Systematic interactome analysis identifies OCRL as a DENV 2 NS4A interactor that inhibits viral replication through a mechanism independent of global actin cytoskeleton rearrangement

Dengue virus (DENV) is a medically important human pathogen belonging to the genus Orthoflavivirus. DENV encodes seven non-structural (NS) proteins which exert multiple cellular effects. NS4A is a 16 kDa membrane-associated protein; however, its functions in orthoflavivirus infection remain poorly characterized. In this study, we aimed to elucidate the functions of DENV 2 NS4A by identifying its cellular interactome using EGFP immunoprecipitation coupled with label-free quantitative mass spectrometry. A total of 212 DENV 2 NS4A interacting proteins were identified, with a notable enrichment of proteins regulating endosomal trafficking, proteins possessing ATP-dependent activity acting on RNA, and proteins with helicase activity. Inositol polyphosphate 5-phosphatase oculocerebrorenal syndrome of Lowe protein 1 (OCRL), DEAD-box helicases, DDX18 and DDX41 (DDX18 and DDX41) were shown to interact with DENV 2 NS4A and have inhibitory roles in DENV infection. Further characterization revealed that DENV infection induces a late-stage upregulation of OCRL mRNA at 48 h post-infection. To further evaluate its functional role, siRNA-mediated knockdown of OCRL was performed, which significantly promoted DENV genome replication, viral protein expression, and infectious virus production. Quantitative texture analysis of the actin cytoskeleton demonstrated that while OCRL depletion modulated local actin bundle contrast, it did not induce global spatial disruption or network fragmentation. The antiviral activity of OCRL is suggested to be related to the cathepsin D-mediated lysosomal degradation pathway. Our findings demonstrate that OCRL functions as a restrictive host factor against DENV 2 infection, whereas DDX18 and DDX41 reduce viral production. Thus, this study provides new insights into the complex interplay between the dengue NS4A protein and host regulatory factors, highlighting a novel mechanism of viral restriction through the modulation of lysosomal trafficking.