Picornaviral polymerase domain exchanges reveal a modular basis for distinct biochemical activities of viral RNA-dependent RNA polymerases.

Picornaviral RNA-dependent RNA polymerases (RdRPs) have low replication fidelity that is essential for viral fitness and evolution. Their global fold consists of the classical "cupped right hand" structure with palm, fingers, and thumb domains, and these RdRPs also possess a unique contact between the fingers and thumb domains. This interaction restricts movements of the fingers, and RdRPs use a subtle conformational change within the palm domain to close their active sites for catalysis. We have previously shown that this core RdRP structure and mechanism provide a platform for polymerases to fine-tune replication rates and fidelity to optimize virus fitness. Here, we further elucidated the structural basis for differences in replication rates and fidelity among different viruses by generating chimeric RdRPs from poliovirus and coxsackievirus B3. We designed these chimeric polymerases by exchanging the fingers, pinky finger, or thumb domains. The results of biochemical, rapid-quench, and stopped-flow assays revealed that differences in biochemical activity map to individual modular domains of this polymerase. We found that the pinky finger subdomain is a major regulator of initiation and that the palm domain is the major determinant of catalytic rate and nucleotide discrimination. We further noted that thumb domain interactions with product RNA regulate translocation and that the palm and thumb domains coordinately control elongation complex stability. Several RdRP chimeras supported the growth of infectious poliovirus, providing insights into enterovirus species-specific protein-protein interactions required for virus replication.

Inhibition of RNA Helicase Activity Prevents Coxsackievirus B3-Induced Myocarditis in Human iPS Cardiomyocytes.

AIMS: Coxsackievirus B3 (CVB3) is known to be an important cause of myocarditis and dilated cardiomyopathy. Enterovirus-2C (E2C) is a viral RNA helicase. It inhibits host protein synthesis. Based on these facts, we hypothesize that the inhibition of 2C may suppress virus replication and prevent enterovirus-mediated cardiomyopathy. METHODS AND RESULTS: We generated a chemically modified enterovirus-2C inhibitor (E2CI). From the in vitro assay, E2CI was showed strong antiviral effects. For in vivo testing, mice were treated with E2CI intraperitoneally injected daily for three consecutive days at a dose of 8mg/kg per day, after CVB3 post-infection (p.i) (CVB3 + E2CI, n = 33). For the infected controls (CVB3 only, n = 35), mice were injected with PBS (phosphate buffered saline) in a DBA/2 strain to establish chronic myocarditis. The four-week survival rate of E2CI-treated mice was significantly higher than that of controls (92% vs. 71%; p < 0.05). Virus titers and myocardial damage were significantly reduced in the E2CI treated group. In addition, echocardiography indicated that E2CI administration dramatically maintained mouse heart function compared to control at day 28 p.i chronic stage (LVIDD, 3.1 ± 0.08 vs. 3.9 ± 0.09, p < 0.01; LVDS, 2.0 ± 0.07 vs. 2.5 ± 0.07, p < 0.001; FS, 34.8 ± 1.6% vs. 28.5 ± 1.5%; EF, 67. 9 ± 2.9% vs. 54.7 ± 4.7%, p < 0.05; CVB3 + E2CI, n = 6 vs. CVB3, n = 4). Moreover, E2CI is effectively worked in human iPS (induced pluripotent stem cell) derived cardiomyocytes. CONCLUSION: Enterovirus-2C inhibitor (E2CI) was significantly reduced viral replication, chronic myocardium damage, and CVB3-induced mortality in DBA/2 mice. These results suggested that E2CI is a novel therapeutic agent for the treatment of enterovirus-mediated diseases.

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection.

Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cproin vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner.IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection.

Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication.

Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.

Understanding the Mechanism of the Broad-Spectrum Antiviral Activity of Favipiravir (T-705): Key Role of the F1 Motif of the Viral Polymerase.

Favipiravir (T-705) is a broad-spectrum antiviral agent that has been approved in Japan for the treatment of influenza virus infections. T-705 also inhibits the replication of various RNA viruses, including chikungunya virus (CHIKV). We demonstrated earlier that the K291R mutation in the F1 motif of the RNA-dependent RNA polymerase (RdRp) of CHIKV is responsible for low-level resistance to T-705. Interestingly, this lysine is highly conserved in the RdRp of positive-sense single-stranded RNA (+ssRNA) viruses. To obtain insights into the unique broad-spectrum antiviral activity of T-705, we explored the role of this lysine using another +ssRNA virus, namely, coxsackievirus B3 (CVB3). Introduction of the corresponding K-to-R substitution in the CVB3 RdRp (K159R) resulted in a nonviable virus. Replication competence of the K159R variant was restored by spontaneous acquisition of an A239G substitution in the RdRp. A mutagenesis analysis at position K159 identified the K159M variant as the only other viable variant which had also acquired the A239G substitution. The K159 substitutions markedly decreased the processivity of the purified viral RdRp, which was restored by the introduction of the A239G mutation. The K159R A239G and K159M A239G variants proved, surprisingly, more susceptible than the wild-type virus to T-705 and exhibited lower fidelity in polymerase assays. Furthermore, the K159R A239G variant was found to be highly attenuated in mice. We thus demonstrate that the conserved lysine in the F1 motif of the RdRp of +ssRNA viruses is involved in the broad-spectrum antiviral activity of T-705 and that it is a key amino acid for the proper functioning of the enzyme.IMPORTANCE In this study, we report the key role of a highly conserved lysine residue of the viral polymerase in the broad-spectrum antiviral activity of favipiravir (T-705) against positive-sense single-stranded RNA viruses. Substitutions of this conserved lysine have a major negative impact on the functionality of the RdRp. Furthermore, we show that this lysine is involved in the fidelity of the RdRp and that the RdRp fidelity influences the sensitivity of the virus for the antiviral efficacy of T-705. Consequently, these results provide insights into the mechanism of the antiviral activity of T-705 and may lay the basis for the design of novel chemical scaffolds that may be endowed with a more potent broad-spectrum antiviral activity than that of T-705.

Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo.

Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stopped-flow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg(2+) ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe(364) and Pro(357), which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe(364) to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.

The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.

ATP Is an Allosteric Inhibitor of Coxsackievirus B3 Polymerase.

The RNA-dependent RNA polymerases from positive-strand RNA viruses, such as picornaviruses and flaviviruses, close their active sites for catalysis via a unique NTP-induced conformational change in the palm domain. Combined with a fully prepositioned templating nucleotide, this mechanism is error-prone and results in a distribution of random mutations in the viral progeny often described as a quasi-species. Here we examine the extent to which noncognate NTPs competitively inhibit single-cycle elongation by coxsackievirus B3 3D(pol), a polymerase that generates three to four mutations per 10 kb of RNA synthesized during viral infection. Using an RNA with a templating guanosine combined with 2-aminopurine fluorescence as a reporter for elongation, we find that the cognate CTP has a Km of 24 µM and the three noncognate nucleotides competitively inhibit the reaction with Kic values of 500 µM for GTP, 1300 µM for ATP, and 3000 µM for UTP. Unexpectedly, ATP also acted as an uncompetitive inhibitor with a Kiu of 1800 µM, resulting in allosteric modulation of 3D(pol) that slowed the polymerase elongation rate ≈4-fold. ATP uncompetitive inhibition required the ß- and γ-phosphates, and its extent was significantly diminished in two previously characterized low-fidelity polymerases. This led to further mutational analysis and the identification of a putative allosteric binding site below the NTP entry channel at the interface of conserved motifs A and D, although cocrystallization failed to reveal any density for bound ATP in this pocket. The potential role of an ATP allosteric effect during the virus life cycle is discussed.

Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.

UNLABELLED: Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity. IMPORTANCE: Positive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

Heterogeneous Nuclear Ribonucleoprotein M Facilitates Enterovirus Infection.

UNLABELLED: Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoproteinM(hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNPMis cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNPMrelocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNPMusing small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNPMis a general strategy utilized by picornaviruses to facilitate viral infection. IMPORTANCE: Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases, including respiratory ailments, dilated cardiomyopathy, and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we identify hnRNPMas a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNPMand redirects it to a step in the viral life cycle. We propose that cleavage of hnRNPMis a general strategy that picornaviruses use to facilitate infection.

Enterovirus 2Apro targets MDA5 and MAVS in infected cells.

UNLABELLED: RIG-I-like receptors (RLRs) MDA5 and RIG-I are key players in the innate antiviral response. Upon recognition of viral RNA, they interact with MAVS, eventually inducing type I interferon production. The interferon induction pathway is commonly targeted by viruses. How enteroviruses suppress interferon production is incompletely understood. MDA5 has been suggested to undergo caspase- and proteasome-mediated degradation during poliovirus infection. Additionally, MAVS is reported to be cleaved during infection with coxsackievirus B3 (CVB3) by the CVB3 proteinase 3C(pro), whereas MAVS cleavage by enterovirus 71 has been attributed to 2A(pro). As yet, a detailed examination of the RLR pathway as a whole during any enterovirus infection is lacking. We performed a comprehensive analysis of crucial factors of the RLR pathway, including MDA5, RIG-I, LGP2, MAVS, TBK1, and IRF3, during infection of CVB3, a human enterovirus B (HEV-B) species member. We show that CVB3 inhibits the RLR pathway upstream of TBK1 activation, as demonstrated by limited phosphorylation of TBK1 and a lack of IRF3 phosphorylation. Furthermore, we show that MDA5, MAVS, and RIG-I all undergo proteolytic degradation in CVB3-infected cells through a caspase- and proteasome-independent manner. We convincingly show that MDA5 and MAVS cleavages are both mediated by CVB3 2A(pro), while RIG-I is cleaved by 3C(pro). Moreover, we show that proteinases 2A(pro) and 3C(pro) of poliovirus (HEV-C) and enterovirus 71 (HEV-A) exert the same functions. This study identifies a critical role of 2A(pro) by cleaving MDA5 and MAVS and shows that enteroviruses use a common strategy to counteract the interferon response in infected cells. IMPORTANCE: Human enteroviruses (HEVs) are important pathogens that cause a variety of diseases in humans, including poliomyelitis, hand, foot, and mouth disease, viral meningitis, cardiomyopathy, and more. Like many other viruses, enteroviruses target the host immune pathways to gain replication advantage. The MDA5/MAVS pathway is responsible for recognizing enterovirus infections in the host cell and leads to expression of type I interferons (IFN-I), crucial antiviral signaling molecules. Here we show that three species of HEVs all employ the viral proteinase 2A (2A(pro)) to proteolytically target MDA5 and MAVS, leading to an efficient blockade upstream of IFN-I transcription. These observations suggest that MDA5/MAVS antagonization is an evolutionarily conserved and beneficial mechanism of enteroviruses. Understanding the molecular mechanisms of enterovirus immune evasion strategies will help to develop countermeasures to control infections with these viruses in the future.

Benzophenone C-glucosides and gallotannins from mango tree stem bark with broad-spectrum anti-viral activity.

The high mutation rate of RNA viruses has resulted in limitation of vaccine effectiveness and increased emergence of drug-resistant viruses. New effective antivirals are therefore needed to control of the highly mutative RNA viruses. The n-butanol fraction of the stem bark of Mangifera indica exhibited inhibitory activity against influenza neuraminidase (NA) and coxsackie virus 3C protease. Bioassay guided phytochemical study of M. indica stem bark afforded two new compounds including one benzophenone C-glycoside (4) and one xanthone dimer (7), together with eleven known compounds. The structures of these isolated compounds were elucidated on the basis of spectroscopic evidences and correlated with known compounds. Anti-influenza and anti-coxsackie virus activities were evaluated by determining the inhibition of anti-influenza neuraminidase (NA) from pandemic A/RI/5+/1957 H2N2 influenza A virus and inhibition of coxsackie B3 virus 3C protease, respectively. The highest anti-influenza activity was observed for compounds 8 and 9 with IC50 values of 11.9 and 9.2µM, respectively. Compounds 8 and 9 were even more potent against coxsackie B3 virus 3C protease, with IC50 values of 1.1 and 2.0µM, respectively. Compounds 8 and 9 showed weak cytotoxic effect against human hepatocellular carcinoma and human epithelial carcinoma cell lines through MTT assay.

The early communication stages between serine proteases and enterovirus capsids in the race for viral disintegration.

Serine proteases are important environmental contributors of enterovirus biocontrol. However, the structural features of molecular interaction accounting for the susceptibility of enteroviruses to proteases remains unexplained. Here, we describe the molecular mechanisms involved in the recruitment of serine proteases to viral capsids. Among the virus types used, coxsackievirus A9 (CVA9), but not CVB5 and echovirus 11 (E11), was inactivated by Subtilisin A in a host-independent manner, while Bovine Pancreatic Trypsin (BPT) only reduced CVA9 infectivity in a host-dependent manner. Predictive interaction models of each protease with capsid protomers indicate the main targets as internal disordered protein (IDP) segments exposed either on the 5-fold vertex (DE loop VP1) or at the 5/2-fold intersection (C-terminal end VP1) of viral capsids. We further show that a functional binding protease/capsid depends on both the strength and the evolution over time of protease-VP1 complexes, and lastly on the local adaptation of proteases on surrounding viral regions. Finally, we predicted three residues on CVA9 capsid that trigger cleavage by Subtilisin A, one of which may act as a sensor residue contributing to enzyme recognition on the DE loop. Overall, this study describes an important biological mechanism involved in enteroviruses biocontrol.

Crystal structures of the 3C proteases from Coxsackievirus B3 and B4.

Enteroviruses cause a wide range of disorders with varying presentations and severities, and some enteroviruses have emerged as serious public health concerns. These include Coxsackievirus B3 (CVB3), an active causative agent of viral myocarditis, and Coxsackievirus B4 (CVB4), which may accelerate the progression of type 1 diabetes. The 3C proteases from CVB3 and CVB4 play important roles in the propagation of these viruses. In this study, the 3C proteases from CVB3 and CVB4 were expressed in Escherichia coli and purified by affinity chromatography and gel-filtration chromatography. The crystals of the CVB3 and CVB4 3C proteases diffracted to 2.10 and 2.01 Šresolution, respectively. The crystal structures were solved by the molecular-replacement method and contained a typical chymotrypsin-like fold and a conserved His40-Glu71-Cys147 catalytic triad. Comparison with the structures of 3C proteases from other enteroviruses revealed high similarity with minor differences, which will guide the design of 3C-targeting inhibitors with broad-spectrum properties.

A Structural Investigation of the Interaction between a GC-376-Based Peptidomimetic PROTAC and Its Precursor with the Viral Main Protease of Coxsackievirus B3.

The conservation of the main protease in viral genomes, combined with the absence of a homologous protease in humans, makes this enzyme family an ideal target for developing broad-spectrum antiviral drugs with minimized host toxicity. GC-376, a peptidomimetic 3CL protease inhibitor, has shown significant efficacy against coronaviruses. Recently, a GC-376-based PROTAC was developed to target and induce the proteasome-mediated degradation of the dimeric SARS-CoV-2 3CLPro protein. Extending this approach, the current study investigates the application of the GC-376 PROTAC to the 3CPro protease of enteroviruses, specifically characterizing its interaction with CVB3 3CPro through X-ray crystallography, NMR (Nuclear Magnetic Resonance) and biochemical techniques. The crystal structure of CVB3 3CPro bound to the GC-376 PROTAC precursor was obtained at 1.9 Å resolution. The crystallographic data show that there are some changes between the binding of CVB3 3CPro and SARS-CoV-2 3CLPro, but the overall similarity is strong (RMSD on C-alpha 0.3 Å). The most notable variation is the orientation of the benzyloxycarbonyl group of GC-376 with the S4 subsite of the proteases. NMR backbone assignment of CVB3 3CPro bound and unbound to the GC-376 PROTAC precursor (80% and 97%, respectively) was obtained. This information complemented the investigation, by NMR, of the interaction of CVB3 3CPro with the GC-376 PROTAC, and its precursor allows us to define that the GC-376 PROTAC binds to CVB3 3CPro in a mode very similar to that of the precursor. The NMR relaxation data indicate that a quench of dynamics of a large part of the protein backbone involving the substrate-binding site and surrounding regions occurs upon GC-376 PROTAC precursor binding. This suggests that the substrate cavity, by sampling different backbone conformations in the absence of the substrate, is able to select the suitable one necessary to covalently bind the substrate, this being the latter reaction, which is the fundamental step required to functionally activate the enzymatic reaction. The inhibition activity assay showed inhibition potency in the micromolar range for GC-376 PROTAC and its precursor. Overall, we can conclude that the GC-376 PROTAC fits well within the binding sites of both proteases, demonstrating its potential as a broad-spectrum antiviral agent.

The coxsackievirus B 3C protease cleaves MAVS and TRIF to attenuate host type I interferon and apoptotic signaling.

The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3C(pro) cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3C(pro), occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3C(pro)-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3C(pro) cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.

Amiloride is a competitive inhibitor of coxsackievirus B3 RNA polymerase.

Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D(pol) in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D(pol) by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg(2+). Docking analysis suggested a binding site for amiloride and EIPA in 3D(pol), located in close proximity to one of the Mg(2+) ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.

Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy.

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy, but the mechanism of this pathology is unknown. Mutations in cytoskeletal proteins such as dystrophin cause hereditary dilated cardiomyopathy, but it is unclear if similar mechanisms underlie acquired forms of heart failure. We demonstrate here that purified Coxsackievirus protease 2A cleaves dystrophin in vitro as predicted by computer analysis. Dystrophin is also cleaved during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function. In vivo, dystrophin and the dystrophin-associated glycoproteins alpha-sarcoglycan and beta-dystroglycan are morphologically disrupted in infected myocytes. We suggest a molecular mechanism through which enteroviral infection contributes to the pathogenesis of acquired forms of dilated cardiomyopathy.

Polyamine Analog Diethylnorspermidine Restricts Coxsackievirus B3 and Is Overcome by 2A Protease Mutation In Vitro.

Enteroviruses, including Coxsackievirus B3 (CVB3), are pervasive pathogens that cause significant disease, including cardiomyopathies. Unfortunately, no treatments or vaccines are available for infected individuals. We identified the host polyamine pathway as a potential drug target, as inhibiting polyamine biosynthesis significantly reduces enterovirus replication in vitro and in vivo. Here, we show that CVB3 is sensitive to polyamine depletion through the polyamine analog diethylnorspermidine (DENSpm), which enhances polyamine catabolism through induction of polyamine acetylation. We demonstrate that CVB3 acquires resistance to DENSpm via mutation of the 2A protease, which enhances proteolytic activity in the presence of DENSpm. Resistance to DENSpm occurred via mutation of a non-catalytic site mutation and results in decreased fitness. These data demonstrate that potential for targeting polyamine catabolism as an antiviral target as well as highlight a potential mechanism of resistance.

Enterovirus type 71 2A protease functions as a transcriptional activator in yeast.

Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME) in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.