Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

Foot-and-mouth disease virus (FMDV) causes economically-damaging infections of cloven-hooved animals, with outbreaks resulting in large financial losses to the agricultural industry. Due to the highly contagious nature of FMDV, research with infectious virus is restricted to a limited number of key facilities worldwide. FMDV subgenomic replicons are therefore important tools for the study of viral translation and genome replication. The type III phosphatidylinositol-4-kinases (PI4K) are a family of enzymes that play a key role in the production of replication complexes (viral factories) of a number of positive-sense RNA viruses and represents a potential target for novel pan-viral therapeutics. Here, we have investigated whether type III PI4Ks also play a role in the FMDV lifecycle, using a combination of FMDV subgenomic replicons and bicistronic IRES-containing reporter plasmids. We have demonstrated that replication of the FMDV replicon was unaffected by inhibitors of either PI4KIIIα or PI4KIIIβ. However, PIK93, an inhibitor previously demonstrated to target PI4KIIIβ, did inhibit IRES-mediated protein translation. Consistent with this, cells transfected with FMDV replicons did not exhibit elevated levels of PI4P lipids. These results are therefore supportive of the hypothesis that FMDV genome replication does not require type III PI4K activity and does not activate these kinases

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

Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part to the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for viral replication as well as locations that can tolerate small genetic insertions. Such insertion sites can be subsequently adapted by the incorporation of commonly used epitope tags and so facilitate their detection with commercial available reagents. In this study, we use random transposon-mediated mutagenesis to produce a library of 15 nucleotide insertions in the FMDV nonstructural polyprotein. Using a replicon-based assay we isolated multiple replication-competent as well as replication-defective insertions. We have adapted the replication competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins, for the use in a variety of downstream assays. Additionally, we show that replication of some of the replication-defective insertion mutants can be rescued by co-transfection of a 'helper' replicon, demonstrating a novel use of random mutagenesis to identify inter-genomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornaviral replication complexes

Producing interventions for AIDS-affected young people in Lesotho's schools: Scalar relations and power differentials

This is the post-print version of the final paper published in Geoforum. The published article is available from the link below. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. Copyright @ 2009 Elsevier B.V.Children and youth are a key target group for interventions to address southern Africa’s AIDS pandemic. Such interventions are frequently implemented through schools, and are often complex products of negotiation between a range of institutional actors including international agencies, NGOs, government departments and individual schools. These institutions not only stand in different (horizontally scaled) spatial relationships to students in schools; they also appear to operate at different hierarchical levels. Empirical research with policy makers and practitioners in Lesotho, however, reveals how interventions are produced through flows of knowledge, funding and personnel within and between institutions that make it difficult to assert that any intervention is manifestly more international or more local than any other. Scale theory offers the metaphor of a network or web which usefully serves to move attention away from discrete organisations, sectors and scalar positionings and onto the relationships and flows between them. Nevertheless, organisations and development interventions are often partly structured in scalar hierarchical ways that express substantive power differentials and shape the forms of interaction that take place, albeit not binding them to strict binaries or nested hierarchies. A modified network metaphor is useful in aiding understanding of how particular interventions are produced through intermeshing scales and diverse fluid interactions, and why they take the form they do.RGS-IB

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

Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part to the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for viral replication as well as locations that can tolerate small genetic insertions. Such insertion sites can be subsequently adapted by the incorporation of commonly used epitope tags and so facilitate their detection with commercial available reagents. In this study, we use random transposon-mediated mutagenesis to produce a library of 15 nucleotide insertions in the FMDV non-structural polyprotein. Using a replicon-based assay we isolated multiple replication-competent as well as replication-defective insertions. We have adapted the replication-competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins, for the use in a variety of downstream assays. Additionally, we show that replication of some of the replication-defective insertion mutants can be rescued by co-transfection of a 'helper' replicon, demonstrating a novel use of random mutagenesis to identify inter-genomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornaviral replication complexes

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

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

Reversion mutations at the 3B-3C boundary restore P3 polyprotein processing.

<p>Plasmid constructs expressing wild-type or mutant P3 polyprotein were used to assemble coupled transcription/translation reactions with [³⁵S] labelled methionine. Reactions were incubated for 40 minutes before addition of excess unlabelled methionine/cysteine, samples taken at regular intervals and reactions stopped by the addition of 2 x Laemmli buffer. <b>(A)</b> Proteins were separated on 12% SDS-PAGE and visualised by autoradiography. The positions of FMDV protein products are indicated by arrows. Control reactions were assembled using empty expression vector alone. The proportion of uncleaved P3 <b>(B)</b> and 3D^pol <b>(C)</b> product was quantified as a percentage of the total 3D^pol containing products (n = 2 ± SD).</p