During flavivirus infection, some viral proteins move to the nucleus and cellular components are relocated from the nucleus to the cytoplasm. Thus, the integrity of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC), was evaluated during infection with dengue virus (DENV) and Zika virus (ZIKV). We found that while during DENV infection the integrity and distribution of at least three nucleoporins (Nup), Nup153, Nup98, and Nup62 were altered, during ZIKV infection, the integrity of TPR, Nup153, and Nup98 were modified. In this work, several lines of evidence indicate that the viral serine protease NS2B3 is involved in Nups cleavage. First, the serine protease inhibitors, TLCK and Leupeptin, prevented Nup98 and Nup62 cleavage. Second, the transfection of DENV and ZIKV NS2B3 protease was sufficient to inhibit the nuclear ring recognition detected in mock-infected cells with the Mab414 antibody. Third, the mutant but not the active (WT) protease was unable to cleave Nups in transfected cells. Thus, here we describe for the first time that the NS3 protein from flavivirus plays novel functions hijacking the nuclear pore complex, the main controller of the nuclear-cytoplasmic transport.

Viruses hijack cellular proteins and components to be replicated in the host cell and to evade the immune response. Although flaviviruses have a cytoplasmic replicative cycle, some viral proteins such as the capsid (C) and the RNA dependent RNA polymerase, NS5, can reach the nucleus of the infected cells. Considering the important roles of NS5 in viral replication and in the control of the immune response, and its striking presence in the nucleus, the possible functions of this protein in some mechanisms orchestrated by the nucleus was analyzed. We isolated and identified nuclear proteins that interact with NS5; one of them, the DEAD-box RNA helicase DDX5 is relocated to the cytoplasm and degraded during infection with DENV, which correlates with its function in IFN dependent response. Since DDX5 and many other proteins are relocated from the nucleus to the cytoplasm during flavivirus infection, the integrity and function of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC) was evaluated. We found that during DENV and ZIKV infection nucleoporins (NUPs) such as TPR, Nup153, Nup98, and Nup62 were cleavaged/degraded. The protease NS2B-NS3 induces NUPs degradation and it causes a dramatic inhibition of mature mRNAs export to the cytoplasm but not the export of DDX5 protein, which is dependent on NS5. Here we describe for the first time that the NS3 and NS5 proteins from flavivirus play novel functions hijacking the NPC and some nuclear proteins relevant in triggering immune response pathways, inducing a favorable environment for viral replication. IMPORTANCE: Viruses, as intracellular obligate parasites, hijack cellular components to enter and replicate in infected cells. Remarkably, in many cases, viruses hijack molecules with crucial functions for the cells. Here it is described how RNA viruses such as DENV and ZIKV, with a cytoplasmic replicative cycle, use NS3 and NS5, two of their unique non-structural proteins with enzymatic activity, to modulate nuclear-cytoplasmic transport. We found that NS3 disrupts the nuclear pore complex, the main regulator in nuclear-cytoplasmic transport, causing a strong reduction in the amount of mature mRNAs in the cytoplasm and an inhibition in innate immune response. Additionally, NS5 induces the relocation of nuclear proteins to the cytoplasm such as DDX5, involved in immune response, which is later degraded by NS3. These findings allow the understanding of crucial mechanisms that viruses use to deal with the control of the immune response to grant the production of new viral particles.

Despite the importance of Dengue virus (DENV) infection in human health, there is not a fully effective vaccine or antiviral treatment against the infection. Since lipids such as cholesterol are required during DENV infection, its uptake and synthesis are increased in infected cells. Ezetimibe is an FDA-approved drug that reduces cholesterol uptake in humans by inhibiting the endocytosis through Niemman-Pick C1-Like 1 (NPC1L1) receptor, expressed on the membrane of enterocytes and hepatocytes. Our results indicate that an increase in the amount of NPC1L1 occurs on the surface of Huh-7 cells during DENV infection, which correlates with an increase in cholesterol levels. Blockage of NPC1L1 with ezetimibe in concentrations up to 50 μM does not reduce cell viability but diminished total cellular cholesterol, the percentage of infected cells, viral yield, viral RNA and protein synthesis without affecting DENV binding and/or entry to Huh-7 cells. Moreover, ezetimibe inhibited DENV replicative complex formation and lipid droplets accumulation. All these results indicate that ezetimibe is an excellent drug to inhibit DENV infection and confirm that cholesterol is a key target to inhibit viral infection.