Discovery of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC 50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k inact /K I of 6.4 x 10 3 M -1 s -1 . LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic. Significance Statement: Chikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.

Enteroviral 2C protein is an RNA-stimulated ATPase and uses a two-step mechanism for binding to RNA and ATP.

The enteroviral 2C protein is a therapeutic target, but the absence of a mechanistic framework for this enzyme limits our understanding of inhibitor mechanisms. Here, we use poliovirus 2C and a derivative thereof to elucidate the first biochemical mechanism for this enzyme and confirm the applicability of this mechanism to other members of the enterovirus genus. Our biochemical data are consistent with a dimer forming in solution, binding to RNA, which stimulates ATPase activity by increasing the rate of hydrolysis without impacting affinity for ATP substantially. Both RNA and DNA bind to the same or overlapping site on 2C, driven by the phosphodiester backbone, but only RNA stimulates ATP hydrolysis. We propose that RNA binds to 2C driven by the backbone, with reorientation of the ribose hydroxyls occurring in a second step to form the catalytically competent state. 2C also uses a two-step mechanism for binding to ATP. Initial binding is driven by the α and ß phosphates of ATP. In the second step, the adenine base and other substituents of ATP are used to organize the active site for catalysis. These studies provide the first biochemical description of determinants driving specificity and catalytic efficiency of a picornaviral 2C ATPase.

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective.

The absence of 'shovel-ready' anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

To multiply and spread from cell to cell, the virus responsible for COVID-19 (also known as SARS-CoV-2) must first replicate its genetic information. This process involves a 'polymerase' protein complex making a faithful copy by assembling a precise sequence of building blocks, or nucleotides. The only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), remdesivir, consists of a nucleotide analog, a molecule whose structure is similar to the actual building blocks needed for replication. If the polymerase recognizes and integrates these analogs into the growing genetic sequence, the replication mechanism is disrupted, and the virus cannot multiply. Most approaches to study this process seem to indicate that remdesivir works by stopping the polymerase and terminating replication altogether. Yet, exactly how remdesivir and other analogs impair the synthesis of new copies of the virus remains uncertain. To explore this question, Seifert, Bera et al. employed an approach called magnetic tweezers which uses a magnetic field to manipulate micro-particles with great precision. Unlike other methods, this technique allows analogs to be integrated under conditions similar to those found in cells, and to be examined at the level of a single molecule. The results show that contrary to previous assumptions, remdesivir does not terminate replication; instead, it causes the polymerase to pause and backtrack (which may appear as termination in other techniques). The same approach was then applied to other nucleotide analogs, some of which were also found to target the SARS-CoV-2 polymerase. However, these analogs are incorporated differently to remdesivir and with less efficiency. They also obstruct the polymerase in distinct ways. Taken together, the results by Seifert, Bera et al. suggest that magnetic tweezers can be a powerful approach to reveal how analogs interfere with replication. This information could be used to improve currently available analogs as well as develop new antiviral drugs that are more effective against SARS-CoV-2. This knowledge will be key at a time when treatments against COVID-19 are still lacking, and may be needed to protect against new variants and future outbreaks.

2'-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase.

The 2'-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir. When appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here we used these observations as a framework for interpreting the effects of 2'-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2'-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been proposed previously, poliovirus RdRp that was bound to RNA with an incorporated 2'-C-methyl nucleotide could still bind to the next incoming NTP. Our results also indicated that incorporation of the 2'-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2'-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.

A naturally occurring antiviral ribonucleotide encoded by the human genome.

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.

Multiple poliovirus-induced organelles suggested by comparison of spatiotemporal dynamics of membranous structures and phosphoinositides.

At the culmination of poliovirus (PV) multiplication, membranes are observed that contain phosphatidylinositol-4-phosphate (PI4P) and appear as vesicular clusters in cross section. Induction and remodeling of PI4P and membranes prior to or concurrent with genome replication has not been well studied. Here, we exploit two PV mutants, termed EG and GG, which exhibit aberrant proteolytic processing of the P3 precursor that substantially delays the onset of genome replication and/or impairs virus assembly, to illuminate the pathway of formation of PV-induced membranous structures. For WT PV, changes to the PI4P pool were observed as early as 30 min post-infection. PI4P remodeling occurred even in the presence of guanidine hydrochloride, a replication inhibitor, and was accompanied by formation of membrane tubules throughout the cytoplasm. Vesicular clusters appeared in the perinuclear region of the cell at 3 h post-infection, a time too slow for these structures to be responsible for genome replication. Delays in the onset of genome replication observed for EG and GG PVs were similar to the delays in virus-induced remodeling of PI4P pools, consistent with PI4P serving as a marker of the genome-replication organelle. GG PV was unable to convert virus-induced tubules into vesicular clusters, perhaps explaining the nearly 5-log reduction in infectious virus produced by this mutant. Our results are consistent with PV inducing temporally distinct membranous structures (organelles) for genome replication (tubules) and virus assembly (vesicular clusters). We suggest that the pace of formation, spatiotemporal dynamics, and the efficiency of the replication-to-assembly-organelle conversion may be set by both the rate of P3 polyprotein processing and the capacity for P3 processing to yield 3AB and/or 3CD proteins.

Single-Cell Virology: On-Chip Investigation of Viral Infection Dynamics.

We have developed a high-throughput, microfluidics-based platform to perform kinetic analysis of viral infections in individual cells. We have analyzed thousands of individual poliovirus infections while varying experimental parameters, including multiplicity of infection, cell cycle, viral genotype, and presence of a drug. We make several unexpected observations masked by population-based experiments: (1) viral and cellular factors contribute uniquely and independently to viral infection kinetics; (2) cellular factors cause wide variation in replication start times; and (3) infections frequently begin later and replication occurs faster than predicted by population measurements. We show that mutational load impairs interaction of the viral population with the host, delaying replication start times and explaining the attenuated phenotype of a mutator virus. We show that an antiviral drug can selectively extinguish the most-fit members of the viral population. Single-cell virology facilitates discovery and characterization of virulence determinants and elucidation of mechanisms of drug action eluded by population methods.

UGGT1 enhances enterovirus 71 pathogenicity by promoting viral RNA synthesis and viral replication.

Positive-strand RNA virus infections can induce the stress-related unfolded protein response (UPR) in host cells. This study found that enterovirus A71 (EVA71) utilizes host UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), a key endoplasmic reticulum protein (ER) involved in UPR, to enhance viral replication and virulence. EVA71 forms replication complexes (RCs) on cellular membranes that contain a mix of host and viral proteins to facilitate viral replication, but the components and processes involved in the assembly and function of RCs are not fully understood. Using EVA71 as a model, this study found that host UGGT1 and viral 3D polymerase co-precipitate along with other factors on membranous replication complexes to enhance viral replication. Increased UGGT1 levels elevated viral growth rates, while viral pathogenicity was observed to be lower in heterozygous knockout mice (Uggt1 +/- mice). These findings provide important insight on the role of UPR and host UGGT1 in regulating RNA virus replication and pathogenicity.

Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases.

The nucleotide incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp) is important for maintaining functional genetic information but, at the same time, is also important for generating sufficient genetic diversity to escape the bottlenecks of the host's antiviral response. We have previously shown that the structural dynamics of the motif D loop are closely related to nucleotide discrimination. Previous studies have also suggested that there is a reorientation of the triphosphate of the incoming nucleotide, which is essential before nucleophilic attack from the primer RNA 3'-hydroxyl. Here, we have used 31P NMR with poliovirus RdRp to show that the binding environment of the triphosphate is different when correct versus incorrect nucleotide binds. We also show that amino acid substitutions at residues known to interact with the triphosphate can alter the binding orientation/environment of the nucleotide, sometimes lead to protein conformational changes, and lead to substantial changes in RdRp fidelity. The analyses of other fidelity variants also show that changes in the triphosphate binding environment are not always accompanied by changes in the structural dynamics of the motif D loop or other regions known to be important for RdRp fidelity, including motif B. Altogether, our studies suggest that the conformational changes in motifs B and D, and the nucleoside triphosphate reorientation represent separable, "tunable" fidelity checkpoints.

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer.

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

Effects of BMS-986094, a Guanosine Nucleotide Analogue, on Mitochondrial DNA Synthesis and Function.

BMS-986094, the prodrug of a guanosine nucleotide analogue (2'-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and specific gene expression. There were no mitochondrial DNA changes at concentrations ≤10 µM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (≥10 µM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak affinity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS-986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3-4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.

Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination.

Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3' non-coding region we have further developed a cell-based assay (the 3'CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.

Long-Range Communication between Different Functional Sites in the Picornaviral 3C Protein.

The 3C protein is a master regulator of the picornaviral infection cycle, responsible for both cleaving viral and host proteins, and interacting with genomic RNA replication elements. Here we use nuclear magnetic resonance spectroscopy and molecular dynamics simulations to show that 3C is conformationally dynamic across multiple timescales. Binding of peptide and RNA lead to structural dynamics changes at both the protease active site and the RNA-binding site, consistent with these sites being dynamically coupled. Indeed, binding of RNA influences protease activity, and likewise, interactions at the active site affect RNA binding. We propose that RNA and peptide binding re-shapes the conformational energy landscape of 3C to regulate subsequent functions, including formation of complexes with other viral proteins. The observed channeling of the 3C energy landscape may be important for regulation of the viral infection cycle.

Expression and Purification of Mitochondrial RNA Polymerase and Transcription Factor A from Drosophila melanogaster.

Mitochondrial gene expression is essential in all organisms. Our understanding of mitochondrial transcription on a biochemical level has been limited by the inability to purify the individual protein components involved in mitochondrial gene expression. Recently, new systems have been identified that permit purification of these proteins from bacteria. However, the generalizability of these systems is not clear. Here, we have applied the technology from the Cameron lab to express and purify mitochondrial RNA polymerase and transcription factor A from Drosophila melanogaster. We show that the use of SUMO system to produce SUMO fusion proteins in bacteria is effective not only for the human and mouse proteins, but also for the fly proteins. The application of this system to produce the mitochondrial proteins from other organisms should permit detailed understanding of mitochondrial transcription from any organism.

Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using (15)N-, (13)C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome.

Cystoviral polymerase complex protein P7 uses its acidic C-terminal tail to regulate the RNA-directed RNA polymerase P2.

In bacteriophages of the cystovirus family, the polymerase complex (PX) encodes a 75-kDa RNA-directed RNA polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a regulatory role in transcription. Deletion of P7 from the PX leads to aberrant plus-strand synthesis suggesting its influence on the transcriptase activity of P2. Here, using solution NMR techniques and the P2 and P7 proteins from cystovirus ϕ12, we demonstrate their largely electrostatic interaction in vitro. Chemical shift perturbations on P7 in the presence of P2 suggest that this interaction involves the dynamic C-terminal tail of P7, more specifically an acidic cluster therein. Patterns of chemical shift changes induced on P2 by the P7 C-terminus resemble those seen in the presence of single-stranded RNA suggesting similarities in binding. This association between P2 and P7 reduces the affinity of the former toward template RNA and results in its decreased activity both in de novo RNA synthesis and in extending a short primer. Given the presence of C-terminal acidic tracts on all cystoviral P7 proteins, the electrostatic nature of the P2/P7 interaction is likely conserved within the family and could constitute a mechanism through which P7 regulates transcription in cystoviruses.

Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides.

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase.

RNA helicases function in numerous aspects of RNA biology. These enzymes are RNA-stimulated ATPases that translocate on RNA and unwind or remodel structured RNA in an ATP-dependent fashion. How ATP and the ATPase cycle fuel the work performed by helicases is not completely clear. The hepatitis C virus RNA helicase, NS3, is an important model system for this class of enzymes. NS3 binding to a single-/double-strand RNA or DNA junction leads to ATP-independent melting of the duplex and formation of a complex capable of ATP-dependent unwinding by using a spring-loaded mechanism. We have established an RNA substrate for NS3 that can be unwound in a single sub-step. Our studies are consistent with a model in which a single ATP binding and/or hydrolysis event sets the unwinding spring and phosphate dissociation contributes to release of the spring, thereby driving the power stroke used for unwinding.

Insight into poliovirus genome replication and encapsidation obtained from studies of 3B-3C cleavage site mutants.

A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.

Purification and characterization of recombinant human respiratory syncytial virus nonstructural protein NS1.

We report here the first biochemical and structural characterization of the respiratory syncytial virus (RSV) NS1 protein. We have used a pET-ubiquitin expression system to produce respiratory syncytial virus (RSV) NS1 protein in E. coli that contains a hexahistidine-tag on either the amino- or carboxyl-terminus (His(6)-NS1 and NS1-His(6), respectively). We have been able to isolate milligram quantities of highly purified His(6)-NS1 and NS1-His(6) by nickel affinity chromatography. Generation of recombinant RSV indicated that addition of the hexahistidine tag to the C-terminus of NS1 slightly decreased viral replication competence whereas addition of the tag to the N-terminus had no observable effect. Therefore, we performed a comprehensive biochemical and biophysical characterization on His(6)-NS1. His(6)-NS1 is monodisperse in solution as determined by dynamic light scattering analysis. Both gel filtration and analytical ultracentrifugation showed that His(6)-NS1 is predominantly a monomer. In agreement with theoretical predictions, circular dichroism spectroscopy showed that His(6)-NS1 contains 21% alpha-helices, 34% beta-sheets, and 45% undefined structure. Immunization with purified His(6)-NS1 generated an antiserum that specifically recognizes NS1 by immunoprecipitation from HEp-2 cells infected by RSV, indicating that His(6)-NS1 resembles native NS1. The availability of purified RSV NS1 will permit biochemical and structural investigations providing insight into the function of NS1 in viral replication and interferon antagonism.