Conformational Changes in the Connector Protein Complex of the Bacteriophage φ29 DNA Packaging Motor

DNA packaging in the bacteriophage φ29 involves a molecular motor. It is proposed that dsDNA is packaged through a channel in a connector located at the 5-fold vertex of a preformed prolate icosahedral capsid. The packaging motor also consists of virally-encoded RNA molecules (pRNA) coupled to ATPases. Data obtained from studies using surface plasmon resonance, fluorescence quenching and circular dichroism are presented to demonstrate the importance of the N-termini of the connector protein subunits in pRNA interaction and in conformational change. Based on our findings, we propose a model of DNA packaging based on connector conformational change

Generation of antibodies against foot-and-mouth-disease virus capsid protein VP4 using hepatitis B core VLPs as a Scaffold

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the eco-nomically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neu-tralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.</p

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

FMDV replicons encoding green fluorescent protein are replication competent

The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (Lpro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the Lpro showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays

Antimicrobial peptide LL-37 facilitates intracellular uptake of RNA aptamer Apt 21-2 without inducing an inflammatory or interferon response

RNA aptamers are synthetic single stranded RNA oligonucleotides that function analogously to antibodies. Recently, they have shown promise for use in treating inflammatory skin disease as, unlike antibody-based biologics, they are able to enter the skin following topical administration. However, it is important to understand the inflammatory milieu into which aptamers are delivered, as numerous immune-modulating mediators will be present at abnormal levels. LL-37 is an important immune-modifying protein upregulated in several inflammatory skin conditions, including psoriasis, rosacea and eczema. This inflammatory antimicrobial peptide is known to complex nucleic acids and induce both inflammatory and interferon responses from keratinocytes. Given the attractive notion of using RNA aptamers in topical medication and the prevalence of LL-37 in these inflammatory skin conditions, we examined the effect LL-37 had on the efficacy and safety of the anti-IL-17A RNA aptamer, Apt 21-2. LL-37 was demonstrated to complex with the RNA aptamer by electrophoretic mobility shift and filter binding assays. In contrast to free Apt 21-2, LL-37-complexed Apt 21-2 was observed to efficiently enter both keratinocytes and fibroblasts by confocal microscopy. Despite internalisation of LL-37-complexed aptamers, measurement of inflammatory mediators and interferon stimulated genes showed LL-37-complexed Apt 21-2 remained immunologically inert in keratinocytes, fibroblasts, and peripheral blood mononuclear cells including infiltrating dendritic cells and monocytes. The findings of this study suggest RNA aptamers delivered into an inflammatory milieu rich in LL-37 may become complexed and subsequently internalised by surrounding cells in the skin. Whilst the results of this study indicate delivery of RNA aptamers into tissue rich in LL-37 should not cause an unwarranted inflammatory of interferon response, these results have significant implications for the efficacy of aptamers with regards to extracellular vs intracellular targets that should be taken into consideration when developing treatment strategies utilising RNA aptamers in inflamed tissue

An upstream protein-coding region in enteroviruses modulates virus infection in gut epithelial cells.

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all of the enteroviral proteins were thought to derive from the proteolytic processing of a polyprotein encoded in a single open reading frame. Here we report that many enterovirus genomes also harbour an upstream open reading frame (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed the expression of uORF protein in infected cells. Through ribosome profiling (a technique for the global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B and C. In differentiated human intestinal organoids, uORF protein-knockout echoviruses are attenuated compared to the wild-type at late stages of infection where membrane-associated uORF protein facilitates virus release. Thus, we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells-the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy and have important implications for the understanding of enterovirus pathogenesis.This work was supported by Wellcome Trust grant [106207] and European Research Council grant [646891] to A.E.F; and Wellcome Trust grants 097997/Z/11/Z and 207498/Z/17/Z to I.G

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

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