Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

The dengue virus (DENV) non-structural protein 1 (NS1) is a glycoprotein highly conserved among mosquito-borne orthoflaviviruses. NS1 is typically localized in the lumen of the endoplasmic reticulum, where it forms part of the replication complexes, and is also exposed at the plasma membrane. In addition, NS1 is secreted as a lipoprotein. Here, using a combination of approaches, including confocal microscopy with deconvolution, in situ analysis, and biochemical cell fractionation, we show that a substantial fraction of NS1 (up to 30%) translocates to the nucleus during infection. We identified a conserved, structurally exposed bipartite nuclear localization signal (NLS) within NS1. Pharmacological inhibition with ivermectin and site-directed mutagenesis of the NLS in recombinant confirmed that nuclear import of NS1 is an active process, dependent on the classical importin /{beta} pathway. Notably, both dimeric and multimeric forms of NS1 were detected in the nucleus in association with nuclear lamin. Introduction of the NLS mutations into DENV2 infectious clones resulted in a non-viable virus. Production of virus progeny and completion of the replicative cycle by the mutant genomes could be rescued by trans-complementation with wild-type NS1, but not with an NLS-mutated NS1, indicating that an NS1 nuclear phase is required for a productive infection. Transcriptomic analysis by RNA-seq further revealed that NS1 functions depend on its subcellular location. Nuclear NS1 induced the overexpression of genes associated with DNA-binding transcription factors, whereas NLS-mutated NS1, retained in the cytoplasm, failed to induce these genes and instead triggered pro-inflammatory and metabolic responses. Together, these findings reveal a previously unrecognized nuclear phase of NS1 that is required for an efficient viral life cycle, redefining NS1 as a modulator of the host transcriptional environment. These findings also suggest new avenues for antiviral and vaccine development. Authors summaryDengue virus NS1 is a glycoprotein of approximately 45-50 kDa that rapidly dimerizes after proteolytic maturation. Dimeric NS1 is located in the lumen of the endoplasmic reticulum where it acts as a scaffold component of the viral replication complexes. In addition, NS1 is secreted from infected cells as a tetramer or hexamer and circulates in the serum of infected individuals during the acute phase of dengue disease. Circulating NS1 is widely used as a diagnostic marker and has also been associated with dengue pathogenesis through several mechanisms. Here, we expand the current understanding of DENV NS1 by identifying a previously unrecognized and essential nuclear location of this protein. We show that NS1 contains a conserved nuclear localization signal that mediates import into the nucleus via the classical import pathway. Using wild-type and NLS-mutated infectious clones, we demonstrated that nuclear localization of NS1 is required for completion of the DENV replicative cycle. Transcriptomic analysis further revealed that nuclear NS1 promotes the expression of host genes involved in nucleic acid metabolism, whereas retention of NS1 in the cytoplasm triggers an antiviral and inflammatory response. Together, these findings identify the nucleus as an important site of dengue virus-host interactions and redefine NS1 as a regulator of the host transcriptional environment during infection.