Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication associated with both a previously un-recognized DDX3 inhibitory role in type I interferon production in arenavirus infected cells and a positive DDX3 effect on arenavirus RNA synthesis that was dependent on its ATPase and Helicase activities. Our results uncover novel mechanisms used by arenaviruses to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses.

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVɸNSs). Unlike rTOSV and the wt virus, rTOSVɸNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.