Biology subjects

Rowlands, D.

Publications and source records attributed to Rowlands, D..

8 recordsLinked to original sources

bioRxiv2026

Enhanced inter-chain hydrogen bonding in the murine norovirus VP1 capsid leads to increased particle stability and delayed viral uncoating

Capsid stability is vital for virion survival as the capsid must withstand varying environmental challenges such as pH and temperature to allow the virus to reach a target cell. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of importance to human health globally, with no approved vaccine or antiviral available. Despite this, the molecular mechanisms behind norovirus capsid stability and capsid rearrangement prior to RNA translocation are understudied. Using murine norovirus as a model, we utilised thermal stress to create a thermally stable virus population. By introducing three identified substitutions in the major capsid protein VP1 from this virus population into an infectious clone, we were able to create a heat and pH stable virus that had delayed viral uncoating during the infectious lifecycle. Cryo-EM reconstructions of the triple substitution virus demonstrated that enhanced inter-chain hydrogen bonding was vital for increased capsid stability. Finally, mutagenesis to remove the enhanced inter-chain hydrogen bonding reverted capsid stability back to wild-type levels. This work contributes to fundamental calicivirus biology by demonstrating areas of importance in capsid stability down to amino acid resolution. Furthermore, this work could inform vaccine design for a thermostable norovirus vaccine in the future. Author SummaryNoroviruses cause gastroenteritis and globally contribute to the death of up to 250,000 people worldwide per annum and an estimated healthcare cost of $4 billion. Despite this, there is no vaccine or antiviral treatment available, thus more work needs to be conducted to understand the mechanisms that underpin the viral life cycle. The norovirus capsid is a meta-stable shell-like structure evolved to protect the viral RNA from the harsh external environment, until cellular triggers allow genome release to initiate infection of a host cell. However, the molecular interactions that are key for controlling this balance are relatively unstudied. In this report, we identify that hydrogen bonding at the capsid protomer-protomer interface are vital for maintaining this balance, with increased inter-molecular hydrogen bonding at three specific amino acids able to increase viral stability whilst still permitting infectious genome release. Furthermore, reducing inter-molecular hydrogen bonding at these crucial amino acids was able to reverse this mechanism. These results should inform future norovirus vaccine studies, where thermostable virus-like-particles are needed to overcome issues with cold-chain storage.

microbiology↗

bioRxiv2025

Interactions between the 2C protein of FMDV and components of the viral replication machinery are mediated by ER-derived membranes

Foot-and-mouth disease virus (FMDV) remains an ever-present threat to the economic stability of the livestock industry and global trade. Despite this, questions remain regarding the fundamental biology underpinning the replication of this virus. Here, we examine components of the FMDV replication machinery (focussing on the viral 2C protein) and investigate the conditions under which they interact. Using a novel 2C antibody in co-immunoprecipitation experiments under different conditions followed by mass spectrometry, we identify membrane-associated proteins (such as the viral proteins 2B and precursors of 3A, which are poorly-characterised proteins involved in viral replication) along with ER-associated host proteins. In addition, our analysis shows that a number of nuclear factors interact with 2C in a membrane-independent manner, potentially being co-opted to support RNA replication of the virus. Furthermore, we demonstrate that the interaction of 2C with several viral proteins (including key members of the replication machinery and viral RNA) is maintained following ultracentrifugation, suggesting that these co-sediment as part of a complex. Our data suggest that the replication complex is ER-derived and highlight several new avenues of investigation for the disruption of the FMDV lifecycle.

microbiology↗

bioRxiv2024

A VLP vaccine platform comprising the core protein of hepatitis B virus with N-terminal antigen capture

Nanoparticle presentation systems offer the potential to develop new vaccines rapidly in response to emerging diseases, a public health need that has become increasingly evident in the wake of the COVID-19 pandemic. Previously, we reported a nanoparticle scaffold system termed VelcroVax. This was constructed by insertion of a high affinity SUMO binding protein (Affimer), able to recognise a SUMO peptide tag, into the major immunodominant region of VLPs assembled from a tandem (fused dimer) form of hepatitis B virus (HBV) core protein (HBc). Here we describe a modified form of VelcroVax, comprising monomeric HBc with the Affimer inserted at the N-terminus (termed N-VelcroVax). In contrast to the tandem form of VelcroVax, N-VelcroVax VLPs were expressed well in E. coli. The VLPs effectively bound SUMO-tagged Junin virus glycoprotein, gp1 as assessed by structural and serological analyses. Cryo-EM characterisation of N-VelcroVax complexed with a SUMO-Junin gp1 showed continuous density attributable to the fused Affimer, in addition to evidence of target antigen capture. Collectively, these data suggest that N-VelcroVax has potential as a versatile next generation vaccine scaffold.

molecular biology↗

bioRxiv2024

Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate.

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design.

microbiology↗

bioRxiv2023

Production of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation. We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilised virus-like particles (VLPs) in Pichia pastoris. The stabilised VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilisation, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralise virus in vitro. Therefore, the anti-EVA71 neutralising antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.

immunology↗

bioRxiv2023

Insights into polyprotein processing and RNA-protein interactions in foot-and-mouth disease virus genome replication.

Foot-and-mouth disease affects cloven hoofed animals and is caused by foot-and-mouth disease virus (FMDV), a picornavirus with a positive-sense RNA genome. The FMDV genome contains a single open reading frame, which is translated to produce a polyprotein that is cleaved by viral proteases to produce the viral structural and non-structural proteins. Initial processing of the polyprotein occurs at three main junctions to generate four primary products; Lpro and the P1, P2 and P3 precursors (also termed 1ABCD, 2BC and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate non-structural proteins that are required for viral replication, including the enzymes 2C, 3Cpro and 3Dpol. These precursors can be processed through both cis and trans (i.e., intra- and inter-molecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction had an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single point mutation at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this point mutation permitted production of some non-enzymatic non-structural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements. ImportanceFoot-and-mouth disease virus (FMDV) is an economically important pathogen of animals that is responsible for foot-and-mouth disease (FMD). FMD is endemic in many parts of the world and can results in major economic losses. Replication of the virus is a highly coordinated event that occurs within membrane-associated compartments in infected cells and requires the viral non-structural proteins. These are all initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans pathways (i.e., intra- and inter-molecular proteolysis). Alternative processing pathways can provide a mechanism to help coordinate viral replication by providing temporal control to protein production. Here, we analyse the consequences of mutations that change temporal control of FMDV polyprotein processing. Our data suggests 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 FMDV genome replication.

microbiology↗

bioRxiv2022

Production and characterisation of stabilised PV-3 virus-like particles using Pichia pastoris

Following the success of global vaccination programmes using the live-attenuated oral and inactivated poliovirus vaccines (OPV and IPV), wild poliovirus (PV) is now only endemic in Afghanistan and Pakistan. However, the continued use of these vaccines poses potential risks to the eradication of PV. The production of recombinant PV virus-like particles (VLPs), which lack the viral genome offer great potential as next-generation vaccines for the post-polio world. We have previously reported production of PV VLPs using Pichia pastoris, however, these VLPs were in the non-native conformation (C Ag), which would not produce effective protection against PV. Here, we build on this work and show that it is possible to produce wt PV-3 and thermally-stabilised PV-3 (referred to as PV-3 SC8) VLPs in the native conformation (D Ag) using Pichia pastoris. We show that the PV-3 SC8 VLPs provide a much-improved D:C antigen ratio as compared to wt PV-3, whilst exhibiting greater thermostability than the current IPV vaccine. Finally, we determine the cryo-EM structure of the yeast-derived PV-3 SC8 VLPs and compare this to previously published PV-3 D Ag structures, highlighting the similarities between these recombinantly-expressed VLPs and the infectious virus, further emphasising their potential as a next-generation vaccine candidate for PV.

microbiology↗

bioRxiv2018

Targeting hepatitis C virus p7 channel activity reveals prospect for bimodal antiviral prophylaxis

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or \"viroporins\", contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery.\n\nWe describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

microbiology↗