Structure of HIV-1 RRE stem-loop II identifies two conformational states of the high-affinity Rev binding site.

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.

High-resolution RNA tertiary structures in Zika virus stem-loop A for the development of inhibitory small molecules.

Flaviviruses such as Zika (ZIKV) and dengue virus (DENV) are positive-sense RNA viruses belonging to Flaviviridae The flavivirus genome contains a 5' end stem-loop promoter sequence known as stem-loop A (SLA) that is recognized by the flavivirus polymerase NS5 during viral RNA synthesis and 5' guanosine cap methylation. The crystal structures of ZIKV and DENV SLAs show a well-defined fold, consisting of a bottom stem, side loop, and top stem-loop, providing unique interaction sites for small molecule inhibitors to disrupt the promoter function. To facilitate the identification of small molecule binding sites in flavivirus SLA, we determined high-resolution structures of the bottom and top stems of ZIKV SLA, which contain a single U- or G-bulge, respectively. Both bulge nucleotides exhibit multiple orientations, from folded back on the adjacent nucleotide to flipped out of the helix, and are stabilized by stacking or base triple interactions. These structures suggest that even a single unpaired nucleotide can provide flexibility to RNA structures, and its conformation is mainly determined by the stabilizing chemical environment. To facilitate discovery of small molecule inhibitors that interfere with the functions of ZIKV SLA, we screened and identified compounds that bind to the bottom and top stems of ZIKV SLA.

Binding of microRNA-122 to the hepatitis C virus 5' untranslated region modifies interactions with poly(C) binding protein 2 and the NS5B viral polymerase.

Hepatitis C virus (HCV) requires two cellular factors, microRNA-122 (miR-122) and poly(C) binding protein 2 (PCBP2), for optimal replication. These host factors compete for binding to the 5' end of the single-stranded RNA genome to regulate the viral replication cycle. To understand how they interact with the RNA, we measured binding affinities of both factors for an RNA probe representing the 5' 45 nucleotides of the HCV genome (HCV45). Isothermal titration calorimetry revealed two, unequal miR-122 binding sites in HCV45, high-affinity (S1) and low-affinity (S2), differing roughly 100-fold in binding affinity. PCBP2 binds a site overlapping S2 with affinity similar to miR-122 binding to S2. PCBP2 circularizes the genome by also binding to the 3' UTR, bridging the 5' and 3' ends of the genome. By competing with PCBP2 for binding at S2, miR-122 disrupts PCBP2-mediated genome circularization. We show that the viral RNA-dependent RNA polymerase, NS5B, also binds to HCV45, and that the binding affinity of NS5B is increased in the presence of miR-122, suggesting miR-122 promotes recruitment of the polymerase. We propose that competition between miR-122 and PCBP2 for HCV45 functions as a translation-to-replication switch, determining whether the RNA genome templates protein synthesis or RNA replication.

Structural basis for cloverleaf RNA-initiated viral genome replication.

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CDpro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CDpro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem-loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

Flavivirus proteases: The viral Achilles heel to prevent future pandemics.

Flaviviruses are important human pathogens and include dengue (DENV), West Nile (WNV), Yellow fever virus (YFV), Japanese encephalitis (JEV) and Zika virus (ZIKV). DENV, transmitted by mosquitoes, causes diseases ranging in severity from mild dengue fever with non-specific flu-like symptoms to fatal dengue hemorrhagic fever and dengue shock syndrome. DENV infections are caused by four serotypes, DENV1-4, which interact differently with antibodies in blood serum. The incidence of DENV infection has increased dramatically in recent decades and the CDC estimates 400 million dengue infections occur each year, resulting in ∼25,000 deaths mostly among children and elderly people. Similarly, ZIKV infections are caused by infected mosquito bites to humans, can be transmitted sexually and through blood transfusions. If a pregnant woman is infected, the virus can cross the placental barrier and can spread to her fetus, causing severe brain malformations in the child including microcephaly and other birth defects. It is noteworthy that the neurological manifestations of ZIKV were also observed in DENV endemic regions, suggesting that pre-existing antibody response to DENV could augment ZIKV infection. WNV, previously unknown in the US (and known to cause only mild disease in Middle East), first arrived in New York city in 1999 (NY99) and spread throughout the US and Canada by Culex mosquitoes and birds. WNV is now endemic in North America. Thus, emerging and re-emerging flaviviruses are significant threat to human health. However, vaccines are available for only a limited number of flaviviruses, and antiviral therapies are not available for any flavivirus. Hence, there is an urgent need to develop therapeutics that interfere with essential enzymatic steps, such as protease in the flavivirus lifecycle as these viruses possess significant threat to future pandemics. In this review, we focus on our E. coli expression of NS2B hydrophilic domain (NS2BH) covalently linked to NS3 protease domain (NS3Pro) in their natural context which is processed by the combined action of both subunits of the NS2B-NS3Pro precursor. Biochemical activities of the viral protease such as solubility and autoproteolysis of NS2BH-NS3Pro linkage depended on the C-terminal portion of NS2BH linked to the NS3Pro domain. Since 2008, we also focus on the use of the recombinant protease in high throughput screens and characterization of small molecular compounds identified in these screens.

Flavivirus enzymes and their inhibitors.

Flaviviruses such as dengue, Japanese encephalitis, West Nile, Yellow Fever and Zika virus, cause viral hemorrhagic fever and encephalitis in humans. However, antiviral therapeutics to treat or prevent flavivirus infections are not yet available. Thus, there is pressing need to develop therapeutics and vaccines that target flavivirus infections. All flaviviruses carry a positive-sense single-stranded RNA genome, which encodes ten proteins; three structural proteins form the virus shell, and seven nonstructural (NS) proteins are involved in replication of the viral genome. While all NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are part of a functional membrane-bound replication complex, enzymatic activities required for flaviviral replication reside in only two NS proteins, NS3 and NS5. NS3 functions as a protease, helicase, and triphosphatase, and NS5 as a capping enzyme, methyltransferase, and RNA-dependent RNA polymerase. In this chapter, we provide an overview of viral replication focusing on the structure and function of NS3 and NS5 replicases. We further describe strategies and examples of current efforts to identify potential flavivirus inhibitors against NS3 and NS5 enzymatic activities that can be developed as therapeutic agents to combat flavivirus infections.

The Role of the Stem-Loop A RNA Promoter in Flavivirus Replication.

An essential challenge in the lifecycle of RNA viruses is identifying and replicating the viral genome amongst all the RNAs present in the host cell cytoplasm. Yet, how the viral polymerase selectively recognizes and copies the viral RNA genome is poorly understood. In flaviviruses, the 5'-end of the viral RNA genome contains a 70 nucleotide-long stem-loop, called stem-loop A (SLA), which functions as a promoter for genome replication. During replication, flaviviral polymerase NS5 specifically recognizes SLA to both initiate viral RNA synthesis and to methylate the 5' guanine cap of the nascent RNA. While the sequences of this region vary between different flaviviruses, the three-way junction arrangement of secondary structures is conserved in SLA, suggesting that viruses recognize a common structural feature to replicate the viral genome rather than a particular sequence. To better understand the molecular basis of genome recognition by flaviviruses, we recently determined the crystal structures of flavivirus SLAs from dengue virus (DENV) and Zika virus (ZIKV). In this review, I will provide an overview of (1) flaviviral genome replication; (2) structures of viral SLA promoters and NS5 polymerases; and (3) and describe our current model of how NS5 polymerases specifically recognize the SLA at the 5' terminus of the viral genome to initiate RNA synthesis at the 3' terminus.

Structures of flavivirus RNA promoters suggest two binding modes with NS5 polymerase.

Flaviviruses use a ~70 nucleotide stem-loop structure called stem-loop A (SLA) at the 5' end of the RNA genome as a promoter for RNA synthesis. Flaviviral polymerase NS5 specifically recognizes SLA to initiate RNA synthesis and methylate the 5' guanosine cap. We report the crystal structures of dengue (DENV) and Zika virus (ZIKV) SLAs. DENV and ZIKV SLAs differ in the relative orientations of their top stem-loop helices to bottom stems, but both form an intermolecular three-way junction with a neighboring SLA molecule. To understand how NS5 engages SLA, we determined the SLA-binding site on NS5 and modeled the NS5-SLA complex of DENV and ZIKV. Our results show that the gross conformational differences seen in DENV and ZIKV SLAs can be compensated by the differences in the domain arrangements in DENV and ZIKV NS5s. We describe two binding modes of SLA and NS5 and propose an SLA-mediated RNA synthesis mechanism.

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors.

Double-stranded DNA viruses use ATP-powered molecular motors to package their genomic DNA. To ensure efficient genome encapsidation, these motors regulate functional transitions between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes oligomerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination. Taken together, these results provide insight into the evolution of functional transitions in viral dsDNA packaging motors.

Identification of the viral RNA promoter stem loop A (SLA)-binding site on Zika virus polymerase NS5.

Zika virus has recently emerged as an important human pathogen that has spread to more than 60 countries. Infection of a pregnant woman with Zika virus can cause severe brain malformations in the child such as microcephaly and other birth defects. Despite the medical importance of Zika virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. Stem-loop A (SLA), located in the 5' untranslated region of the viral genome, acts as a promotor for viral replication and thus is critical for recognition of the viral genome by the viral polymerase NS5. However, how NS5 engages SLA is not clear. We have quantitatively examined the intrinsic affinities between Zika virus SLA and NS5, and identified the SLA-binding site on NS5. Amino acid substitutions in the thumb subdomain of the RNA-dependent RNA polymerase (RdRp) and the methyltransferase (MTase) domain reduced SLA-binding affinity, indicating that they each are part of the SLA-binding site. Furthermore, stopped-flow kinetic analysis of Zika NS5-, RdRp- and MTase-SLA interactions identified distinct intermediates during NS5 and SLA complex formation. These data suggest a model for SLA recognition and the initiation of flaviviral replication by NS5.

A cocrystal structure of dengue capsid protein in complex of inhibitor.

Dengue virus (DENV) was designated as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a "kissing" interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor's inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.

Membrane binding and rearrangement by chikungunya virus capping enzyme nsP1.

Alphavirus genome replication is carried out by the viral replication complex inside modified membrane structures called spherules. The viral nonstructural protein 1 (nsP1) is the only membrane-associated protein that anchors the replication complex to the cellular membranes. Although an internal amphipathic helix of nsP1 is critical for membrane association, the mechanism of nsP1 interaction with membranes and subsequent membrane reorganization is not well understood. We studied the membrane interaction of chikungunya virus (CHIKV) nsP1 and show that both the CHIKV nsP1 protein and the amphipathic peptide specifically bind to negatively charged phospholipid vesicles. Using cryo-electron microscopy, we further show that nsP1 forms a contiguous coat on lipid vesicles and induces structural reorganization, while the amphipathic peptide alone failed to deform the membrane bilayer. This suggests that although amphipathic helix of nsP1 is required for initial membrane binding, the remaining cytoplasmic domain of nsP1 is involved in the subsequent membrane reorganization.

Ehrlichia chaffeensis TRP120 nucleomodulin binds DNA with disordered tandem repeat domain.

Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, secretes several effector proteins that bind host DNA to modulate host gene expression. The tandem repeat protein 120 (TRP120), one of the largest effector proteins, has four nearly identical tandem repeat (TR) regions that each consists of 80 amino acids. In addition to playing a role in ehrlichial binding and internalization, TRP120 translocates to the host nucleus where it is thought to function as a transcription factor that modulates gene expression. However, sequence analysis of TRP120 does not identify the presence of DNA-binding or trans-activation domains typical of classical eukaryotic transcription factors. Thus, the mechanism by which TRP120 binds DNA and modulates gene expression remains elusive. Herein, we expressed the TR regions of the TRP120 protein, and characterized its solution structure and ability to bind DNA. TRP120, expressed as either a one or two TR repeat, is a monomer in solution, and is mostly disordered as determined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Using NMR spectroscopy, we further show that the 1 TR construct selectively binds GC-rich DNA. Although low pH was required for TRP120 TR-DNA interaction, acidic pH alone does not induce any significant structural changes in the TR region. This suggests that TRP120 folds into an ordered structure upon forming a protein-DNA complex, and thus folding of TRP120 TR is coupled with DNA binding.

Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells.

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.

Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication.

Four serotypes of mosquito-borne dengue virus (DENV), evolved from a common ancestor, are human pathogens of global significance for which there is no vaccine or antiviral drug available. The N-terminal domain of DENV NS5 has guanylyltransferase and methyltransferase (MTase), and the C-terminal region has the polymerase (POL), all of which are important for 5'-capping and RNA replication. The crystal structure of NS5 shows it as a dimer, but the functional evidence for NS5 dimer is lacking. Our studies showed that the substitution of DENV2 NS5 MTase or POL for DENV4 NS5 within DENV2 RNA resulted in a severe attenuation of replication in the transfected BHK-21 cells. A replication-competent species was evolved with the acquired mutations in the DENV2 and DENV4 NS5 MTase or POL domain or in the DENV2 NS3 helicase domain in the DENV2 chimera RNAs by repeated passaging of infected BHK-21 or mosquito cells. The linker region of seven residues in NS5, rich in serotype-specific residues, is important for the recovery of replication fitness in the chimera RNA. Our results, taken together, provide genetic evidence for a serotype-specific interaction between NS3 and NS5 as well as specific interdomain interaction within NS5 required for RNA replication. Genome-wide RNAseq analysis revealed the distribution of adaptive mutations in RNA quasispecies. Those within NS3 and NS5 are located at the surface and/or within the NS5 dimer interface, providing a functional significance to the crystal structure NS5 dimer.

Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A.

The process of RNA replication by dengue virus is still not completely understood despite the significant progress made in the last few years. Stem-loop A (SLA), a part of the viral 5' untranslated region (UTR), is critical for the initiation of dengue virus replication, but quantitative analysis of the interactions between the dengue virus polymerase NS5 and SLA in solution has not been performed. Here, we examine how solution conditions affect the size and shape of SLA and the formation of the NS5-SLA complex. We show that dengue virus NS5 binds SLA with a 1:1 stoichiometry and that the association reaction is primarily entropy driven. We also observe that the NS5-SLA interaction is influenced by the magnesium concentration in a complex manner. Binding is optimal with 1 mM MgCl2 but decreases with both lower and higher magnesium concentrations. Additionally, data from a competition assay between SLA and single-stranded RNA (ssRNA) indicate that SLA competes with ssRNA for the same binding site on the NS5 polymerase. SLA70 and SLA80, which contain the first 70 and 80 nucleotides (nt), respectively, bind NS5 with similar binding affinities. Dengue virus NS5 also binds SLAs from different serotypes, indicating that NS5 recognizes the overall shape of SLA as well as specific nucleotides.IMPORTANCE Dengue virus is an important human pathogen responsible for dengue hemorrhagic fever, whose global incidence has increased dramatically over the last several decades. Despite the clear medical importance of dengue virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. In particular, stem-loop A (SLA) and stem-loop B (SLB) located in the 5' untranslated region (UTR) are critical for binding the viral polymerase NS5 to initiate minus-strand RNA synthesis. However, little is known regarding the kinetic and thermodynamic parameters driving these interactions. Here, we quantitatively examine the energetics of intrinsic affinities, characterize the stoichiometry of the complex of NS5 and SLA, and determine how solution conditions such as magnesium and sodium concentrations and temperature influence NS5-SLA interactions in solution. Quantitatively characterizing dengue virus NS5-SLA interactions will facilitate the design and assessment of antiviral therapeutics that target this essential step of the dengue virus life cycle.

Pestivirus Npro Directly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer.

UNLABELLED: Interferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I alpha/beta interferon (IFN-α/ß) in response to viral infection. Upon viral infection, the IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses, such as classical swine fever virus (CSFV), target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein N(pro) that interacts with IRF3, but the molecular details for this interaction are largely unknown. We used recombinant N(pro) and IRF3 proteins and show that N(pro) interacts with IRF3 directly without additional proteins and forms a soluble 1:1 complex. The full-length IRF3 but not merely either of the individual domains is required for this interaction. The interaction between N(pro) and IRF3 is not dependent on the activation state of IRF3, since N(pro) binds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator, CREB-binding protein (CBP). The results indicate that the N(pro)-binding site on IRF3 encompasses a region that is unperturbed by the phosphorylation and subsequent activation of IRF3 and thus excludes the dimer interface and CBP-binding site. IMPORTANCE: The pestivirus N-terminal protease, N(pro), is essential for evading the host's immune system by facilitating the degradation of interferon regulatory factor 3 (IRF3). However, the nature of the N(pro) interaction with IRF3, including the IRF3 species (inactive monomer versus activated dimer) that N(pro) targets for degradation, is largely unknown. We show that classical swine fever virus N(pro) and porcine IRF3 directly interact in solution and that full-length IRF3 is required for interaction with N(pro) Additionally, N(pro) interacts with a constitutively active form of IRF3 bound to its transcriptional cofactor, the CREB-binding protein. This is the first study to demonstrate that N(pro) is able to bind both inactive IRF3 monomer and activated IRF3 dimer and thus likely targets both IRF3 species for ubiquitination and proteasomal degradation.

Dengue Virus Nonstructural Protein 5 (NS5) Assembles into a Dimer with a Unique Methyltransferase and Polymerase Interface.

Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5'-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.

Allosteric inhibitors of Coxsackie virus A24 RNA polymerase.

Coxsackie virus A24 (CVA24), a causative agent of acute hemorrhagic conjunctivitis, is a prototype of enterovirus (EV) species C. The RNA polymerase (3D(pol)) of CVA24 can uridylylate the viral peptide linked to the genome (VPg) from distantly related EV and is thus, a good model for studying this reaction. Once UMP is bound, VPgpU primes RNA elongation. Structural and mutation data have identified a conserved binding surface for VPg on the RNA polymerase (3D(pol)), located about 20Å from the active site. Here, computational docking of over 60,000 small compounds was used to select those with the lowest (best) specific binding energies (BE) for this allosteric site. Compounds with varying structures and low BE were assayed for their effect on formation of VPgU by CVA24-3D(pol). Two compounds with the lowest specific BE for the site inhibited both uridylylation and formation of VPgpolyU at 10-20µM. These small molecules can be used to probe the role of this allosteric site in polymerase function, and may be the basis for novel antiviral compounds.