and the heterodimeric processivity factor formed by D4 and A20. The A20 protein links D4 to the catalytic subunit. High-resolution structures have been obtained for the VACV D4 protein in complex with an N-terminal fragment of A20 as well as for E9. In addition, biochemical studies provided evidence that a poxvirus-specific insertion (insert 3) in E9 interacts with the C-terminal residues of A20. Here, we provide solution structures of two different VACV A20 C-terminal constructs containing residues 304-426, fused at their C-terminus to either a BAP (Biotin Acceptor Peptide)-tag or a short peptide containing the helix of E9 insert 3. Together with results from titration studies, these structures shed light on the molecular interface between the catalytic subunit and the processivity factor component A20. The interface comprises hydrophobic residues conserved within the Chordopoxvirinae subfamily. Finally, we constructed a HADDOCK model of the VACV A20304-426-E9 complex, which is in excellent accordance with previous experimental data.

Intermediate-resolution crystal structure of the human adenovirus B serotype 3 fibre knob in complex with the EC2-EC3 fragment of desmoglein 2.

The cryo-electron microscopy (cryo-EM) structure of the complex between the trimeric human adenovirus B serotype 3 fibre knob and human desmoglein 2 fragments containing cadherin domains EC2 and EC3 has been published, showing 3:1 and 3:2 complexes. Here, the crystal structure determined at 4.5 Šresolution is presented with one EC2-EC3 desmoglein fragment bound per fibre knob monomer in the asymmetric unit, leading to an apparent 3:3 stoichiometry. However, in concentrated solution the 3:2 complex is predominant, as shown by small-angle X-ray scattering (SAXS), while cryo-EM at lower concentrations showed a majority of the 3:1 complex. Substitution of the calcium ions bound to the desmoglein domains by terbium ions allowed confirmation of the X-ray model using their anomalous scattering and shows that at least one binding site per cluster of calcium ions is intact and exchangeable and, combined with SAXS data, that the cadherin domains are folded even in the distal part that is invisible in the cryo-EM reconstruction.

The vaccinia virus DNA polymerase structure provides insights into the mode of processivity factor binding.

Vaccinia virus (VACV), the prototype member of the Poxviridae, replicates in the cytoplasm of an infected cell. The catalytic subunit of the DNA polymerase E9 binds the heterodimeric processivity factor A20/D4 to form the functional polymerase holoenzyme. Here we present the crystal structure of full-length E9 at 2.7 Å resolution that permits identification of important poxvirus-specific structural insertions. One insertion in the palm domain interacts with C-terminal residues of A20 and thus serves as the processivity factor-binding site. This is in strong contrast to all other family B polymerases that bind their co-factors at the C terminus of the thumb domain. The VACV E9 structure also permits rationalization of polymerase inhibitor resistance mutations when compared with the closely related eukaryotic polymerase delta-DNA complex.

Structural analysis of point mutations at the Vaccinia virus A20/D4 interface.

The Vaccinia virus polymerase holoenzyme is composed of three subunits: E9, the catalytic DNA polymerase subunit; D4, a uracil-DNA glycosylase; and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase cofactor, the function of which is essential for processive DNA synthesis. The recent crystal structure of D4 bound to the first 50 amino acids of A20 (D4/A201-50) revealed the importance of three residues, forming a cation-π interaction at the dimerization interface, for complex formation. These are Arg167 and Pro173 of D4 and Trp43 of A20. Here, the crystal structures of the three mutants D4-R167A/A201-50, D4-P173G/A201-50 and D4/A201-50-W43A are presented. The D4/A20 interface of the three structures has been analysed for atomic solvation parameters and cation-π interactions. This study confirms previous biochemical data and also points out the importance for stability of the restrained conformational space of Pro173. Moreover, these new structures will be useful for the design and rational improvement of known molecules targeting the D4/A20 interface.

Production and characterisation of Epstein-Barr virus helicase-primase complex and its accessory protein BBLF2/3.

The helicase-primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein-Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase-primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase-primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases.

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 µm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.

Structure determination of feline calicivirus virus-like particles in the context of a pseudo-octahedral arrangement.

The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P213. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA) domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion) and S (shell) domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination.

Crystal structure of the vaccinia virus DNA polymerase holoenzyme subunit D4 in complex with the A20 N-terminal domain.

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase E9, the uracil-DNA glycosylase D4 and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase co-factor whose function is essential for processive DNA synthesis. Genetic and biochemical data have established that residues located in the N-terminus of A20 are critical for binding to D4. However, no information regarding the residues of D4 involved in A20 binding is yet available. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A201₋50). We showed that whereas D4 forms homodimers in solution when expressed alone, D4/A201₋50 clearly behaves as a heterodimer. The crystal structure of D4/A201₋50 solved at 1.85 Å resolution reveals that the D4/A20 interface (including residues 167 to 180 and 191 to 206 of D4) partially overlaps the previously described D4/D4 dimer interface. A201₋50 binding to D4 is mediated by an α-helical domain with important leucine residues located at the very N-terminal end of A20 and a second stretch of residues containing Trp43 involved in stacking interactions with Arg167 and Pro173 of D4. Point mutations of the latter residues disturb D4/A201₋50 formation and reduce significantly thermal stability of the complex. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A201₋50 interaction. Finally, we propose a model of D4/A201₋50 in complex with DNA and discuss a number of mutants described in the literature, which affect DNA synthesis. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase cofactor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface.

Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: implication of the PKA pathway.

Ubinuclein-1 is a NACos (Nuclear and Adhesion junction Complex components) protein which shuttles between the nucleus and tight junctions, but its function in the latter is not understood. Here, by co-immunoprecipitation and confocal analysis, we show that Ubinuclein-1 interacts with the 14-3-3ɛ protein both in HT29 colon cells, and AGS gastric cells. This interaction is mediated by an Ubinuclein-1 phosphoserine motif. We show that the arginine residues (R56, R60 and R132) which form the 14-3-3ɛ ligand binding site are responsible for the binding of 14-3-3ɛ to phosphorylated Ubinuclein-1. Furthermore, we demonstrate that in vitro Ubinuclein-1 can be directly phosphorylated by cAMP-dependent protein kinase A. This in vitro phosphorylation allows binding of wildtype 14-3-3ɛ. Moreover, treatment of the cells with inhibitors of the cAMP-dependent protein kinase, KT5720 or H89, modifies the subcellular localization of Ubinuclein-1. Indeed, KT5720 and H89 greatly increase the staining of Ubinuclein-1 at the tight junctions in AGS gastric cells. In the presence of the kinase inhibitor KT5720, the amount of Ubinuclein-1 in the NP40 insoluble fraction is increased, together with actin. Moreover, treatment of the cells with KT5720 or H89 induces the concentration of Ubinuclein-1 at tricellular intersections of MDCK cells. Taken together, our findings demonstrate novel cell signaling trafficking by Ubinuclein-1 via association with 14-3-3ɛ following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A.

Low-resolution structure of vaccinia virus DNA replication machinery.

Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.

The structural basis for the integrity of adenovirus Ad3 dodecahedron.

During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59-61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1-47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

Allosteric competitive inactivation of hematopoietic CSF-1 signaling by the viral decoy receptor BARF1.

Hematopoietic human colony-stimulating factor 1 (hCSF-1) is essential for innate and adaptive immunity against viral and microbial infections and cancer. The human pathogen Epstein-Barr virus secretes the lytic-cycle protein BARF1 that neutralizes hCSF-1 to achieve immunomodulation. Here we show that BARF1 binds the dimer interface of hCSF-1 with picomolar affinity, away from the cognate receptor-binding site, to establish a long-lived complex featuring three hCSF-1 at the periphery of the BARF1 toroid. BARF1 locks dimeric hCSF-1 into an inactive conformation, rendering it unable to signal via its cognate receptor on human monocytes. This reveals a new functional role for hCSF-1 cooperativity in signaling. We propose a new viral strategy paradigm featuring an allosteric decoy receptor of the competitive type, which couples efficient sequestration and inactivation of the host growth factor to abrogate cooperative assembly of the cognate signaling complex.

The "Bridge" in the Epstein-Barr virus alkaline exonuclease protein BGLF5 contributes to shutoff activity during productive infection.

Replication of the human herpesvirus Epstein-Barr virus drastically impairs cellular protein synthesis. This shutoff phenotype results from mRNA degradation upon expression of the early lytic-phase protein BGLF5. Interestingly, BGLF5 is the viral DNase, or alkaline exonuclease, homologues of which are present throughout the herpesvirus family. During productive infection, this DNase is essential for processing and packaging of the viral genome. In contrast to this widely conserved DNase activity, shutoff is only mediated by the alkaline exonucleases of the subfamily of gammaherpesviruses. Here, we show that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation. Furthermore, shutoff by BGLF5 induces nuclear relocalization of the cytosolic poly(A) binding protein. Guided by the recently resolved BGLF5 structure, mutants were generated and analyzed for functional consequences on DNase and shutoff activities. On the one hand, a point mutation destroying DNase activity also blocks RNase function, implying that both activities share a catalytic site. On the other hand, other mutations are more selective, having a more pronounced effect on either DNA degradation or shutoff. The latter results are indicative of an oligonucleotide-binding site that is partially shared by DNA and RNA. For this, the flexible "bridge" that crosses the active-site canyon of BGLF5 appears to contribute to the interaction with RNA substrates. These findings extend our understanding of the molecular basis for the shutoff function of BGLF5 that is conserved in gammaherpesviruses but not in alpha- and betaherpesviruses.

Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1).

We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.

Structure des enzymes de réplication du virus Epstein-Barr.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen which establishes life-long persistent infection in the large majority of the human population. During viral latency, the cellular machinery takes care of the replication of the viral episome. But EBV, as well as herpesviruses in general, codes for numerous enzymes required for lytic DNA replication which allow viral replication in resting cells. Recently, several tridimensional structures of these enzymes became available for EBV as well as for other herpesviruses so that structural information now exists for most of them. The replication process and the structures of the proteins involved in replication are reviewed in the light of potential drug development and of herpesvirus evolution. The structures of the proteins involved in lytic replication show the relationship between herpesviruses and tailed bacteriophages, furthermore they show that EBV proteins tend to be more complex than their counterparts in other organisms. In this review, we could show the phylogenetic position of the herpesvirus helicase close to the Dda helicases involved in initiation of replication of the caudovirales.

EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection.

Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.

Structure of RavA MoxR AAA+ protein reveals the design principles of a molecular cage modulating the inducible lysine decarboxylase activity.

The MoxR family of AAA+ ATPases is widespread throughout bacteria and archaea but remains poorly characterized. We recently found that the Escherichia coli MoxR protein, RavA (Regulatory ATPase variant A), tightly interacts with the inducible lysine decarboxylase, LdcI/CadA, to form a unique cage-like structure. Here, we present the X-ray structure of RavA and show that the αßα and all-α subdomains in the RavA AAA+ module are arranged as in magnesium chelatases rather than as in classical AAA+ proteins. RavA structure also contains a discontinuous triple-helical domain as well as a ß-barrel-like domain forming a unique fold, which we termed the LARA domain. The LARA domain was found to mediate the interaction between RavA and LdcI. The RavA structure provides insights into how five RavA hexamers interact with two LdcI decamers to form the RavA-LdcI cage-like structure.