CHIMERAS CONTAINING INFLUENZA NS1 AND HIV-1 REV PROTEIN SEQUENCES - MECHANISM OF THEIR INHIBITION OF NUCLEAR EXPORT OF REV PROTEIN RNA COMPLEXES

Nuclear RNA export mediated by the HIV-I Rev protein is inhibited by c himeric proteins in which the wild-type Rev protein is covalently link ed to amino acid sequences of the NS1 protein of influenza A virus (NS 1A protein), a protein that inhibits nuclear RNA export. These chimeri c molecules function not only in cis but also in trans: they inhibit n uclear RNA export mediated by Rev protein molecules that are not coval ently linked to the NS1A protein sequence. Here we show that inhibitio n occurs with a NS1-Rev chimera in which the 78 amino-terminal amino a cids of the Ns1A protein comprising its entire RNA-binding domain is d eleted, thereby establishing that this carboxyl portion of the NS1A pr otein can function as an independent effector domain. The mechanism by which this NS1-Rev chimera inhibits Rev function in trans was determi ned. The Rev sequence in this chimera oligomerizes with Rev molecules in trans, and the resulting mixed oligomers are retained in the nucleu s because the nuclear retention activity of the NS1 effector domain is dominant over the nuclear transport activity of the Rev effector doma in. Binding of the FG-containing nucleoporin-like Rab protein to this NS1-Rev chimera, as measured in yeast two-hybrid assays, is much stron ger than that to the Rev protein itself, yet nuclear export does not o ccur in the presence of the chimera. Unexpectedly, the introduction of specific mutations into the NS1A portion of this NS1-Rev chimera not only restores Rev-mediated nuclear export of RNA but also eliminates d etectable Rab binding, indicating that this nuclear export can occur w ithout detectable Rab binding. A different NS1-Rev chimera, one in whi ch the NS1A protein is full-length but contains a mutated RNA-binding domain, effectively inhibits Rev-mediated nuclear export of RNA withou t blocking the nuclear export of the Rev protein, indicating that nucl ear export of the carrier Rev protein can be uncoupled from nuclear ex port of its passenger RNA. (C) 1998 Academic Press.