Characterization of RNABP, an RNA binding protein that associates with RNase L

The 2',5'-oligoadenylate (2-5A)/RNase L pathway is one of several enzymatic pathways induced by interferons (IFN), RNase L is a latent endoribonucleas e that is activated on its binding by 2-5A and inhibited by the ribonucleas e L inhibitor (RLI), We have shown previously by coimmunoprecipitation that RNase L may be associated with a 90-kDa RNA binding protein (RNABP), ident ified with a monoclonal antibody (mAb) raised against an RNase L complex pu rified under native conditions on 2-5A-sepharose. Here we confirm, by gelfi ltration and pull-down analysis, the association of RNase L and RNABP, and we demonstrate that this association is significantly increased in the pres ence of 2-5A, Moreover, we found that RNABP protein levels decrease during terminal differentiation in various cell lines but do not vary during vesic ular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) infection or following IFN-alpha/beta treatment. In this latter case, although total cellular RNABP levels do not vary, the amount of RNABP found in the cytopla sm increases in comparison to that found in the nucleus, indicating a cytop lasmic localization of RNABP after IFN-alpha/beta treatment. Finally, we de monstrate the interaction between RNase L and RNABP in intact cells. Microi njection of an mAb against RNABP into HeLa cells inhibits RNase L antiviral activity and partially inhibits the IFN-alpha/beta-induced antiviral activ ity.