Identification and characterization of Risp
Beschreibung
vor 23 Jahren
The aim of this study was to characterize the role of
Rev-interacting cellular proteins in controlling the function of
Rev in the host cell. The HIV-1 protein Rev plays an essential role
in the temporal regulation of the virus gene expression by
stimulating the expression of viral structural proteins. Rev
enhances the nucleocytoplasmic transport and the translation of
unspliced and single spliced viral mRNAs by binding with high
affinity to a specific target element on the HIV-RNA. It was
assumed that interaction with cellular factors is essential for Rev
function. At the onset of this study, only a few potential
cofactors were known with no clearly defined functional relevance.
So we decided to search for new Rev-interacting factors using the
yeast two-hybrid system. In this work a new Rev-interacting protein
has been identified, by screening a Jurkat T cell cDNA library. The
protein was termed Risp (Rev-interacting shuttle protein), because
it shuttles between the nuclear and the cytoplasmic compartments.
The Risp gene is widely expressed in human cells and conserved
among various species, most probably as part of a larger gene. High
amino acid homology (99%) with the C-terminal part of a large brain
cDNA clone for KIAA0592 protein has been found, whereas no obvious
homology to proteins with known function was observed. However, a
weak and partial similarity appeared with several RNA-/DNA-binding
and shuttle proteins. This might indicate that the Risp protein -
or the larger protein containing it - could be a member of a new
family of nucleocytoplasmic shuttle proteins with RNA-/DNA-binding
function. Next, the intracellular localization and shuttling of
Risp was investigated. In HeLa cells Risp- GFP localized in both
nuclear and cytoplasmic compartments, but clearly accumulated in
the cytoplasm, indicating the presence of a strong nuclear export
signal (NES). The identification of a NES sequence was confirmed by
deletion analysis of Risp and by nuclear microinjection of
BSA-fusion proteins conjugated to peptides from the C-terminal part
of Risp. Treatment with leptomycin B, a drug which has been shown
to specifically block Crm1 (exportin) mediated export, resulted in
nuclear accumulation of Risp-GFP, showing that the nuclear export
of Risp, like that of Rev, is Crm1-dependent. Using bioinformatic
tools able to detect weak homologies with high specificity,
sequence comparisons between Risp and all currently known Rev
interacting factors were performed. This analysis for the first
time revealed a common motif shared between Rev and Rev-interacting
cellular factors, termed RIP. The region of Risp harboring the RIP
motif was neither essential nor sufficient for the Rev-binding in
the yeast two hybrid system, suggesting no direct correlation
between RIP and the Rev-binding ability. Preliminary experiments
suggested, that Risp, as a Rev-interacting protein, is able to
inhibit Revtrans- activation, while Risp does not interfere with
Tat in a Tat-trans-activation assay. The overexpression of Risp-GFP
reduced the production of the Rev-dependent structural viral
protein p24gag up to 70%. In addition a previously unrecognized
sequence motif in the activation domain of Rev with intrinsic
nuclear import activity was found and tested in transfection and
microinjection assays. This motif (“PPXXR”) is conserved in various
RNA-binding proteins and was proposed to mediate nuclear
translocation of the cellular functional homologue of HIV-1 Rev
Sam68.
Rev-interacting cellular proteins in controlling the function of
Rev in the host cell. The HIV-1 protein Rev plays an essential role
in the temporal regulation of the virus gene expression by
stimulating the expression of viral structural proteins. Rev
enhances the nucleocytoplasmic transport and the translation of
unspliced and single spliced viral mRNAs by binding with high
affinity to a specific target element on the HIV-RNA. It was
assumed that interaction with cellular factors is essential for Rev
function. At the onset of this study, only a few potential
cofactors were known with no clearly defined functional relevance.
So we decided to search for new Rev-interacting factors using the
yeast two-hybrid system. In this work a new Rev-interacting protein
has been identified, by screening a Jurkat T cell cDNA library. The
protein was termed Risp (Rev-interacting shuttle protein), because
it shuttles between the nuclear and the cytoplasmic compartments.
The Risp gene is widely expressed in human cells and conserved
among various species, most probably as part of a larger gene. High
amino acid homology (99%) with the C-terminal part of a large brain
cDNA clone for KIAA0592 protein has been found, whereas no obvious
homology to proteins with known function was observed. However, a
weak and partial similarity appeared with several RNA-/DNA-binding
and shuttle proteins. This might indicate that the Risp protein -
or the larger protein containing it - could be a member of a new
family of nucleocytoplasmic shuttle proteins with RNA-/DNA-binding
function. Next, the intracellular localization and shuttling of
Risp was investigated. In HeLa cells Risp- GFP localized in both
nuclear and cytoplasmic compartments, but clearly accumulated in
the cytoplasm, indicating the presence of a strong nuclear export
signal (NES). The identification of a NES sequence was confirmed by
deletion analysis of Risp and by nuclear microinjection of
BSA-fusion proteins conjugated to peptides from the C-terminal part
of Risp. Treatment with leptomycin B, a drug which has been shown
to specifically block Crm1 (exportin) mediated export, resulted in
nuclear accumulation of Risp-GFP, showing that the nuclear export
of Risp, like that of Rev, is Crm1-dependent. Using bioinformatic
tools able to detect weak homologies with high specificity,
sequence comparisons between Risp and all currently known Rev
interacting factors were performed. This analysis for the first
time revealed a common motif shared between Rev and Rev-interacting
cellular factors, termed RIP. The region of Risp harboring the RIP
motif was neither essential nor sufficient for the Rev-binding in
the yeast two hybrid system, suggesting no direct correlation
between RIP and the Rev-binding ability. Preliminary experiments
suggested, that Risp, as a Rev-interacting protein, is able to
inhibit Revtrans- activation, while Risp does not interfere with
Tat in a Tat-trans-activation assay. The overexpression of Risp-GFP
reduced the production of the Rev-dependent structural viral
protein p24gag up to 70%. In addition a previously unrecognized
sequence motif in the activation domain of Rev with intrinsic
nuclear import activity was found and tested in transfection and
microinjection assays. This motif (“PPXXR”) is conserved in various
RNA-binding proteins and was proposed to mediate nuclear
translocation of the cellular functional homologue of HIV-1 Rev
Sam68.
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