Biology subjects

Carr, J. M.

Publications and source records attributed to Carr, J. M..

4 recordsLinked to original sources

bioRxiv2026

Proteomic mapping of the dengue virus NS1 microenvironment in infected cells identifies novel host dependency factors including TM9SF3

Dengue virus (DENV) is endemic in over 100 countries and causes approximately 100 million symptomatic infections annually, with symptoms ranging from mild febrile illness to life-threatening severe vascular leakage and haemorrhagic fever. There are currently no approved antiviral therapies available to treat DENV infections. The DENV non-structural protein 1 (NS1) is essential for viral RNA replication and infectious virus particle production, while secreted NS1 contributes to immune evasion and pathogenicity. Towards the identification of novel NS1-host protein interactions that are critical to these functions, an APEX2 proximity labelling-coupled quantitative proteomics approach was employed to map the proteomic composition of the NS1 microenvironment in live infected cells. Our analysis identified a panel of 51 NS1-proximal host proteins, including established DENV host dependency factors (HDFs) involved in NS1 folding and N-glycosylation, as well as previously unrecognised host factors. Loss-of-function approaches were used to determine the importance of these NS1-proximal host proteins to DENV infection, identifying several novel HDFs, including transmembrane 9 superfamily member 3 (TM9SF3). Importantly, the knockout of TM9SF3 was shown to impair DENV infectious virion production and intracellular NS1 abundance and secretion, consistent with the recently described roles of TM9SF3 in Golgi integrity and glycosylation fidelity. Together, this study demonstrates the successful application of APEX2 proximity labelling-coupled quantitative proteomics to the identification of functionally relevant NS1-associated host proteins that may inform the development of future antiviral therapies. IMPORTANCEThe DENV NS1 protein is a non-enzymatic, multifunctional glycoprotein that plays multiple distinct roles in viral replication organelle formation, viral RNA replication and infectious virus particle production. It is also secreted from infected cells as an oligomeric lipoparticle that participates in immune evasion and vascular damage. Many of its enigmatic roles are thought to be mediated via its interactions with other viral proteins and host proteins. Here, we have employed an infectious NS1-tagged DENV reporter virus and proximity biotinylation-coupled mass spectrometry to characterise the protein microenvironment of NS1 during viral infection. We have then employed functional genomics approaches to identify NS1-proximal host factors that contribute to the viral replication cycle. Amongst the novel host factors that were identified was TM9SF3, which has recently emerged as a Golgi-resident Golgiphagy receptor that is important for maintenance of Golgi integrity and glycosylation fidelity and may represent a future DENV antiviral drug target.

microbiology↗

bioRxiv2025

Monocrotaline treatment of the rat predisposes to altered lipopolysaccharide-induced lung responses.

There is a need to understand pathogen driven lung disease and the rat is a laboratory model, widely used to study acute lung injury (ALI). Here the monocrotaline (MCT) rat has been investigated as a model of an inflammatory lung with developing pulmonary hypertension (PH) on which an ALI is superimposed. 14 days following a single systemic dose of MCT, the lung is functionally normal, but stimulation with lipopolysaccharide (LPS) results in an altered response. The MCT/LPS lung is morphologically similar to LPS alone, with respiratory mechanics showing increased elastance, reduced compliance and increased tissue resistance. Bronchioalveolar lavage (BAL) fluid demonstrated a cellular infiltrate, with large macrophage-like cells, increased secreted angiotensin converting enzyme-2 (ACE2) and total protein. The lung transcriptome is pushed towards a pro-inflammatory M1 phenotype, (interferon (IFN)-{Upsilon}, interleukin 6 (IL6), CD68 and CD80) compared to LPS alone. Additionally, the MCT/LPS lung has gene transcription signatures for enhanced cell death and DNA damage responses, higher levels of multiple complement components, dysregulation of the renin-angiotensin pathway with reduced ACE2 and increased AGTR1, increased factors such as Erythroferrone that would increase iron levels, and increased fibrinogen and A2M, that would promote thrombosis. Thus, the MCT-treated rat represents an animal with no overt clinical distinction but following LPS stimulation elicits an exacerbated pathogenic lung response. The MCT-treated rat is a simple model that might be beneficial for understanding an M1 lung pathology with activated complement, low ACE2, high iron, and a propensity to clot in the context of developing cardiovascular disease.

physiology↗

bioRxiv2024

Coatomer protein complex I is required for efficient secretion of dengue virus non-structural protein 1

Secreted non-structural protein 1 (sNS1) is an important orthoflavivirus pathogenic factor that can induce vascular leakage; a key symptom of severe dengue disease. Given the role of sNS1 in dengue pathogenesis, defining the molecular mechanisms of NS1 secretion may contribute towards development of NS1-targeting antiviral therapies. To this end, we performed a customised membrane-trafficking siRNA screen to identify human host factors involved in NS1 secretion. Our screen identified COPA, COPB2, and COPG1 as the top-ranking hits. These proteins are three of the seven subunits of the coatomer protein complex I (COPI) that coat transport vesicles that operate within the early secretory pathway, implicating COPI machinery as being involved in NS1 secretion. Validation studies employing host gene knockdown in dengue virus (DENV)-infected cells confirmed that COPI components are required for efficient NS1 secretion but are dispensable for infectious virus egress. Similar reductions in NS1 secretion were observed when COPI components were depleted in cells infected with West Nile virus Kunjin subtype (WNV/KUNV), indicating that the molecular mechanisms exploited to achieve NS1 secretion may be a conserved feature within the Orthoflavivirus genus. Heterologous expression of wildtype and pathogenic COPI variants in DENV NS1-NS5 polyprotein expressing cells resulted in altered NS1 secretion profiles, suggesting that allelic variants and altered expression levels of COPI components may indirectly influence the severity of dengue disease. The identification of COPI components as important determinants of NS1 secretion efficiency may aid in the identification of novel targets for anti-orthoflaviviral therapies. IMPORTANCEOver half of the worlds population is at risk of infection with mosquito-borne pathogenic orthoflaviviruses such as DENV. Although the secreted form of the viral NS1 protein has been identified as a major determinant of the pathogenic effects of DENV and related orthoflaviviruses, the exact mechanisms involved in NS1 secretion are poorly understood. Here we interrogated host factors involved in secretion of NS1 from infected cells using a customised membrane trafficking siRNA screen. This revealed 3 components of the COPI complex that regulates vesicular transport in the early secretory pathway as important factors in NS1 secretion. The involvement of COPI components in NS1 secretion was further validated using wildtype DENV and WNV/KUNV infection, overexpression approaches and chemical inhibition studies. Together, this study demonstrates the importance of COPI machinery in NS1 secretion and suggests that exploitation of this machinery in NS1 secretion may represent a future target of antiviral drug development.

cell biology↗

bioRxiv2024

A pumpless and tubeless microfluidic device enables extended in vitro development of Cryptosporidium parvum

The enteric parasite Cryptosporidium remains a treatment challenge for drinking water utilities globally due to its resistance to chlorine disinfection. However, the lack of an in vitro culture system for Cryptosporidium that is both cost-effective and reliable remains a key bottleneck in Cryptosporidium research. Here we report that the microfluidic culture of HCT-8 cells under fluid shear stress enables the extended development of Cryptosporidium parvum. Specifically, the growth of C. parvum in a user-friendly pumpless microfluidic device was assessed using immunofluorescence assays, scanning electron microscopy and quantitative PCR, which revealed that development peaked at six days post-infection but continued for ten days in total. Oocysts produced within the microfluidic device were infective to fresh HCT-8 monolayers, however these oocysts were only present at low levels. We anticipate that such microfluidic approaches will facilitate a wide range of in vitro studies on Cryptosporidium and may have the potential to be further developed as a routine infectivity assessment tool for the water industry.

microbiology↗