Non-structural protein 1 (NS1) is one of the most enigmatic proteins of the Dengue virus (DENV), playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E) and precursor Membrane (prM). Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles. Dengue virus (DENV) is a major arthropod-borne human pathogen, infecting more than 400 million individuals annually worldwide; however, neither a therapeutic drug nor a prophylactic vaccine is currently available. Amongst the DENV proteins, non-structural protein 1 (NS1) is one of the most enigmatic, being required for RNA replication, but also secreted from infected cells to counteract antiviral immune response, thus contributing to pathogenesis. Despite its essential role at early stages of the viral replication cycle, the molecular determinants governing NS1 functions are unknown. Here, we used a combination of genetic, high-resolution imaging and biochemical approaches and found that NS1 additionally plays an important role for the production of infectious virus particles. By using a novel trans-complementation system with fully functional epitope-tagged NS1, we show that NS1 interacts with the structural proteins residing in the envelope of the virus particle. An NS1 variant retained in the endoplasmic reticulum still supported efficient DENV particle production, demonstrating that secretion of NS1 is dispensable for virion production. This study expands the list of functions exerted by NS1 for the DENV replication cycle. Given this multi-functional nature, NS1 appears to be an attractive target for antiviral therapy.

Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro . ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.