Author: Stephen Johnson
Johnson, Stephen, 2025 Identifying and interrogating human host factors associated with dengue virus non-structural protein 1 secretion and internalisation, Flinders University, College of Medicine and Public Health
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As the most prevalent arthropod-borne viral pathogen, dengue virus (DENV) is estimated to infect nearly 400 million people each year. Clinical manifestations encompass a wide disease spectrum ranging from mild febrile illness to more serious complications including haemorrhage, shock and death. No DENV-specific therapeutics are currently available to prevent dengue disease progression, thus necessitating a deeper understanding of DENV-human host molecular interactions. A key DENV virulence factor is its non-structural protein 1 (NS1). This multifunctional viral protein performs a variety of roles that are indispensable to DENV and critical for dengue disease. Within infected cells, intracellular NS1 is essential for viral genome replication and virion morphogenesis. NS1 is also secreted from infected cells, and this secreted NS1 (sNS1) form has been identified as a key mediator of dengue pathogenesis. In the extracellular environment, the highly immunogenic sNS1 can elicit the production of both protective and pathogenic antibodies. sNS1 can also interfere with components of the complement system and modulate their activity. Importantly, sNS1 can bind to immune cells and potently induce the production of proinflammatory and vasoactive cytokines that can influence endothelial cell permeability and contribute to vascular leakage – a key hallmark of severe dengue disease. Moreover, sNS1 can bind and internalise into a variety of uninfected target host cell types and enhance their susceptibility to infection. The binding and internalisation of sNS1 by endothelial cells directly contributes to endothelial cell hyperpermeability and vascular leakage. While much research has been conducted on the synthesis, structure, and key functional residues of this viral virulence factor, major gaps exist in our understanding of the molecular mechanisms that are exploited by DENV to achieve NS1 secretion and sNS1 internalisation. As such, the focus of this work was to identify the human host molecular machinery that are involved in NS1 secretion and sNS1 internalisation. First, to identify and interrogate the human host factors involved in NS1 secretion, we employed a customised membrane-trafficking siRNA screen targeting ~180 human host factors in cells infected with an NS1-tagged luciferase reporter virus. Our screen identified COPA, COPB2, and COPG1 as the top ranking host determinants of NS1 secretion efficiency. These proteins are three of the seven subunits of the coatomer protein complex I (COPI) that coat intracellular transport vesicles, implicating COPI machinery and associated pathways as being involved in NS1 secretion. Validation studies employing COPI gene knockdown in DENV-infected cells confirmed that COPI components are required for efficient NS1 secretion but are dispensable for infectious virus secretion. Similar reductions in NS1 secretion were observed when COPI components were depleted in cells infected with the related West Nile virus Kunjin subtype (WNV/KUNV), indicating that the exploitation of COPI to achieve efficient NS1 secretion may be a feature conserved within the Orthoflavivirus genus. Overexpression of wildtype and pathogenic COPI variants in DENV NS1-NS5 polyprotein expressing cells altered NS1 secretion profiles suggesting that allelic variants or COPI expression levels influence NS1 secretion. To functionally inhibit the formation of COPI vesicles, we employed Golgicide A, a potent and specific inhibitor of GBF1 – a master regulator of COPI vesicle biogenesis. Our results revealed that when GCA is applied late in infection, the catalytic activity of GBF1 is dispensable for DENV genome replication but is required for infectious virus production and efficient NS1 secretion. Interestingly, while virion secretion was reduced by GCA treatment in a dose-dependent manner, NS1 secretion was only reduced at the highest dose applied, indicating that multiple mechanisms may be exploited by DENV to achieve NS1 secretion. Next, to identify the human host molecular machinery associated with sNS1 internalisation, we employed an APEX2-based proximity labelling strategy. APEX2 is an engineered plant peroxidase that can catalyse the biotinylation of proximal proteins within live cells. We utilised our previously characterised DENV2-NS1-APEX2 virus that secretes sNS1-APEX2 fusion protein from infected cells. sNS1-APEX2-containing cell culture supernatants were inoculated onto naïve human cells to allow sNS1-directed host cell binding and internalisation. Following APEX2-catalysed biotinylation of sNS1 proximal proteins, enriched biotinylated proteins were identified by mass spectrometry, revealing a broad range of candidate human host proteins associated with the early events of sNS1 internalisation. Comparisons of these sNS1 proximal proteins with previous NS1 proteomic studies revealed substantial overlap, confirming the validity of our approach. The identification of multiple proteins that are known to directly interact with sNS1 to induce important biological effects, or to associate with sNS1 in DENV infected patients, confirmed the biological and physiological relevance of our novel data set. Gene ontology analyses revealed an enrichment of host factors associated with extracellular vesicles, suggesting that sNS1 may hijack these intercellular communication vehicles to facilitate sNS1 host cell binding and internalisation. Protein-protein interaction network analyses revealed several functional and/or physical associations that connect many of our identified proteins involved in intracellular trafficking pathways, suggesting that these host factors may be involved in the early events of internalised sNS1 trafficking. Taken together, this work has revealed COPI as a key factor in NS1 secretion and identified a range of host factors associated with sNS1 internalisation. Together, this understanding may aid in the identification of novel targets for NS1-targeting antiviral drug development.
Keywords: dengue virus, orthoflavivirus, flavivirus, non-structural protein 1, NS1, secreted NS1, sNS1
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Dr Nicholas Eyre