Biology subjects

Dobson, S. J.

Publications and source records attributed to Dobson, S. J..

6 recordsLinked to original sources

bioRxiv2026

Chikungunya virus live-cell reporter systems reveal cell-type-dependent antiviral responses to ribavirin

Chikungunya virus (CHIKV) remains a significant global public health threat, yet antiviral discovery is limited by experimental systems that provide little mechanistic resolution and rely heavily on transformed cell lines of uncertain physiological relevance. Here, we developed complementary live-cell CHIKV reporter systems comprising a dual-fluorescent subgenomic replicon and fluorescent infectious viruses that enable real-time monitoring of distinct stages of the viral replication cycle. By coupling an nsP3-tagged reporter to genomic RNA translation and replication with a subgenomic reporter of structural gene expression, we achieved temporal resolution of early and late replication events in living cells. Functional validation using replication-defective and subgenomic RNA-deficient mutants confirmed that the system distinguishes between viral RNA replication and downstream structural gene expression. Application of these reporters revealed distinct antiviral responses across transformed and physiologically relevant human cell models. Ribavirin and the nsP2 inhibitor RA-0002034 potently inhibited CHIKV replication in transformed cell lines. Unexpectedly, ribavirin activity was markedly diminished in primary human skeletal muscle myoblasts and dermal fibroblasts, despite robust antiviral effects in commonly used screening cell lines. Mechanistic studies showed that ribavirin-mediated inhibition was rescued by guanosine but not inosine supplementation and was recapitulated by mycophenolic acid, consistent with guanine nucleotide depletion as the predominant antiviral mechanism under the conditions tested. Notably, inhibition through this pathway was substantially less effective in primary human cells. Together, these findings establish versatile live-cell CHIKV reporter systems for mechanistic studies of viral replication and reveal striking cell-type-dependent differences in response to guanine nucleotide depletion. Our results demonstrate that host-cell context can profoundly influence both CHIKV replication and antiviral susceptibility, highlighting the importance of physiologically relevant models for understanding virus-host interactions and antiviral mechanism.

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↗

bioRxiv2023

Genetic evidence of a functional linkage between the RNA-dependent RNA polymerase and the highly structured S fragment located at the 5' end of the genome of foot-and-mouth disease virus.

Secondary and tertiary RNA structures play key roles in genome replication of single stranded positive sense RNA viruses. Complex, functional structures are particularly abundant in the untranslated regions of picornaviruses, where they are involved in initiation of translation, priming of new strand synthesis and genome circularisation. The 5' UTR of foot-and-mouth disease virus (FMDV) is predicted to include a c. 360 nucleotide-long stem-loop, termed the short (S) fragment. This structure is highly conserved and essential for viral replication, but the precise function(s) are unclear. Here, we used selective 2' hydroxyl acetylation analysed by primer extension (SHAPE) to experimentally-determine aspects of the structure, alongside comparative genomic analyses to confirm structure conservation from a wide range of field isolates. To examine its role in virus replication, we introduced a series of deletions to the distal and proximal regions of the stem loop. These truncations affected genome replication in a size-dependent and, in some cases, host cell-dependent manner. Furthermore, during passage of viruses incorporating the largest tolerated deletion from the proximal region of the S fragment stem loop, an additional mutation was selected in the viral RNA-dependent RNA polymerase, 3Dpol.These data suggest that the S fragment and 3Dpol interact in the formation of the FMDV replication complex.

molecular biology↗

bioRxiv2023

The virus-encoded ion channel "viroporin" activity of the agnoprotein is required for BK Polyomavirus release from infected kidney cells

BK polyomavirus (BKPyV) is a common opportunistic pathogen and the causative agent of several diseases in transplant patients and the immunosuppressed. Despite its importance, aspects of the virus lifecycle such as how the virus exits an infected cells, remain poorly understood. The late region of the BKPyV genome encodes an auxillery protein called agnoprotein. We and others have shown that agnoprotein is an essential factor in virus release, and the loss of agnoprotein results in an accumulation of virus particles within the nucleus of an infected cell. The functions of agnoprotein necessary for this egress phenotype are not known. Here we demonstrate that agnoprotein shows properties associated with viroporins, a group of virus-encoded membrane spanning proteins that play key roles in virus infection and release. We demonstrate that agnoprotein oligomerises and perturbs membranes in cells. The development of a novel recombinant agnoprotein expression system permitted the identification of the first small molecules targeting agnoprotein. These compounds abrogated agnoprotein viroporin activity in vitro and reduced virus release, indicating that viroporin activity contributes to the phenotype observed in agnoprotein knockout viruses. The identification of channel activity should enhance the future understanding of the physiological function of agnoprotein and could represent an important target for antiviral intervention.

microbiology↗

bioRxiv2023

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication

Foot-and-mouth-disease virus (FMDV), the etiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the Picornavirus family. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, however, the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as qPCR preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific qPCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis-acting replication elements and provide direct evidence for their roles in negative-strand synthesis.

molecular biology↗

bioRxiv2019

A requirement for Potassium and Calcium Channels during the Endosomal Trafficking of Polyomavirus Virions

Following internalisation viruses have to escape the endocytic pathway and deliver their genomes to initiate replication. Members of the Polyomaviridae transit through the endolysosomal network and through the endoplasmic reticulum (ER), from which heavily degraded capsids escape into the cytoplasm prior to nuclear entry. Acidification of endosomes and ER entry are essential in the lifecycle of polyomaviruses, however many mechanistic requirements are yet to be elucidated. Alteration of endocytic pH relies upon the activity of ion channels. Using two polyomaviruses with differing capsid architecture, namely Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we firstly describe methods to rapidly quantify infection using an IncuCyte ZOOM instrument, prior to investigating the role of K+ and Ca2+ channels during early stages of infection. Broad spectrum inhibitors identified that MCPyV, but not SV40, is sensitive to K+ channel modulation. In contrast, both viruses are restricted by the broad spectrum Ca2+ channel inhibitor verapamil, however specific targeting of transient or long lasting Ca2+ channel subfamilies had no detrimental effect. Further investigation revealed that tetrandrine blockage of two-pore channels (TPCs), the activity of which is essential for endolysosomal-ER fusion, ablates infectivity of both MCPyV and SV40 by preventing disassembly of the capsid, which is required for the exposure of minor capsid protein nuclear signals necessary for nuclear transport. This study therefore identifies a novel target to restrict the entry of polyomaviruses.\n\nIMPORTANCEPolyomaviruses establish ubiquitous, asymptomatic infection in their host. However, in the immunocompromised these viruses can cause a range of potentially fatal diseases. Through the use of SV40 and MCPyV, two polyomaviruses with different capsid organisation, we have investigated the role of ion channels during infection. Here, we show that Ca2+ channel activity is essential for both polyomaviruses and that MCPyV is also sensitive to K+ channel blockage, highlighting new mechanistic requirements of ion channels during polyomavirus infection. In particular, tetrandrine blockage of endolysosomal-ER fusion is highlighted as an essential modulator of both SV40 and MCPyV. Given that the role of ion channels in disease have been well characterised, there is a large panel of clinically available therapeutics that could be repurposed to restrict persistent polyomavirus infection and may ultimately prevent polyomavirus-associated disease.

microbiology↗