ABSTRACT
DEAD-box helicases play central roles in the metabolism of many RNAs and ribonucleoproteins by assisting their synthesis, folding, function and even their degradation or disassembly. They have been implicated in various phenomena, and it is often difficult to rationalize their molecular roles from in vivo studies. Once purified in vitro, most of them only exhibit a marginal activity and poor specificity. The current model is that they gain specificity and activity through interaction of their intrinsically disordered domains with specific RNA or proteins. DDX3 is a DEAD-box cellular helicase that has been involved in several steps of the HIV viral cycle, including transcription, RNA export to the cytoplasm and translation. In this study, we investigated DDX3 biochemical properties in the context of a biological substrate. DDX3 was overexpressed, purified and its enzymatic activities as well as its RNA binding properties were characterized using both model substrates and a biological substrate, HIV-1 gRNA. Biochemical characterization of DDX3 in the context of a biological substrate identifies HIV-1 gRNA as a rare example of specific substrate and unravels the extent of DDX3 ATPase activity. Analysis of DDX3 binding capacity indicates an unexpected dissociation between its binding capacity and its biochemical activity. We further demonstrate that interaction of DDX3 with HIV-1 gRNA relies both on specific RNA determinants and on the disordered N- and C-terminal regions of the protein. These findings shed a new light regarding the potentiality of DDX3 biochemical activity supporting its multiple cellular functions.
