2 FUNCTIONAL DOMAINS OF THE INFLUENZA-VIRUS NS1 PROTEIN ARE REQUIRED FOR REGULATION OF NUCLEAR EXPORT OF MESSENGER-RNA

The influenza virus NS1 protein is the only known example of a protein that inhibits the nuclear export of mRNA. To identify the functional domains of this protein, we introduced 18 2- or 3-amino-acid substitut ions at approximately equally spaced locations along the entire length of the protein. Two functional domains were identified. The domain ne ar the amino end (amino acids 19 through 38) was shown to be the RNA-b inding domain, by using a gel shift assay with purified NS1 protein an d spliced viral NS2 mRNA as the RNA target. The second domain, which i s in the carboxy half of the molecule, was presumed to be the effector domain that interacts with host nuclear proteins to carry out the nuc lear RNA export function, by analogy with the effector domain of the R ev proteins of human immunodeficiency virus (HIV) and other lentivirus es which facilitate rather than inhibit nuclear RNA export. The NS1 pr otein has a 10-amino-acid sequence that is similar to the consensus se quence in the effector domains of lentivirus Rev proteins, specificall y including two crucial leucines at positions 7 and 9 of this sequence . However, the effector domains of the NS1 and Rev (HIV type 1 [HIV-1] ) proteins differed in several significant ways including the followin g: (i) unlike the HIV-1 Rev protein, NS1 effector domain mutants were negative recessive rather than negative dominant, (ii) the NS1 effecto r domain is about three times larger than the effector domain of the H IV-1 Rev protein, and (iii) unlike the HIV-1 protein, NS1 effector dom ain mutants exhibited a surprising property, a changed intracellular/i ntranuclear distribution, compared with the wild-type protein. These d ifferences strongly suggest that the effector domains of the NS1 and R ev proteins interact with different nuclear protein targets, which lik ely explains the opposite effects of these two proteins on nuclear mRN A export.