The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication, but essential for the production of infectious virus.

Non-coding regions of viral RNA (vRNA) genomes are critically important in the regulation of gene expression. In particular, pseudoknot (PK) structures, which are present in a wide range of RNA molecules, have a variety of roles. The 5' untranslated region (5' UTR) of foot-and-mouth disease virus (FMDV) vRNA is considerably longer than in other viruses from the picornavirus family and consists of a number of distinctive structural motifs that includes multiple (2, 3 or 4 depending on the virus strain) putative PKs linked in tandem. The role(s) of the PKs in the FMDV infection are not fully understood. Here, using bioinformatics, sub-genomic replicons and recombinant viruses we have investigated the structural conservation and importance of the PKs in the FMDV lifecycle. Our results show that despite the conservation of two or more PKs across all FMDVs, a replicon lacking PKs was replication competent, albeit at reduced levels. Furthermore, in competition experiments, GFP FMDV replicons with less than two (0 or 1) PK structures were outcompeted by a mCherry FMDV wt replicon that had 4 PKs, whereas GFP replicons with 2 or 4 PKs were not. This apparent replicative advantage offered by the additional PKs correlates with the maintenance of at least two PKs in the genomes of FMDV field isolates. Despite a replicon lacking any PKs retaining the ability to replicate, viruses completely lacking PK were not viable and at least one PK was essential for recovery of infections virus, suggesting a role for the PKs in virion assembly. Thus, our study points to roles for the PKs in both vRNA replication and virion assembly, thereby improving understanding the molecular biology of FMDV replication and the wider roles of PK in RNA functions.

Functional advantages of triplication of the 3B coding region of the FMDV genome.

For gene duplication to be maintained, particularly in the small genomes of RNA viruses, this should offer some advantages. We have investigated the functions of a small protein termed VPg or 3B, which acts as a primer in the replication of foot-and-mouth disease virus (FMDV). Many related picornaviruses encode a single copy but uniquely the FMDV genome includes three (nonidentical) copies of the 3B coding region. Using sub-genomic replicons incorporating nonfunctional 3Bs and 3B fusion products in competition and complementation assays, we investigated the contributions of individual 3Bs to replication and the structural requirements for functionality. We showed that a free N-terminus is required for 3B to function as a primer and although a single 3B can support genome replication, additional copies provide a competitive advantage. However, a fourth copy confers no further advantage. Furthermore, we find that a minimum of two 3Bs is necessary for trans replication of FMDV replicons, which is unlike other picornaviruses where a single 3B can be used for both cis and trans replication. Our data are consistent with a model in which 3B copy number expansion within the FMDV genome has allowed evolution of separate cis and trans acting functions, providing selective pressure to maintain multiple copies of 3B.

Dynamics in the murine norovirus capsid revealed by high-resolution cryo-EM.

Icosahedral viral capsids must undergo conformational rearrangements to coordinate essential processes during the viral life cycle. Capturing such conformational flexibility has been technically challenging yet could be key for developing rational therapeutic agents to combat infections. Noroviruses are nonenveloped, icosahedral viruses of global importance to human health. They are a common cause of acute gastroenteritis, yet no vaccines or specific antiviral agents are available. Here, we use genetics and cryo-electron microscopy (cryo-EM) to study the high-resolution solution structures of murine norovirus as a model for human viruses. By comparing our 3 structures (at 2.9- to 3.1-Å resolution), we show that whilst there is little change to the shell domain of the capsid, the radiating protruding domains are flexible, adopting distinct states both independently and synchronously. In doing so, the capsids sample a range of conformational space, with implications for maintaining virion stability and infectivity.

Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using Pichia pastoris.

For enteroviruses such as poliovirus (PV), empty capsids, which are antigenically indistinguishable from mature virions, are produced naturally during viral infection. The production of such capsids recombinantly, in heterologous systems such as yeast, have great potential as virus-like particle (VLP) vaccine candidates. Here, using PV as an exemplar, we show the production of VLPs in Pichia pastoris by coexpression of the structural precursor protein P1 and the viral protease 3CD. The level of expression of the potentially cytotoxic protease relative to that of the P1 precursor was modulated by three different approaches: expression of the P1 precursor and protease from different transcription units, separation of the P1 and protease proteins using the Thosea asigna virus (TaV) 2A translation interruption sequence, or separation of the P1 and protease-coding sequences by an internal ribosome entry site sequence from Rhopalosiphum padi virus (RhPV). We also investigate the antigenicity of VLPs containing previously characterized mutations when produced in Pichia Finally, using transmission electron microscopy and two-dimensional classification, we show that Pichia-derived VLPs exhibited the classical icosahedral capsid structure displayed by enteroviruses.IMPORTANCE Although the current poliovirus immunization program has been extremely successful in reducing the number of cases of paralytic polio worldwide, now more cases are caused by vaccine-derived polioviruses than by wild poliovirus. Switching to inactivated poliovirus vaccines will reduce this over time; however, their production requires the growth of large amounts of virus. This biosafety concern can be addressed by producing just the virus capsid. The capsid serves to protect the genetic material, which causes disease when introduced into a cell. Therefore, empty capsids (virus-like particles [VLPs]), which lack the viral RNA genome, are safe both to make and to use. We exploit yeast as a versatile model expression system to produce VLPs, and here we specifically highlight the potential of this system to supply next-generation poliovirus vaccines to secure a polio-free world for the future.

The broad-spectrum antiviral drug arbidol inhibits foot-and-mouth disease virus genome replication.

Arbidol (ARB, also known as umifenovir) is used clinically in several countries as an anti-influenza virus drug. ARB inhibits multiple enveloped viruses in vitro and the primary mode of action is inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. ARB is also an effective inhibitor of non-enveloped poliovirus types 1 and 3. In the current report, we evaluate the antiviral potential of ARB against another picornavirus, foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus and an important veterinary pathogen. ARB inhibits the replication of FMDV RNA sub-genomic replicons. ARB inhibition of FMDV RNA replication is not a result of generalized inhibition of cellular uptake of cargo, such as transfected DNA, and ARB can be added to cells up to 3 h post-transfection of FMDV RNA replicons and still inhibit FMDV replication. ARB prevents the recovery of FMDV replication upon withdrawal of the replication inhibitor guanidine hydrochloride (GuHCl). Although restoration of FMDV replication is known to require de novo protein synthesis upon GuHCl removal, ARB does not suppress cellular translation or FMDV internal ribosome entry site (IRES)-driven translation. ARB also inhibits infection with the related Aphthovirus, equine rhinitis A virus (ERAV). Collectively, the data demonstrate that ARB can inhibit some non-enveloped picornaviruses. The data are consistent with inhibition of picornavirus genome replication, possibly via the disruption of intracellular membranes on which replication complexes are located.

The Oxysterol 25-Hydroxycholesterol Inhibits Replication of Murine Norovirus.

Cholesterol, an essential component of mammalian cells, is also an important factor in the replicative-cycles of several human and animal viruses. The oxysterol, 25-hydroxycholesterol, is produced from cholesterol by the enzyme, cholesterol 25-hydroxylase. 25-hydroxycholesterol (25-HC) has been shown to have anti-viral activities against a wide range of viruses, including a range of positive-sense RNA viruses. In this study, we have investigated the role of 25-HC in norovirus replication using murine norovirus (MNV) as a model system. As a control, we employed herpes simplex virus-1 (HSV-1), a pathogen previously shown to be inhibited by 25-HC. Consistent with previous studies, 25-HC inhibited HSV-1 replication in the MNV-susceptible cell line, RAW264.7. Treating RAW264.7 cells with sub-cytotoxic concentrations of 25-HC reduced the MNV titers. However, other sterols such as cholesterol or the oxysterol, 22-S-hydroxycholesterol (22-S-HC), did not inhibit MNV replication. Moreover, treating MNV-infected RAW264.7 cells with 25-HC-stimulated caspase 3/7 activity, which leads to enhanced apoptosis and increased cell death. Our study adds noroviruses to the list of viruses inhibited by 25-HC and begins to offer insights into the mechanism behind this inhibition.

Involvement of a Nonstructural Protein in Poliovirus Capsid Assembly.

Virus capsid proteins must perform a number of roles. These include self-assembly and maintaining stability under challenging environmental conditions, while retaining the conformational flexibility necessary to uncoat and deliver the viral genome into a host cell. Fulfilling these roles could place conflicting constraints on the innate abilities encoded within the protein sequences. In a previous study, we identified a number of mutations within the capsid-coding sequence of poliovirus (PV) that were established in the population during selection for greater thermostability by sequential treatment at progressively higher temperatures. Two mutations in the VP1 protein acquired at an early stage were maintained throughout this selection procedure. One of these mutations prevented virion assembly when introduced into a wild-type (wt) infectious clone. Here we show, by sequencing beyond the capsid-coding region of the heat-selected virions, that two mutations had arisen within the coding region of the 2A protease. Both mutations were maintained throughout the selection process. Introduction of these mutations into a wt infectious clone by site-directed mutagenesis considerably reduced replication. However, they permitted a low level of assembly of infectious virions containing the otherwise lethal mutation in VP1. The 2Apro mutations were further shown to slow the kinetics of viral polyprotein processing, and we suggest that this delay improves the correct folding of the mutant capsid precursor protein to permit virion assembly.IMPORTANCE RNA viruses, including poliovirus, evolve rapidly due to the error-prone nature of the polymerase enzymes involved in genome replication. Fixation of advantageous mutations may require the acquisition of complementary mutations which can act in concert to achieve a favorable phenotype. This study highlights a compensatory role of a nonstructural regulatory protein, 2Apro, for an otherwise lethal mutation of the structural VP1 protein to facilitate increased thermal resistance. Studying how viruses respond to selection pressures is important for understanding mechanisms which underpin emergence of resistance and could be applied to the future development of antiviral agents and vaccines.

Photodynamic inactivation of non-enveloped RNA viruses.

We recently reported the photodynamic inactivation (PDI) of bacteriophage MS2 with a photosensitiser- 5, 10, 15, 20-tetrakis (1-methyl-4-pyridinio) porphyrin- tetra- p-toluene sulfonate (TMPyP) in solution and concluded that the A-protein of the virus is the main target of inactivation. Here, we have extended these studies and carried out PDI of bacteriophage Qß, bovine enterovirus 2 (BEV-2) and type 1 murine norovirus (MNV-1). The rate of inactivation observed was in the order MS2 > Qß > MNV-1 > BEV-2. Data suggested that TMPyP-treatment could also target the viral genome as well as result in disintegration/disassembly of viral particles. Although emergence of viral drug resistance is a well-documented phenomenon, it was not possible to generate PDI-resistant MS2. However, emergence of a mutation in the lysis protein was detected after serial exposure to PDI.

Photodynamic inactivation of bacteriophage MS2: The A-protein is the target of virus inactivation.

Singlet oxygen mediated oxidation has been shown to be responsible for photodynamic inactivation (PDI) of viruses in solution with photosensitisers such as 5, 10, 15, 20-tetrakis (1-methyl-4-pyridinio) porphyrin tetra p-toluenesulfonate (TMPyP). The capsids of non-enveloped viruses, such as bacteriophage MS2, are possible targets for viral inactivation by singlet oxygen oxidation. Within the capsid (predominantly composed of coat protein), the A-protein acts as the host recognition and attachment protein. The A-protein has two domains; an α-helix domain and a ß-sheet domain. The α-helix domain is attached to the viral RNA genome inside the capsid while the ß-sheet domain, which is on the surface of the capsid, is believed to be the site for attachment to the host bacteria pilus during infection. In this study, 4 sequence-specific antibodies were raised against 4 sites on the A-protein. Changes induced by the oxidation of singlet oxygen were compared to the rate of PDI of the virus. Using these antibodies, our results suggest that the rate of PDI is relative to loss of antigenicity of two sites on the A-protein. Our data further showed that PDI caused aggregation of MS2 particles and crosslinking of MS2 coat protein. However, these inter- and intra-capsid changes did not correlate to the rate of PDI we observed in MS2. Possible modes of action are discussed as a means to gaining insight to the targets and mechanisms of PDI of viruses.

A multi-template multiplex PCR assay for hepatitis B virus and human ß-globin.

The Hepatitis B surface antigen (HBsAg) is the hallmark of HBV infection. Detection of antibodies to HBs and the core (ie, HBsAg and HBcAb) are primary serological algorithms in the laboratory diagnosis of HBV. Detection of HBsAg DNA is an important supplement to serological diagnosis especially in clinical cases. Simultaneous amplification of internal cellular controls is a good indicator of sample quality. Human ß-globin is a well characterized housekeeping gene (HKG) that is often applied as internal controls (IC) in molecular diagnosis. In this study, individual plasmid clones of the human ß-globin and HBs genes were constructed. These plasmid constructs have been applied to characterize a multiplex PCR assays for HBs and ß-globin genes. The findings suggest detection limits of less than 10 genome copies of either template In vitro using conventional and multiplex PCR conditions. Under the multiplex conditions, co-amplification of ß-globin and HBsAg DNA had a resultant effect on assay sensitivity. This study further highlights the importance of molecular diagnosis in HBV infectious individuals. If fully optimized, this assay could provide a possible diagnostic complement to serological detection in developing countries.

Increasing Type 1 Poliovirus Capsid Stability by Thermal Selection.

Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunization programs using live-attenuated oral (OPV) and/or inactivated (IPV) PV vaccines have systematically reduced its spread and paved the way for eradication. Immunization will continue posteradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. They could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines that mimic the "empty" capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert into an alternative conformation unsuitable for vaccine purposes. Stabilized ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we selected thermally stable ECs of type 1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild-type (wt) virions. We have identified mutations on the capsid surface and in internal networks that are responsible for EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilized VLPs could be used as novel vaccines. IMPORTANCE: Poliomyelitis is a highly infectious disease caused by PV and is on the verge of eradication. There are biosafety concerns about reintroduction of the disease from current vaccines that require live virus for production. Recombinantly expressed virus-like particles (VLPs) could address these inherent problems. However, the genome-free capsids (ECs) of wt PV are unstable and readily change antigenicity to a form not suitable as a vaccine. Here, we demonstrate that the ECs of type 1 PV can be stabilized by selecting heat-resistant viruses. Our data show that some capsid mutations stabilize the ECs and could be applied as candidates to synthesize stable VLPs as future genome-free poliovirus vaccines.