A single point mutation in the nuclear localization domain of Sam68 blocks the Rev/RRE-mediated transactivation.
We have previously demonstrated that overexpression of Sam68 functionally substitutes for, as well as synergizes with, HIV-1 Rev in RRE-mediated gene expression and virus replication. In addition, we have shown that the C-terminal deletion mutants of Sam68 act with a transdominant negative phenotype in HIV replication. Previously, an Arginine429 mutation within the C-terminal domain of Sam68 has been reported to be critical for the localization of Sam68 in the nucleus. However, these studies were done in the context of truncated protein in which C-terminal amino acids 420 - 443 of Sam68 were fused to GFP. In contrast, we now report that the full length Sam68 protein having the same mutation (Arginine429-->Alanine) is completely localized in the nucleus while another Sam68 (Proline439-->Arginine) mutant is found in the cytoplasm. The localization of these Sam68 mutant proteins also correlates with their function in RRE-mediated reporter gene expression, i.e. Sam68 mutant protein that is localized in the cytoplasm failed to enhance RRE-mediated transactivation. Furthermore, we demonstrate that Sam68 P439-->R inhibited the transactivation of RRE-mediated gene expression by both wild type Sam68 and Rev. These results indicate that the proline residue at position 439 unlike arginine at position 429, may play a critical role in targeting Sam68 protein to nucleus. We propose that these negative dominant mutants of Sam68 may have potential as anti-viral agents to combat AIDS.
Reddy
T R
TR
Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California, CA 92093-0665, USA.
D048868:Adaptor Proteins, Signal Transducing; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D017930:Genes, Reporter; D005822:Genetic Vectors; D054303:HIV Reverse Transcriptase / Q000502:physiology*; D015497:HIV-1 / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000502:physiology*; D017354:Point Mutation; D016601:RNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014779:Virus Replication / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489559:KHDRBS1 protein, human; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C069802:nuclear factor, RRE binding; D015500:Chloramphenicol O-Acetyltransferase; D054303:HIV Reverse Transcriptase