Chimeras Containing Influenza NS1 and HIV-1 Rev Protein Sequences: Mechanism of Their Inhibition of Nuclear Export of Rev Protein–RNA Complexes

Abstract

Nuclear RNA export mediated by the HIV-1 Rev protein is inhibited by chimeric proteins in which the wild-type Rev protein is covalently linked to amino acid sequences of the NS1 protein of influenza A virus (NS1A protein), a protein that inhibits nuclear RNA export. These chimeric molecules function not only incisbut also intrans:they inhibit nuclear RNA export mediated by Rev protein molecules that are not covalently linked to the NS1A protein sequence. Here we show that inhibition occurs with a NS1-Rev chimera in which the 78 amino-terminal amino acids of the NS1A protein comprising its entire RNA-binding domain is deleted, thereby establishing that this carboxyl portion of the NS1A protein can function as an independent effector domain. The mechanism by which this NS1-Rev chimera inhibits Rev function intranswas determined. The Rev sequence in this chimera oligomerizes with Rev molecules intrans,and the resulting mixed oligomers are retained in the nucleus because the nuclear retention activity of the NS1 effector domain is dominant over the nuclear transport activity of the Rev effector domain. Binding of the FG-containing nucleoporin-like Rab protein to this NS1-Rev chimera, as measured in yeast two-hybrid assays, is much stronger than that to the Rev protein itself, yet nuclear export does not occur 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 detectable Rab binding, indicating that this nuclear export can occur without detectable Rab binding. A different NS1-Rev chimera, one in which the NS1A protein is full-length but contains a mutated RNA-binding domain, effectively inhibits Rev-mediated nuclear export of RNA without blocking the nuclear export of the Rev protein, indicating that nuclear export of the carrier Rev protein can be uncoupled from nuclear export of its passenger RNA.