Proviral NUP153 binding to viral proteins and RNA regulates structural–nonstructural protein ratios in orthoflavivirus infection

Abstract Orthoflaviviruses are RNA viruses that cause serious diseases in humans, with currently no antivirals available. Targeting host factors is emerging as an attractive antiviral approach. However, as a first step, there is a need to understand which host proteins are hijacked and for what purpose. Here, using a combination of fluorescence microscopy, knock-down, crosslinking immunoprecipitation sequencing, mass spectrometry, and in vitro and biophysical assays, we identify nucleoporin-153 (NUP153) as a proviral factor during orthoflavivirus infection. We show that NUP153 is recruited to the virus amplification site on the endoplasmic reticulum to impact the structural to nonstructural viral protein ratios. We find that NUP153 interacts with both the viral proteins NS3 and NS5, and a highly conserved G-rich motif on the viral RNA. These interactions specifically promote the production of viral structural proteins, leading to an efficient virion assembly, virus release and spread to new cells. We propose that NUP153 acts as a key regulator in viral protein ratios, a mechanism that appears conserved among orthoflaviviruses.