Long-Range Communication between Different Functional Sites in the Picornaviral 3C Protein.
ABSTRACT: 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.
Project description:Picornaviruses replicate their RNA genomes through a highly conserved mechanism that involves an interaction between the principal viral protease (3C(pro)) and the 5'-UTR region of the viral genome. The 3C(pro) catalytic site is the target of numerous replication inhibitors. This paper describes the first structural model of a complex between a picornaviral 3C(pro) and a region of the 5'-UTR, stem-loop D (SLD). Using human rhinovirus as a model system, we have combined NMR contact information, small-angle X-ray scattering (SAXS) data, and previous mutagenesis results to determine the shape, position and relative orientation of the 3C(pro) and SLD components. The results clearly identify a 1:1 binding stoichiometry, with pronounced loops from each molecule providing the key binding determinants for the interaction. Binding between SLD and 3C(pro) induces structural changes in the proteolytic active site that is positioned on the opposite side of the protease relative to the RNA/protein interface, suggesting that subtle conformational changes affecting catalytic activity are relayed through the protein.
Project description:Human enterovirus 71 (EV71) is the major pathogen that causes hand, foot and mouth disease that particularly affects young children. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date. The genome of EV71 consists of a positive sense, single-stranded RNA of ?7400 bp, encoding a large precursor polyprotein that requires proteolytic processing to generate mature viral proteins. The proteolytic processing mainly depends on EV71 3C protease (3C(pro)) that possesses both proteolysis and RNA binding activities, which enable the protease to perform multiple tasks in viral replication and pathogen-host interactions. The central roles played by EV71 3C(pro) make it an appealing target for antiviral drug development. We determined the first crystal structure of EV71 3C(pro) and analyzed its enzymatic activity. The crystal structure shows that EV71 3C(pro) has a typical chymotrypsin-like fold that is common in picornaviral 3C(pro). Strikingly, we found an important surface loop, also denoted as ?-ribbon, which adopts a novel open conformation in EV71 3C(pro). We identified two important residues located at the base of the ?-ribbon, Gly123 and His133, which form hinges that govern the intrinsic flexibility of the ribbon. Structure-guided mutagenesis studies revealed that the hinge residues are important to EV71 3C(pro) proteolytic activities. In summary, our work provides the first structural insight into EV71 3C(pro), including a mobile ?-ribbon, which is relevant to the proteolytic mechanism. Our data also provides a framework for structure-guided inhibitor design against EV71 3C(pro).
Project description:Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3C(pro)). However, PABP is cleaved by HAV 3C(pro) in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5' nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.
Project description:Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases and peptide substrates. To deal with the disadvantages associated with in vitro assays, we modified a cell-based protease assay for picornavirus proteases. The assay is based on the induction of expression of a firefly luciferase reporter by a chimeric transcription factor in which the viral protease and cleavage sites are inserted between the GAL4 binding domain and the VP16 activation domain. Firefly luciferase expression is dependent on cleavage of the transcription factor by the viral protease. This biosafe assay enables testing the effect of compounds on protease activity in cells while circumventing the need for infection. We designed the assay for 3C proteases (3C(pro)) of various enteroviruses as well as of viruses of several other picornavirus genera, and show that the assay is amenable for use in a high-throughput setting. Furthermore, we show that the spectrum of activity of 3C(pro) inhibitor AG7088 (rupintrivir) not only encompasses enterovirus 3C(pro) but also 3C(pro) of foot-and-mouth disease virus (FMDV), an aphthovirus. In contrary, AG7404 (compound 1), an analogue of AG7088, had no effect on FMDV 3C(pro) activity, for which we provide a structural explanation.
Project description:During viral infections, some viruses subvert the host proteins to promote the translation or RNA replication with their protease-mediated cleavage. Poly (A)-binding protein (PABP) is a target for several RNA viruses; however, the impact of duck hepatitis A virus (DHAV) on PABP remains unknown. In this study, we demonstrated for the first time that DHAV infection stimulates a decrease in endogenous PABP and generates two cleavage fragments. On the basis of in vitro cleavage assays, an accumulation of PABP cleavage fragments was detected in duck embryo fibroblast (DEF) cell extracts incubated with functional DHAV 3C protease. In addition, DHAV 3C protease was sufficient for the cleavage of recombinant PABP without the assistance of other eukaryotic cellular cofactors. Furthermore, using site-directed mutagenesis, our data demonstrated a 3C protease cleavage site located between Q367 and G368 in duck PABP. Moreover, the knockdown of PABP inhibited the production of viral RNA, and the C-terminal domain of PABP caused a reduction in viral replication compared to the N-terminal domain. Taken together, these findings suggested that DHAV 3C protease mediates the cleavage of PABP, which may be a strategy to manipulate viral replication.
Project description:Oligonucleotides as short as 6?nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C(pro)). Inhibition assays in vitro identified the hexanucleotide 5'-GGGGGT-3' (G(5)T) as a 3C(pro) protease inhibitor. Using (1)H NMR spectroscopy, G(5)T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of (1)H, (15)N-HSQC experiments the binding site for G(5)T was located to the C-terminal ?-barrel of HAV 3C(pro). Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G(5)T-binding site, nor does G(5)T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid-protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.
Project description:Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3Dpol. Over the past decade we have made tremendous advances in our understanding of 3Dpol structure and function, including the discovery of a novel mechanism for closing the active site that allows these viruses to easily fine tune replication fidelity and quasispecies distributions. This review summarizes current knowledge of picornaviral polymerase structure and how the enzyme interacts with RNA and other viral proteins to form stable and processive elongation complexes. The picornaviral RdRPs are among the smallest viral polymerases, but their fundamental molecular mechanism for catalysis appears to be generally applicable as a common feature of all positive strand RNA virus polymerases.
Project description:A series of pyrazolone compounds as possible SARS-CoV 3CL protease inhibitors were designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide in which several showed potent inhibition against the 3CL protease. Interestingly, one of the inhibitors was also active against 3C protease from coxsackievirus B3. These inhibitors could be potentially developed into anti-coronaviral and anti-picornaviral agents.
Project description:3C proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. In addition, 3C proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. The activity of 3C proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. To date, cytotoxic effects have been studied for several 3C proteases, all of which induce apoptosis. This study for the first time describes the cytotoxic effect of 3C protease of human hepatitis A virus (3Cpro), the only proteolytic enzyme of the virus.Individual expression of 3Cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor (z-VAD-fmk) and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3C proteases. The cell survival was also not affected by the inhibitors of cysteine proteases (z-FA-fmk) and RIP1 kinase (necrostatin-1), critical enzymes involved in non-apoptotic cell death. A substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. The lysosomal protein Lamp1 and GTPases Rab5, Rab7, Rab9, and Rab11 were associated with the vacuolar membranes. The vacuolization was completely blocked by the vacuolar ATPase inhibitor (bafilomycin A1) and did not depend on the activity of the principal factors of endosomal transport, GTPases Rab5 and Rab7, as well as on autophagy and macropinocytosis.3Cpro, apart from other picornaviral 3C proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3Cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. The data obtained demonstrate previously undocumented morphological characters of the 3Cpro-induced cell death, which can reflect unknown aspects of the human hepatitis A virus-host cell interaction.
Project description:Enterovirus 71 (EV71) is an emerging pathogen causing hand, foot, and mouth disease (HFMD) and fatal neurological diseases in infants and young children due to their underdeveloped immunocompetence. EV71 infection can induce cellular apoptosis through a variety of pathways, which promotes EV71 release. The viral protease 3C plays an important role in EV71-induced apoptosis. However, the molecular mechanism responsible for 3C-triggered apoptosis remains elusive. Here, we found that EV71 3C directly interacted with PinX1, a telomere binding protein. Furthermore, 3C cleaved PinX1 at the site of Q50-G51 pair through its protease activity. Overexpression of PinX1 reduced the level of EV71-induced apoptosis and EV71 release, whereas depletion of PinX1 by small interfering RNA promoted apoptosis induced by etoposide and increased EV71 release. Taken together, our study uncovered a mechanism that EV71 utilizes to promote host cell apoptosis through cleavage of cellular protein PinX1 by 3C. IMPORTANCE:EV71 3C plays an important role in processing viral proteins and interacting with host cells. In this study, we showed that 3C promoted apoptosis through cleaving PinX1, a telomere binding protein, and that this cleavage facilitated EV71 release. Our study demonstrated that PinX1 plays an important role in EV71 release and revealed a novel mechanism that EV71 utilizes to induce apoptosis. This finding is important in understanding EV71-host cell interactions and has potential impact on understanding other enterovirus-host cell interactions.