Biology subjects

Courtney, D. G.

Publications and source records attributed to Courtney, D. G..

5 recordsLinked to original sources

bioRxiv2026

A host ATPase essential for rhinovirus replication is an antiviral target with a high barrier to resistance

Rhinoviruses are the leading cause of acute respiratory illnesses and comprise more than 170 types that constantly circulate in humans worldwide. Beyond common colds, rhinoviruses can trigger severe symptoms, particularly in young children, older adults and people with asthma or chronic obstructive pulmonary disease. Despite their clinical and socio-economic impact, no approved vaccine or antiviral treatment exist. Here, we uncovered the interaction of the host AAA+ ATPase RUVBL1/2 with rhinovirus non-structural protein 2C and we demonstrated that RUVBL1/2 is strictly and specifically required for the replication of the viral RNA of the most prevalent and pathogenic rhinovirus species. Pharmacological inhibition of RUVBL1/2 ATPase activity efficiently inhibited rhinovirus replication in a human nasal epithelium model, even post-infection. Moreover, serial viral passaging in the presence of a RUVBL1/2 inhibitor did not lead to the emergence of resistance. These findings reveal an unexpected and strong host dependency with promising potential for antiviral targeting.

microbiology↗

bioRxiv2024

The cold-inducible RNA-binding protein RBM3 stabilises viral mRNA at the cooler temperatures of the upper respiratory tract.

Temperature is a critical determinant of host-pathogen interactions in the respiratory tract, where influenza A virus (IAV) has adapted to the cooler environment of the upper respiratory tract (URT) to enable efficient replication and transmission. The cold-inducible RNA-binding protein RBM3 is highly expressed in nasopharyngeal tissue and is known to stabilise mRNAs under hypothermic conditions; however, its role during viral infection has not been defined. Here, we identify RBM3 as a key host factor that facilitates IAV replication at sub-physiological temperatures. siRNA-mediated knockdown of RBM3 significantly impaired viral replication, while its constitutive overexpression at 37{degrees}C restored replication to levels typically observed at 33{degrees}C. Mechanistically, RBM3 binds directly to viral nucleoprotein (NP) mRNA, prolonging mRNA half-life and inevitably leading to increased viral production. This effect was abolished in an RNA-binding-deficient RBM3 mutant, confirming the requirement for direct RNA interaction, visualised by smiFISH-PLA. Crucially, this positive regulation of NP mRNA and interaction was validated in well-differentiated primary nasal epithelial cells, highlighting the physiological relevance of RBM3 in the human URT. These results reveal a temperature-sensitive host-virus interaction that promotes IAV replication in the cooler URT, a key site for viral shedding and transmission. By linking environmental temperature, host RNA-binding proteins, and viral mRNA stability, this study uncovers a novel mechanism of respiratory virus adaptation and identifies RBM3 as a potential therapeutic target for limiting early-stage viral replication and transmission. Author SummaryTo establish productive infection and ensure transmission, respiratory viruses such as influenza A virus (IAV) must adapt to the diverse environments of the human respiratory tract. One key challenge is the temperature gradient that exists between the upper and lower respiratory tract. The upper respiratory tract (URT), the main site of viral entry and transmission, maintains a lower physiological temperature ([~]33{degrees}C) than the lower respiratory tract ([~]37{degrees}C, LRT), creating a distinct cellular environment. In this study, we investigated how this cooler URT temperature alters the landscape of RNA-binding proteins (RBPs), which play a central role in regulating gene expression after transcription. Using mass spectrometry-based profiling and molecular virology approaches, we identified a cold-inducible RBP, RBM3, as significantly enriched at 33{degrees}C and acting as a key proviral host factor during IAV infection. We show that RBM3 binds directly to viral nucleoprotein (NP) mRNA, mainly in the cytoplasm, in both immortalised cell models and primary nasal epithelial cells. This interaction promotes the stability of NP mRNA thereby enhancing the overall production of infectious virions. Importantly, disrupting RBM3s RNA-binding ability abolished this proviral effect. These findings reveal that RBM3, elevated in the cooler URT environment, directly supports IAV replication. This work highlights how subtle physiological differences in host tissue can reshape post-transcriptional regulation and influence the outcome of respiratory virus infection.

microbiology↗

bioRxiv2024

Myoferlin is an essential component of late-stage vRNP trafficking vesicles for enveloped RNA viruses.

The Rab11a endosomal recycling pathway is exploited by important respiratory RNA viruses such as IAV and RSV, aiding viral egress from the apical surface of polarized epithelial cells. Late in infection, Rab11a-containing vesicles specifically transport viral ribonucleoprotein (vRNP) complexes towards the cell surface before packaging and budding. Rather than employing traditional Rab11a-positive recycling endosomes, virus-infected cells generate remodelled Rab11a-containing vesicles, as observed during IAV infection. Besides Rab11a, no other conserved host co-factors have been identified among these various vRNP trafficking vesicles. Here we discovered and confirmed myoferlins association with IAV vRNPs in the cytoplasm and colocalisation with Rab11a during late stages of infection. We also found that this role was conserved in late-stage vRNP trafficking of other viruses, including RSV and SeV. Myoferlin likely recruits the EHD family of proteins, which are involved in endosomal biogenesis, to these unique vRNP trafficking endosomes, highlighting myoferlins pivotal role in viral replication.

microbiology↗

bioRxiv2023

Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking

Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA likely through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which we suspect leads to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.

microbiology↗

bioRxiv2021

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

Pseudouridine ({Psi}) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes [~]7% of the total uridine level. However, {Psi} constitutes only [~]0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. {Psi} residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of {Psi} residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based {Psi} mapping technique called photo-crosslinking assisted {Psi} sequencing (PA-{Psi}-seq) and use it to map {Psi} residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe several 293T-derived cell lines in which human PUS enzymes previously reported to add {Psi} residues to human mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of {Psi} addition on cellular mRNAs to each of these three PUS enzymes, the {Psi} sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of {Psi} residues detected on total human mRNA below the [~]0.1% level seen in wild type cells, thus implying that the PUS enzyme(s) that adds the bulk of {Psi} residues to human mRNAs remains to be defined.

molecular biology↗