Interaction of the HIV-1 rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5 - PubMed (original) (raw)

Interaction of the HIV-1 rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5

O Schatz et al. Proc Natl Acad Sci U S A. 1998.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1

Isolation of a ribosomal protein L5 cDNA by yeast two-hybrid assay. (A) A cDNA library derived from HeLaS3 cells was used to identify proteins that bind to human eIF-5A. Cell growth on His− medium was observed by the interaction of the GAL4 binding domain–eIF-5A and the L5–GAL4 activation domain fusion proteins. Yeast cells were transformed with the following constructs: Regions: I (positive control), pVA3 (p53/GAL4 binding domain hybrid) + pTD1 (simian virus 40 large tumor antigen–GAL4 activation domain hybrid); II (negative control), pGB5A (eIF-5A–GAL4 binding domain hybrid) + pGAD424 (GAL4 activation domain); III, pGB5A + pGADL5 (L5–GAL4 activation domain hybrid). (B) Amino acid sequence of ribosomal protein L5. The isolated cDNA encodes a protein that perfectly matches the recently published sequence and displays strong homology to the L5 protein from rat (43, 47) (indicated on the lower line).

Figure 2

Interaction of ribosomal protein L5 with eIF-5A in reticulocyte extracts. (A) Binding specificity of affinity-purified rabbit polyclonal anti-eIF-5A antibodies (α-eIF-5A; lanes 1 and 2) and human anti-L5 protein antiserum (α-L5; lanes 3 and 4) (42). Protein extracts of untransfected COS cells and cultures transiently transfected with peIF-5A (26) or pL5 were subjected to Western blot analysis. Both the anti-eIF-5A and anti-L5 antibodies show high specificity for their homologous antigen. Molecular mass standards (in kilodaltons) are indicated on the left. The location of L5 and eIF-5A protein are indicated on the right. (B) Coprecipitation of L5 protein with anti-eIF-5A antibody. Sequences encoding either the human L5 gene (L5) or the corresponding antisense sequence (L5as) were expressed in an in vitro transcription/translation system, followed by immunoprecipitation with anti-eIF-5A or anti-L5 antibodies shown in A. The reticulocyte extract used in lane 7 was first depleted of L5 protein using the anti-L5 serum and then subjected to eIF-5A-specific immunoprecipitation.

Figure 3

Binding of L5 to eIF-5A in vivo and in vitro. (A) Coprecipitation of eIF-5A and ribosomal protein L5 using total cell lysate. Equal amounts of HeLa cell extracts were subjected to immunoprecipitation analyses with preimmune IgG (lane 1, negative control), anti-eIF-5A antibodies (lane 2, α-eIF–5A), or anti-L5 antibodies (α-pL5, lane 3, positive control). The precipitated complexes were resolved on SDS/polyacrylamide gels, immobilized on membranes, and subjected to Western blot analysis with anti-L5 antibodies (α-pL5). The location of L5 protein is indicated on the left. (B) Specific interaction of recombinant His-tagged L5 with GST–eIF-5A fusion proteins. Bacterial S100 extracts containing either His-tagged L5 (lanes 2–5) or His-tagged prolactin (PRL, lanes 6–9) protein were incubated in vitro with various GST fusion proteins (indicated at the bottom). The binding reactions were then immobilized with metal affinity resin and eluted with imidazole. Eluted proteins were separated on SDS/polyacrylamide gels, blotted, and subjected to Western blot analysis with anti-GST antibodies. For comparison, recombinant GST–eIF-5A wild-type protein (WT) was loaded directly in lane 1 of the SDS/polyacrylamide gel (indicated at the left). The GST–M9 (49, 50) protein served as negative control (lanes 2 and 6).

Figure 4

Rev export in human somatic cells. Nuclei of HeLa cells were microinjected with the indicated GST Rev (A–G) or GST L5 (H) fusion proteins, antibodies, and rabbit IgG. About 20 min after microinjection, the cells were fixed and subjected to double-label indirect immunofluorescence analysis [Texas Red, Rev or L5; fluorescein isothiocyanate, IgG (internal injection control)], and nuclei were visualized by DNA staining (4′,6-diamidino-2-phenylindole). Control experiments using Rev or RevM32 (Rev activation domain mutant) confirmed the requirement of the activation domain for Rev nuclear export (A and B).

References

1. Melchior F, Gerace L. Curr Opin Cell Biol. 1995;7:310–318. - PubMed
1. Görlich D, Mattaj I W. Science. 1996;271:1513–1518. - PubMed
1. Corbett A H, Silver P A. Microbiol Mol Biol Rev. 1997;61:193–211. - PMC - PubMed
1. Izaurralde E, Mattaj I W. Cell. 1995;81:153–159. - PubMed
1. Cullen B R, Malim M H. Trends Biochem Sci. 1991;16:346–350. - PubMed

Interaction of the HIV-1 rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5 - PubMed (original) (raw)