Structural diversity and biological role of the 5' untranslated regions of picornavirus.

The genomic arrangement of most picornavirus of the Picornaviridae family shares a similar monocistronic genomic pattern and a defining organizational feature. A defining feature of picornavirus is the presence of evolutionarily conserved and highly-structured RNA elements in untranslated regions (UTRs) at the genome' 5'and 3' ends, essential for viral replication and translation. Given the diversity and complexity of RNA structure and the limitations of molecular biology techniques, the functional characterization and biological significance of UTRs remain to be fully elucidated, especially for 5' UTR. Here, we summarize the current knowledge of the 5' UTR of picornavirus. This review focuses on the structural characterization and the biological function of the RNA secondary and tertiary structures in the 5' UTR of picornavirus. Understanding the role of the 5' UTR of picornavirus can provide a deep insight into the viral replication cycle and pathogenic mechanisms.

Structure of Senecavirus A 3C Protease Revealed the Cleavage Pattern of 3C Protease in Picornaviruses.

Senecavirus A (SVA) is an emerging picornavirus infecting porcine of all age groups and causing foot and mouth disease (FMD)-like symptoms. One of its key enzymes is the 3C protease (3Cpro), which is similar to other picornaviruses and essential for virus maturation by controlling polyprotein cleavage and RNA replication. In this study, we reported the crystal structure of SVA 3Cpro at a resolution of 1.9 Å and a thorough structural comparison against all published picornavirus 3Cpro structures. Using statistical and graphical visualization techniques, we also investigated the sequence specificity of the 3Cpro. The structure revealed that SVA 3Cpro adopted a typical chymotrypsin-like fold with the S1 subsite as the most conservative site among picornavirus 3Cpro. The surface loop, A1-B1 hairpin, adopted a novel conformation in SVA 3Cpro and formed a positively charged protrusion around S' subsites. Correspondingly, SVA scissile bonds preferred Asp rather than neutral amino acids at P3' and P4'. Moreover, SVA 3Cpro showed a wide range tolerance to P4 residue volume (acceptable range: 67 Å3 to 141 Å3), such as aromatic side chain, in contrast to other picornaviruses. In summary, our results provided valuable information for understanding the cleavage pattern of 3Cpro. IMPORTANCE Picornaviridae is a group of RNA viruses that harm both humans and livestock. 3Cpro is an essential enzyme for picornavirus maturation, which makes it a promising target for antiviral drug development and a critical component for virus-like particle (VLP) production. However, the current challenge in the development of antiviral drugs and VLP vaccines includes the limited knowledge of how subsite structure determines the 3Cpro cleavage pattern. Thus, an extensive comparative study of various picornaviral 3Cpro was required. Here, we showed the 1.9 Å crystal structure of SVA 3Cpro. The structure revealed similarities and differences in the substrate-binding groove among picornaviruses, providing new insights into the development of inhibitors and VLP.

Virus structure and structure-based antivirals.

Structure-based antiviral developments in the past two years have been dominated by the structure determination and inhibition of SARS-CoV-2 proteins and new lead molecules for picornaviruses. The SARS-CoV-2 spike protein has been targeted successfully with antibodies, nanobodies, and receptor protein mimics effectively blocking receptor binding or fusion. The two most promising non-structural proteins sharing strong structural and functional conservation across virus families are the main protease and the RNA-dependent RNA polymerase, for which design and reuse of broad range inhibitors already approved for use has been an attractive avenue. For picornaviruses, the increasing recognition of the transient expansion of the capsid as a critical transition towards RNA release has been targeted through a newly identified, apparently widely conserved, druggable, interprotomer pocket preventing viral entry. We summarize some of the key papers in these areas and ponder the practical uses and contributions of molecular modeling alongside empirical structure determination.

The Picornavirus Precursor 3CD Has Different Conformational Dynamics Compared to 3Cpro and 3Dpol in Functionally Relevant Regions.

Viruses have evolved numerous strategies to maximize the use of their limited genetic material, including proteolytic cleavage of polyproteins to yield products with different functions. The poliovirus polyprotein 3CD is involved in important protein-protein, protein-RNA and protein-lipid interactions in viral replication and infection. It is a precursor to the 3C protease and 3D RNA-dependent RNA polymerase, but has different protease specificity, is not an active polymerase, and participates in other interactions differently than its processed products. These functional differences are poorly explained by the known X-ray crystal structures. It has been proposed that functional differences might be due to differences in conformational dynamics between 3C, 3D and 3CD. To address this possibility, we conducted nuclear magnetic resonance spectroscopy experiments, including multiple quantum relaxation dispersion, chemical exchange saturation transfer and methyl spin-spin relaxation, to probe conformational dynamics across multiple timescales. Indeed, these studies identified differences in conformational dynamics in functionally important regions, including enzyme active sites, and RNA and lipid binding sites. Expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not observed in 3C and 3D alone despite having nearly identical lowest-energy structures.

Picornaviruses: A View from 3A.

Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3Dpol (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the Picornaviridae and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.

The In Silico Prediction of Hotspot Residues that Contribute to the Structural Stability of Subunit Interfaces of a Picornavirus Capsid.

The assembly of picornavirus capsids proceeds through the stepwise oligomerization of capsid protein subunits and depends on interactions between critical residues known as hotspots. Few studies have described the identification of hotspot residues at the protein subunit interfaces of the picornavirus capsid, some of which could represent novel drug targets. Using a combination of accessible web servers for hotspot prediction, we performed a comprehensive bioinformatic analysis of the hotspot residues at the intraprotomer, interprotomer and interpentamer interfaces of the Theiler's murine encephalomyelitis virus (TMEV) capsid. Significantly, many of the predicted hotspot residues were found to be conserved in representative viruses from different genera, suggesting that the molecular determinants of capsid assembly are conserved across the family. The analysis presented here can be applied to any icosahedral structure and provides a platform for in vitro mutagenesis studies to further investigate the significance of these hotspots in critical stages of the virus life cycle with a view to identify potential targets for antiviral drug design.

Structural insights into Acyl-coenzyme A binding domain containing 3 (ACBD3) protein hijacking by picornaviruses.

Many picornaviruses hijack the Golgi resident Acyl-coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4-kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4-phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small-angle X-ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.

Hepatitis A Virus Capsid Structure.

Hepatitis A virus (HAV) has been enigmatic, evading detailed structural analysis for many years. Its recently determined high-resolution structure revealed an angular surface without the indentations often characteristic of receptor-binding sites. The viral protein 1 (VP1) ß-barrel shows no sign of a pocket factor and the amino terminus of VP2 displays a "domain swap" across the pentamer interface, as in a subset of mammalian picornaviruses and insect picorna-like viruses. Structure-based phylogeny confirms this placement. These differences suggest an uncoating mechanism distinct from that of enteroviruses. An empty capsid structure reveals internal differences in VP0 and the VP1 amino terminus connected with particle maturation. An HAV/Fab complex structure, in which the antigen-binding fragment (Fab) appears to act as a receptor-mimic, clarifies some historical epitope mapping data, but some remain difficult to reconcile. We still have little idea of the structural features of enveloped HAV particles.

Genetic characterization of a second novel picornavirus from an amphibian host, smooth newt (Lissotriton vulgaris).

In this study, a novel picornavirus was identified in faecal samples from smooth newts (Lissotriton vulgaris). The complete genome of picornavirus strain newt/II-5-Pilis/2014/HUN (KX463670) is 7755 nt long with type-IV IRES and has 39.6% aa sequence identity in the protein P1 to the corresponding protein of bat picornavirus (KJ641686, unassigned) and 42.7% and 53.5% aa sequence identity in the 2C and 3CD protein, respectively, to oscivirus (GU182410, genus Oscivirus). Interestingly, the L-protein of newt/II-5-Pilis/2014/HUN has conserved aa motifs that are similar to those found in phosphatase-1 catalytic (PP1C) subunit binding region (pfam10488) proteins. This second amphibian-origin picornavirus could represent a novel species and could be a founding member of a potential novel picornavirus genus.

The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs.

Many nonenveloped viruses engage host receptors that initiate capsid conformational changes necessary for genome release. Structural studies on the mechanisms of picornavirus entry have relied on in vitro approaches of virus incubated at high temperatures or with excess receptor molecules to trigger the entry intermediate or A-particle. We have induced the coxsackievirus B3 entry intermediate by triggering the virus with full-length receptors embedded in lipid bilayer nanodiscs. These asymmetrically formed A-particles were reconstructed using cryo-electron microscopy and a direct electron detector. These first high-resolution structures of a picornavirus entry intermediate captured at a membrane with and without imposing icosahedral symmetry (3.9 and 7.8 Å, respectively) revealed a novel A-particle that is markedly different from the classical A-particles. The asymmetric receptor binding triggers minimal global capsid expansion but marked local conformational changes at the site of receptor interaction. In addition, viral proteins extrude from the capsid only at the site of extensive protein remodeling adjacent to the nanodisc. Thus, the binding of the receptor triggers formation of a unique site in preparation for genome release.

Intra-family differences in efficacy of inactivation of small, non-enveloped viruses.

The use of specific model viruses for validating viral purification process steps and for assessing the efficacies of viral disinfectants is based, in part, on the assumption that viral susceptibilities to such treatments will be similar for different members, including different genera, within a given viral family. This assumption is useful in cases where cell-based infectivity assays or laboratory strains for the specific viruses of interest might not exist. There are some documented cases, however, where exceptions to this assumption exist. In this paper, we discuss some of the more striking cases of intra-family differences in susceptibilities to inactivation steps used for downstream viral purification steps in biologics manufacture (e.g. heat inactivation, low pH, and guanidinium hydrochloride inactivation) and to specific viral disinfectants (e.g. alcohols, hydrogen peroxide, and quaternary ammonium-containing disinfectants) that might be employed for facility/equipment disinfection. The results suggest that care should be taken when extrapolating viral inactivation susceptibilities from specific model viruses to different genera or even to different members of the same genus. This should be taken into consideration by regulatory agencies and biologics manufacturers designing viral clearance and facility disinfection validation studies, and developers and evaluators of viral disinfectants.

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.

A diarrheic chicken simultaneously co-infected with multiple picornaviruses: Complete genome analysis of avian picornaviruses representing up to six genera.

In this study all currently known chicken picornaviruses including a novel one (chicken phacovirus 1, KT880670) were identified by viral metagenomic and RT-PCR methods from a single specimen of a diarrheic chicken suffering from a total of eight picornavirus co-infections, in Hungary. The complete genomes of six picornaviruses were determined and their genomic and phylogenetic characteristics and UTR RNA structural models analyzed in details. Picornaviruses belonged to genera Sicinivirus (the first complete genome), Gallivirus, Tremovirus, Avisivirus and "Orivirus" (two potential genotypes). In addition, the unassigned phacoviruses were also detected in multiple samples of chickens in the USA. Multiple co-infections promote and facilitate the recombination and evolution of picornaviruses and eventually could contribute to the severity of the diarrhea in chicken, in one of the most important food sources of humans.

NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII.

Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap-binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut-off host-cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot-and-mouth disease virus (FMDV) leader proteinase (Lb(pro)), human rhinovirus 2 (HRV2) 2A proteinase (2A(pro)) and coxsackievirus B4 (CVB4) 2A(pro) with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed (13)C/(15) N sequential backbone assignment of human eIF4GII residues 551-745 and examined their binding to murine eIF4E. eIF4GII551-745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain-like Lb(pro) only forms a stable complex with eIF4GII(551-745) in the presence of eIF4E, with KD values in the low nanomolar range; Lb(pro) contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin-like 2A(pro) from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K(D) values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut-off.

Secondary structure analysis of swine pasivirus (family Picornaviridae) RNA reveals a type-IV IRES and a parechovirus-like 3' UTR organization.

The potential RNA structures of the 5' and 3' untranslated regions (UTRs) and cis-acting replication elements (CREs) of a novel pasivirus (PaV) genotype (family Picornaviridae) were analysed. PaV-A3 (KM259923) was identified in a faecal sample from a domestic pig in Hungary with posterior paraplegia of unknown etiology. Based on likely structural features of the 5' UTR, the pasiviruses were inferred to possess Hepacivirus/Pestivirus-like type-IV IRES. The pasivirus CRE was mapped to the 2B genome region, similar to Ljungan virus. The secondary RNA structure of the pasivirus 3' UTR was structurally similar to that of human parechoviruses. The genome, CRE, and 3' UTR of pasiviruses provide further evidence of the common origin of the members of the genera Parechovirus and Pasivirus, although their different 5' UTR IRES types suggest that a recombination event occurred during the divergence these viruses.

Hepatitis A virus and the origins of picornaviruses.

Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2 'domain swap' characteristic of insect picorna-like viruses, and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between 'modern' picornaviruses and the more 'primitive' precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.

A sialic acid binding site in a human picornavirus.

The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.

[Structure and function of 3'- untranslated region in picornavirus].

Both sides of the picornavirus genome have 5'-untranslated region (5'UTR) and 3'- untranslated region (3'UTR). This study demontrated that both the 5'-and 3'-UTR can form complex structures, such as stem-loop, clover and pseudoknot structure, These structures play an important role in the regulaton of the replication and translation of the viruses. This article reviewed the progress of research on the structure and function of picornavirus' 3'-UTR over recent years.

Coxsackievirus cloverleaf RNA containing a 5' triphosphate triggers an antiviral response via RIG-I activation.

Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5' triphosphate (5'ppp)-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3), a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5'ppp. We show here that a 5'ppp-containing cloverleaf (CL) RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand.