It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.The transport of proteins and ribonucleoprotein (RNP) particles into and out of the cell nucleus is mediated by nuclear pore complexes, which are an integral part of the nuclear envelope. Over the past few years, significant advances in the understanding of receptor-mediated nuclear import of proteins have been made (for reviews, see refs. 1-3). In contrast, the mechanisms mediating the transport of RNA, although expected to exploit similar processes, are still poorly understood. Nevertheless, several candidate proteins conceivably involved in RNP-mediated export of RNA have been identified (4). Among them are nucleocytoplasmic shuttle proteins such as the heterogeneous nuclear RNP A1 protein that is presumably involved in the export of mRNA, glyceraldehyde-3-phosphate dehydrogenase that is possibly involved in the transport of tRNA, and also proteins mediating the export of 5S rRNA, such as the transcription factor IIIA (TFIIIA) or ribosomal protein L5. Furthermore, certain viral proteins have been described as affecting the intracellular distribution of viral mRNA. In particular, the regulatory protein Rev of HIV-1 appears to be a specific RNA export factor (5).The activity of Rev is essential for HIV-1 replication. In the absence of Rev, only fully spliced HIV-1 mRNAs, encoding viral regulatory proteins, accumulate in the cytoplasm. In the presence of Rev, incompletely spliced and unspliced viral transcripts, encoding the viral structural proteins or serving as progeny virus genomes, are transported across the nuclear envelope (6-9). In accordance with its activity in RNA export, Rev has been shown to be a nucleolar protein that shuttles constantly between the nucleus and cytoplasm (10)(11)(12). A series of studies demonstrated that Rev binds in a sequencespecific manner to the Rev response element (RRE) (13-16), a cis-acting target sequence within the env gene (17, 18), resulting in the transport of viral mRNA independent of pre-mRNA splicing (19).Functional analyses of the Rev protein revealed a leucinerich carbo...