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.