Structural basis for cloverleaf RNA-initiated viral genome replication.

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

A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication.

As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions.

CD155 immunoregulation as a target for natural killer cell immunotherapy in glioblastoma.

Natural killer (NK) cells are powerful immune effectors, modulating their anti-tumor function through a balance activating and inhibitor ligands on their cell surface. Though still emerging, cancer immunotherapies utilizing NK cells are proving promising as a modality for the treatment of a number of solid tumors, including glioblastoma (GBM) and other gliomas, but are often limited due to complex immunosuppression associated with the GBM tumor microenvironment which includes overexpression of inhibitory receptors on GBM cells. CD155, or poliovirus receptor (PVR), has recently emerged as a pro-tumorigenic antigen, overexpressed on GBM and contributing to increased GBM migration and aggressiveness. CD155 has also been established as an immunomodulatory receptor, able to both activate NK cells through interactions with CD226 (DNAM-1) and CD96 and inhibit them through interaction with TIGIT. However, NK cell TIGIT expression has been shown to be upregulated in cancer, establishing CD155 as a predominantly inhibitory receptor within the context of GBM and other solid tumors, and rendering it of interest as a potential target for antigen-specific NK cell-based immunotherapy. This review will explore the function of CD155 within GBM as it relates to tumor migration and NK cell immunoregulation, as well as pre-clinical and clinical targeting of CD155/TIGIT and the potential that this pathway holds for the development of emerging NK cell-based immunotherapies.

Bio-redox potential of Hyphaene thebaica in bio-fabrication of ultrafine maghemite phase iron oxide nanoparticles (Fe2O3 NPs) for therapeutic applications.

Maghemite (Fe2O3-NPs) nanoparticles were synthesized by a convenient, green and cost effective method using aqueous fruit extracts of Hyphaene thebaica. Different techniques like FTIR, XRD, UV-Vis, Raman, HR-TEM, EDS. SAED, Zeta potential were used to establish the nature of Fe2O3-NPs, while the therapeutic properties were studied using different biological assays including antiviral, antibacterial, antifungal, antioxidant and enzyme inhibition assays. XRD pattern revealed sharp peaks and a crystalline nature of Fe2O3-NPs. HR-TEM revealed quasi-spherical and cuboidal morphologies, while the particle size in ~10 nm. FTIR indicated a sharp peak centered at ~444 cm-1 which is the characteristic FeO band vibration. SAED pattern indicated the crystalline nature while EDS also confirmed the synthesis of Fe2O3 NPs. Zeta potential was obtained in different solvents and physiological buffers indicating highest value in water (-26.5 mV) and lowest in DMSO (-15.8 mV). Tested bacterial strains, Bacillus subtilis was found to be inhibited significantly. Aspergillus flavus appeared to be susceptible to all of the tested concentration of Fe2O3 NPs. Maximum 40.78% FRSA was obtained at 400 µg/mL. Cell culture based studies on RD cells and L20B cells indicated reduction in viability of cells with increase concentration of Fe2O3 NPs. Moderate inhibition of polio virus-1 and polio virus-2 was observed, after culturing the virus in the L20B cells. Excellent Protein Kinase (PK) inhibition was revealed. Hemolytic potential and cytotoxic potential was indicated to be dose dependent. In conclusion, the present report for the first time reports the synthesis of Fe2O3 NPs from H. thebaica fruits and reveals their biomedical potential including antiviral potential.

A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1.

The replication of many positive-strand RNA viruses [(+)RNA viruses] depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with a severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that the Arf-activating property, but not the primary structure of the Sec7 domain, is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, which is dependent not only on direct interaction of the protein with the viral protein 3A but also on determinants located in the noncatalytic C-terminal domains of GBF1. Such a double-targeting mechanism explains the previous observations of the remarkable tolerance of different levels of GBF1-3A interaction by the virus and likely constitutes an important element of the resilience of viral replication.IMPORTANCE Enteroviruses are a vast group of viruses associated with diverse human diseases, but only two of them could be controlled with vaccines, and effective antiviral therapeutics are lacking. Here, we investigated in detail the contribution of a cellular protein, GBF1, in the replication of poliovirus, a representative enterovirus. GBF1 supports the functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our results demonstrate that the virus requires a limited subset of the normal GBF1 functions and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad-spectrum antiviral therapeutics.

Oncolytic Activity of the Vaccine Strain of Type 3 Poliovirus on the Model of Rat Glioma C6 Cells.

Rat glioma cell line C6 expressing human poliovirus receptor (PVR) and susceptible to polioviruses (C6-PVR-BFP) was used to produce a clone with knockout of IFNα/ß (Ifnar1) receptor subunit 1 gene (Ifnar1). The sensitivity of C6-PVR-BFP cells to the vaccine strain of poliovirus type 3 (PV3) depended on the signaling pathways of the cell response to type 1 IFN. Using the model of subcutaneous tumor xenografts, we demonstrated oncolytic activity of PV3 against C6-PVR-BFP cells that depended on the expression of PVR and increased considerably upon disturbances in IFN response pathways.

Poliovirus Evolution toward Independence from the Phosphatidylinositol-4 Kinase III ß/Oxysterol-Binding Protein Family I Pathway.

Phosphatidylinositol-4 kinase III ß (PI4KB) and oxysterol-binding protein (OSBP) family I provide a conserved host pathway required for enterovirus replication. Here, we analyze the role and essentiality of this pathway in enterovirus replication. Phosphatidylinositol 4-phosphate (PI4P) production and cholesterol accumulation in the replication organelle (RO) are severely suppressed in cells infected with a poliovirus (PV) mutant isolated from a PI4KB-knockout cell line (RD[Δ PI4KB]). Major determinants of the mutant for infectivity in RD(Δ PI4KB) cells map to the A5270U(3A-R54W) and U3881C(2B-F17L) mutations. The 3A mutation is required for PI4KB-independent development of RO. The 2B mutation rather sensitizes PV to PI4KB/OSBP inhibitors by itself but confers substantially complete resistance to the inhibitors with the 3A mutation. The 2B mutation also confers hypersensitivity to interferon alpha treatment on PV. These suggest that the PI4KB/OSBP pathway is not necessarily essential for enterovirus replication in vitro. This work supports a two-step resistance model of enterovirus to PI4KB/OSBP inhibitors involving unique recessive epistasis of 3A and 2B and offers insights into a potential evolutionary pathway of enterovirus toward independence from the PI4KB/OSBP pathway.

Eligibility of Feline Calicivirus for a Surrogate of Human Norovirus in Comparison with Murine Norovirus, Poliovirus and Coxsackievirus.

Feline calicivirus (FCV) is frequently used as a surrogate of human norovirus. We investigated eligibility of FCV for anti-viral assay by investigating the stability of infectivity and pH sensitivity in comparison with other viruses. We found that infectivities of FCV and murine norovirus (MNV) are relatively unstable in infected cells compared with those of coxsackievirus (CoV) and poliovirus (PoV) , suggesting that FCV and MNV have vulnerability. Western blotting indicated that inactivation of FCV was not due to viral protein degradation. We also demonstrated sensitivity of FCV to low pH, the 50% inhibitory pH value being ca. 3.9. Since human norovirus is thought to persist longer, in infectivity and to be a resistant virus, CoV, which is robust and not restrained in use as PoV, may be more appropriate as a test virus for disinfectants, rather than FCV and MNV.

Replicase-mediated shielding of the poliovirus replicative double-stranded RNA to avoid recognition by MDA5.

Replication of the positive-strand RNA viruses generates double-stranded RNAs (dsRNAs) that are recognized by host pattern recognition receptors (PRRs) to trigger innate immune responses. Formation of the viral replication complex (RC) has been thought to shield dsRNA from being recognized by innate sensors. To elucidate the RC-mediated evasion of innate recognition, we selected poliovirus (PV) as a model. We first found that RNAs generated during PV replication were potent interferon (IFN) inducers upon transfection, while there was no obvious IFN production detected in PV-replicating cells. PV replication did not interfere with IFN production when IFN agonists were synchronously introduced with the replicating PV RNAs, and in PV-infected cells, IFN agonist-induced IFN production was only moderately impaired but not completely abolished. When PV-infected cells were in situ permeabilized by digitonin, viral dsRNAs were readily detected by an anti-dsRNA antibody and were resistant to RNase III digestion. When digitonin-permeabilized cells were further solubilized by 1 % triton X-100, the dsRNAs of PV became sensitive to RNase III digestion. A co-localization study showed that PV dsRNA did not co-localize with MDA5 in virally infected cells. Given that the PV replication complex is protruding single-membrane and tubular in form, viral replicative dsRNAs are probably shielded by the replication complex or the viral replicase to avoid being accessed by RNase III and MDA5. We propose that the replication complex- or replicase-mediated shielding of dsRNA may act as a means for innate evasion.

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.

Risk factors and short-term projections for serotype-1 poliomyelitis incidence in Pakistan: A spatiotemporal analysis.

BACKGROUND: Pakistan currently provides a substantial challenge to global polio eradication, having contributed to 73% of reported poliomyelitis in 2015 and 54% in 2016. A better understanding of the risk factors and movement patterns that contribute to poliovirus transmission across Pakistan would support evidence-based planning for mass vaccination campaigns. METHODS AND FINDINGS: We fit mixed-effects logistic regression models to routine surveillance data recording the presence of poliomyelitis associated with wild-type 1 poliovirus in districts of Pakistan over 6-month intervals between 2010 to 2016. To accurately capture the force of infection (FOI) between districts, we compared 6 models of population movement (adjacency, gravity, radiation, radiation based on population density, radiation based on travel times, and mobile-phone based). We used the best-fitting model (based on the Akaike Information Criterion [AIC]) to produce 6-month forecasts of poliomyelitis incidence. The odds of observing poliomyelitis decreased with improved routine or supplementary (campaign) immunisation coverage (multivariable odds ratio [OR] = 0.75, 95% confidence interval [CI] 0.67-0.84; and OR = 0.75, 95% CI 0.66-0.85, respectively, for each 10% increase in coverage) and increased with a higher rate of reporting non-polio acute flaccid paralysis (AFP) (OR = 1.13, 95% CI 1.02-1.26 for a 1-unit increase in non-polio AFP per 100,000 persons aged <15 years). Estimated movement of poliovirus-infected individuals was associated with the incidence of poliomyelitis, with the radiation model of movement providing the best fit to the data. Six-month forecasts of poliomyelitis incidence by district for 2013-2016 showed good predictive ability (area under the curve range: 0.76-0.98). However, although the best-fitting movement model (radiation) was a significant determinant of poliomyelitis incidence, it did not improve the predictive ability of the multivariable model. Overall, in Pakistan the risk of polio cases was predicted to reduce between July-December 2016 and January-June 2017. The accuracy of the model may be limited by the small number of AFP cases in some districts. CONCLUSIONS: Spatiotemporal variation in immunization performance and population movement patterns are important determinants of historical poliomyelitis incidence in Pakistan; however, movement dynamics were less influential in predicting future cases, at a time when the polio map is shrinking. Results from the regression models we present are being used to help plan vaccination campaigns and transit vaccination strategies in Pakistan.

Oncolytic Virotherapy for the Treatment of Malignant Glioma.

Malignant glioma is the most common primary brain tumor and carries a grim prognosis, with a median survival of just over 14 months. Given the poor outcomes with standard-of-care treatments, novel treatment strategies are needed. The concept of virotherapy for the treatment of malignant tumors dates back more than a century and can be divided into replication-competent oncolytic viruses and replication-deficient viral vectors. Oncolytic viruses are designed to selectively target, infect, and replicate in tumor cells, while sparing surrounding normal brain. A host of oncolytic viruses has been evaluated in early phase human trials with promising safety results, but none has progressed to phase III trials. Despite the 25 years that has passed since the initial publication of genetically engineered oncolytic viruses for the treatment of glioma, much remains to be learned about the use of this therapy, including its mechanism of action, optimal treatment paradigm, appropriate targets, and integration with adjuvant agents. Oncolytic viral therapy for glioma remains promising and will undoubtedly impact the future of patient care.

Glutathione is a highly efficient thermostabilizer of poliovirus Sabin strains.

Glutathione (GSH) is the most abundant thiol peptide in animal cells and has a critical role in antioxidation. GSH was reported to be essential for stabilization of some enteroviruses, including poliovirus (PV), during viral morphogenesis. Here, we explored the potential use of GSH as a thermostabilizer of oral poliomyelitis vaccine (OPV) formulations. GSH significantly protected the three types of PV from heat-inactivation in a concentration-dependent manner. At a GSH concentration of 20mM, nearly complete protection was observed against heating temperatures up to 53°C for 2min.GSH also markedly protected PV1 from heat-inactivation and this up to 6 h at temperatures of 44°C and 46°C and 3 h at 48°C. The fact that GSH is naturally present at high concentration in the human body makes it an efficient candidate stabilizer for OPV formulations.

Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

Cytotoxicity and antiviral activity of electrochemical - synthesized silver nanoparticles against poliovirus.

Silver nanoparticles (AgNPs) have been proven to have noticeable cytotoxicity in vitro and antiviral activity against some types of enveloped viruses. This paper presents the cytotoxicity and antiviral activity of pure AgNPs synthesized by the electrochemical method, towards cell culture and poliovirus (a non-enveloped virus). Prepared AgNPs were characterized by ultraviolet-visible spectroscopy, energy-dispersive X-ray spectroscopy and transmission electron microscopy. Before incubation with poliovirus, different concentrations of AgNPs were added to human rhabdomyosarcoma (RD) cell monolayers seeded in 96 well plates for testing their cytotoxicity. The in vitro cytotoxicity and anti-poliovirus activity of AgNPs were daily assessed for cytopathic effect (CPE) through inverted light microscopy. CPE in the tested wells was determined in comparison with those in wells of negative and positive control. Structure analysis showed that AgNPs were formed with a quasi-spherical shape with mean size about 7.1nm and high purity. No CPE of RD cells was seen in wells at the time point of 48h post-incubation with AgNPs at concentration up to 100ppm. The anti-poliovirus activity of AgNPs was determined at 3.13ppm corresponding to the viral concentration of 1TCID50 (Tissue Culture Infective Dose) after 30min, and 10TCID50 after 60min, the cell viability was found up to 98% at 48h post-infection, with no CPE found. Whereas, a strong CPE of RD cells was found at 48h post-infection with the mixture of AgNPs and poliovirus at concentration of 100TCID50, and in wells of positive controls. With mentioned advantages, electrochemical-synthesized AgNPs are promising candidate for advanced biomedical and disinfection applications.

A comparative analysis of virucidal efficiency of biocide agents.

The main groups of biocide agents used for inactivation of bacteria and viruses were studied for their virucidal activity against enveloped (HIV, viral hepatitis C, influenza virus A) and non-enveloped viruses (poliovirus, adenovirus). Their efficiency was analyzed. Quarterly ammonium compounds (QAC) themselves are not able to properly inactivate non-enveloped viruses. However, they can be successfully applied in combination with other biocides (guanidines, aldehydes). Effective composition of QAC with amines and guanidines provided inactivation of viruses (4.0 lgTCID50) in concentrations of 0.166-0.280% for non-enveloped viruses and 0.080-00.185% for enveloped viruses. The combination of QAC with aldehydes is especially effective (0.04-0.64% for non-enveloped viruses). The virucidal efficiency does not directly depend on the QAC concentration in the chemical disinfectants.

Sensitivity of C6 Glioma Cells Carrying the Human Poliovirus Receptor to Oncolytic Polioviruses.

A humanized line of rat C6 glioma cells expressing human poliovirus receptor was obtained and tested for the sensitivity to oncolytic effects of vaccine strains of type 1, 2, and 3 polioviruses. Presentation of the poliovirus receptor on the surface of C6 glioma cells was shown to be a necessary condition for the interaction of cells with polioviruses, but insufficient for complete poliovirus oncolysis.

Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections.

UNLABELLED: Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5' tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg) and the 5' end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis) in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections. IMPORTANCE: Picornaviruses are one of the most prevalent groups of viruses that infect humans and livestock worldwide. These viruses include the human pathogens belonging to the Enterovirus genus, such as poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus. Diseases caused by enteroviruses pose a major problem for public health and have significant economic impact. Poliovirus can cause paralytic poliomyelitis. CVB3 can cause hand, foot, and mouth disease and myocarditis. Human rhinovirus is the causative agent of the common cold, which has a severe economic impact due to lost productivity and severe health consequences in individuals with respiratory dysfunction, such as asthma. By gaining a better understanding of the enterovirus replication cycle, antiviral drugs against enteroviruses may be developed. Here, we report that the absence of the cellular enzyme TDP2 can significantly decrease viral yields of poliovirus, CVB3, and human rhinovirus, making TDP2 a potential target for an antiviral against enterovirus infections.

Conformational Ensemble of the Poliovirus 3CD Precursor Observed by MD Simulations and Confirmed by SAXS: A Strategy to Expand the Viral Proteome?

The genomes of RNA viruses are relatively small. To overcome the small-size limitation, RNA viruses assign distinct functions to the processed viral proteins and their precursors. This is exemplified by poliovirus 3CD protein. 3C protein is a protease and RNA-binding protein. 3D protein is an RNA-dependent RNA polymerase (RdRp). 3CD exhibits unique protease and RNA-binding activities relative to 3C and is devoid of RdRp activity. The origin of these differences is unclear, since crystal structure of 3CD revealed "beads-on-a-string" structure with no significant structural differences compared to the fully processed proteins. We performed molecular dynamics (MD) simulations on 3CD to investigate its conformational dynamics. A compact conformation of 3CD was observed that was substantially different from that shown crystallographically. This new conformation explained the unique properties of 3CD relative to the individual proteins. Interestingly, simulations of mutant 3CD showed altered interface. Additionally, accelerated MD simulations uncovered a conformational ensemble of 3CD. When we elucidated the 3CD conformations in solution using small-angle X-ray scattering (SAXS) experiments a range of conformations from extended to compact was revealed, validating the MD simulations. The existence of conformational ensemble of 3CD could be viewed as a way to expand the poliovirus proteome, an observation that may extend to other viruses.