Trans-activation response (TAR) RNA-binding protein (TRBP), is a cellular protein that binds to the human immunodeficiency virus type 1 (HIV-1) TAR RNA and activates viral expression. TRBP also binds the interferon induced, double-stranded RNA-activated protein kinase, PKR, that exerts antiviral activities. Activated PKR inhibits translational initiation by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIf-2alpha). TRBP blocks the inhibitory effects of PKR, whereas PKR activating protein (PACT), another cellular protein, heterodimerizes with and activates PKR in vitro and in vivo in the absence of viral infection. TRBP and PACT are highly homologous but have opposite effects on PKR. / We studied the interplay between TRBP and PACT on PKR activation and on the regulation of HIV expression. By the yeast two-hybrid method, we showed an interaction between TRBP and PACT and we characterized domains implicated in this interaction. We confirmed this interaction in the human cellular context by coimmunoprecipitation. Immunofluorescence studies showed that TRBP and PACT colocalize in the cytoplasm. In the context of HIV-1 expression, PKR mediates an inhibition of LTR (long terminal repeat) expression, but no further decrease was obtained with PACT. Interestingly, in coimmunoprecipitation studies, we found that the concentration of PACT protein is decreased when PACT and TRBP were cotransfected, whereas PACT mRNA level remains unchanged. This result suggests that TRBP either blocks PACT translation or induces its degradation, which open new ways of PKR and viral regulation.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.80311 |
| Date | January 2004 |
| Creators | Laraki, Ghislaine |
| Contributors | Gatignol, Anne (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002031527, proquestno: AAIMQ98680, Theses scanned by UMI/ProQuest. |
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