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.
Chikungunya virus (CHIKV) is a pathogenic arbovirus spread by Aedes spp. mosquitos. CHIKV has a wide global prevalence and represents a significant health burden in affected populations. Symptoms of ...CHIKV infection include fever, rashes and debilitating joint and muscle pain, which can persist for several months to years in some patients. To date, there remains no vaccine or specific antiviral therapy against this important human pathogen. Based on our previously published structural and phenotypic analysis of the 5′ region of the CHIKV genome, we designed a panel of locked nucleic acid oligonucleotides to bind structured RNA replication elements within the virus genome, which are essential for efficient CHIKV replication. Using electromobility shift assays, we confirmed the relative binding efficiencies of each LNA to target CHIKV genomic RNA. We then went on to demonstrate, using both sub-genomic replicon and infectious virus systems, that targeting individual RNA replication elements inhibits CHIKV genome replication and production of infectious virus. Time course assays demonstrated that LNAs can access the CHIKV replication complex and virus genome, during active virus replication. For the first time, these findings show that functional RNA elements can be specifically targeted during the CHIKV lifecycle and consequently represent potential novel antiviral targets.
Author summary: Chikungunya virus (CHIKV) is a mosquito-transmitted virus, first isolated in Tanzania in 1952. In the last 20 years the virus has caused epidemics in over 70 regions of the world. Infection with CHIKV causes an acute febrile illness associated with rash and fever, which can progress to chronic debilitating arthralgia and persist from months to years. Despite causing high morbidity and financial burden, there are no vaccines or specific antiviral agents against CHIKV, making the development of CHIKV specific therapies a priority for research. The virus has an RNA genome and we demonstrate that RNA stem loops within with the CHIKV genome, that we previously demonstrated are essential to virus replication, can be targeted. Binding of complementary and stabilised nucleic acids disrupted the RNA secondary structure and prevented the RNA stem loops from carrying out their essential role during viral replication - leading to inhibition of CHIKV replication. These findings provide a proof of concept for the development of specific anti-CHIKV agents targeting RNA structures.
The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein ...processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.
The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major ...insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.
Acute gastroenteritis (AGE) is the highest cause of mortality worldwide in children under the age of 5 years, with the highest mortalities occurring in low-to-middle income countries. Treatment can ...involve use of unregulated herbal medication and antibiotics. A cross sectional study was carried out to investigate the use of antibiotics and traditional herbal medications in the management of AGE among Yòrùbá-speaking communities in Kwara State, Nigeria. Our findings suggest habitual use of antibiotics (54.6%) and herbal medication (42.5%) in the management of AGE with high levels of self-prescription of antibiotics (21.7%) and herbal medications (36.2%) within the community. Ethanolic extracts of selected herbal plants reported (i.e. Aristolochia ringens, Azadirachta indica, Chromolaena odorata, Etanda Africana, Ficus capensis, Ficus vogelii, Mangifera indica, Momordica charantia, Ocimum gratisimum, Senna alata, Sorghum bicolor and Vernonia amygdalina) were investigated for antibacterial properties, using bacteria known to be causative agents of AGE. Our findings showed that, with exception of Ficus vogelii, which enhanced bacterial growth, the plant extracts reported all showed some antibacterial activity. We further discuss our findings within a regulatory context, with the aim to guide the use of traditional and herbal medication in low-to medium income countries (LMICs) and reduce the potential risks associated with the development of antimicrobial resistance.
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.
Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, ...a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.
Foot-and-mouth disease virus (FMDV) is a picornavirus, which infects cloven-hoofed animals to cause foot-and-mouth disease (FMD). The positive-sense RNA genome contains a single open reading frame, ...which is translated as a polyprotein that is cleaved by viral proteases to produce the viral structural and nonstructural proteins. Initial processing occurs at three main junctions to generate four primary precursors; L
and P1, P2, and P3 (also termed 1ABCD, 2BC, and 3AB
CD). The 2BC and 3AB
CD precursors undergo subsequent proteolysis to generate the proteins required for viral replication, including the enzymes 2C, 3C
, and 3D
. These precursors can be processed through both
and
(i.e., intra- and intermolecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B
-3C junction has an important role in controlling 3AB
CD processing. Here, we use
based assays to show that a single amino acid substitution at the 3B
-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this amino acid substitution enhanced production of some nonenzymatic nonstructural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in
acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements.
Foot-and-mouth disease virus (FMDV) is responsible for foot-and-mouth disease (FMD), an important disease of farmed animals, which is endemic in many parts of the world and can results in major economic losses. Replication of the virus occurs within membrane-associated compartments in infected cells and requires highly coordinated processing events to produce an array of nonstructural proteins. These are initially produced as a polyprotein that undergoes proteolysis likely through both
and
alternative pathways (i.e., intra- and intermolecular proteolysis). The role of alternative processing pathways may help coordination of viral replication by providing temporal control of protein production and here we analyze the consequences of amino acid substitutions that change these pathways in FMDV. Our data suggest that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of RNA genome replication.
Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and newly emerging pathogens. Virus-like particles (VLPs) form the basis of ...two of the most successful licensed vaccines (against hepatitis B virus HBV and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which assemble into immunogenic nanoparticles. VLPs can be modified to present unrelated antigens, and here we describe a universal "bolt-on" platform (termed VelcroVax) where the capturing VLP and the target antigen are produced separately. We utilize a modified HBV core (HBcAg) VLP with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus Junín virus (JUNV). Using this model system, we have solved the first high-resolution structures of VelcroVax VLPs and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the noncomplexed viral protein. We propose that this system could be modified to present a range of antigens and therefore form the foundation of future rapid-response vaccination strategies.
The hepatitis B core protein (HBc) forms noninfectious virus-like particles, which can be modified to present a capturing molecule, allowing suitably tagged antigens to be bound on their surface. This system can be adapted and provides the foundation for a universal "bolt-on" vaccine platform (termed VelcroVax) that can be easily and rapidly modified to generate nanoparticle vaccine candidates.
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