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 to the development of NS1-targeting antiviral therapies. To this end, we performed a customized 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 wild-type 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 world's 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 the secretion of NS1 from infected cells using a customized membrane-trafficking siRNA screen. This revealed three components of the COPI complex that regulate 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 wild-type 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.