Probing the structure of HIV‐1 Rev by protein footprinting of multiple monoclonal antibody‐binding sites

Jensen, Torben Heick; Jensen, Allan; Szilvay, Anne Marie; Kjems, Jørgen

doi:10.1016/s0014-5793(97)00988-5

Cited by 22 publications

(15 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The C-terminal region of Rev is thought to be more flexible. However, a discontinuous epitope of a Rev-specific monoclonal antibody was mapped to aa 10 to 20 and 95 to 105 by protein foot printing, suggesting that the α-helices are in close proximity to the Rev C-terminus [34,35], and suggesting a role for the C-terminus in stabilizing native Rev structure. Thus, aa changes occurring at the Rev C-terminus or elsewhere such as Pro-74, Leu-104 and/or Pro-106 could potentially affect Rev structure and thus, Rev/RRE binding.…”

Section: Resultsmentioning

confidence: 99%

Persistence of attenuated HIV-1 rev alleles in an epidemiologically linked cohort of long-term survivors infected with nef-deleted virus

et al. 2007

View full text Add to dashboard Cite

Background: The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with nef-deleted, attenuated strains of human immunodeficiency virus type 1 (HIV-1). Although the cohort members have experienced differing clinical courses and now comprise slow progressors (SP) as well as long-term nonprogressors (LTNP), longitudinal analysis of nef/long-terminal repeat (LTR) sequences demonstrated convergent nef/LTR sequence evolution in SBBC SP and LTNP. Thus, the in vivo pathogenicity of attenuated HIV-1 strains harboured by SBBC members is dictated by factors other than nef/LTR. Therefore, to determine whether defects in other viral genes contribute to attenuation of these HIV-1 strains, we characterized dominant HIV-1 rev alleles that persisted in 4 SBBC subjects; C18, C64, C98 and D36.

show abstract

Section: Resultsmentioning

confidence: 99%

Persistence of attenuated HIV-1 rev alleles in an epidemiologically linked cohort of long-term survivors infected with nef-deleted virus

et al. 2007

View full text Add to dashboard Cite

show abstract

“…We have previously used this assay successfully to map the binding sites of the RRE, monoclonal antibodies, and short peptides on Rev (40,48,49). In this approach radioactively end-labeled protein is digested partially by a variety of endoproteinases in the absence and presence of a particular ligand.…”

Section: Addition Of Rangtp To Rev/crm1 Results In a Lmb-sensitive Tementioning

confidence: 99%

“…Finally, the samples were applied to Bio-Spin™ 6 columns (Bio-Rad), and Ran was eluted in Ran buffer (50 mM Tris/HCl, pH 7.5, 200 mM NaCl, 2 mM MgCl 2 , 2 mM dithiothreitol, 10% glycerol) containing 2 mM of either GDP or GTP. Radiolabeling of purified proteins containing a HMK phosphorylation site was performed in solution with HMK enzyme (Sigma) as described earlier (40). Unincorporated nucleotides were removed by affinity purification of His-tagged Rev derivatives on Ni-NTA agarose or by gel filtration of CRM1 using Bio-Spin™ 6 columns (BioRad).…”

Section: Methodsmentioning

confidence: 99%

The Specificity of the CRM1-Rev Nuclear Export Signal Interaction Is Mediated by RanGTP

Askjaer¹,

Jensen²,

Nilsson³

et al. 1998

Journal of Biological Chemistry

Self Cite

203

189

View full text Add to dashboard Cite

Nuclear export of intron-containing human immunodeficiency virus type 1 (HIV-1) RNA is mediated by the viral Rev protein that contains both an RNA binding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process that also requires the GTP bound form of the Ran GTPase. Using purified recombinant factors, we show by co-precipitation, gel mobility shift and protein footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leucine residues of the NES and the presence of the cytotoxin leptomycin B (LMB). Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP complex that is sensitive to LMB, NES mutations, and Ran being charged with GTP. Within this complex, CRM1 is readily cross-linked to Cys 89 near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and two regions in CRM1 become inaccessible to endoproteinases upon binding suggesting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export receptor for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a preformed Rev-CRM1 complex and specifies a functional interaction with the NES. Human immunodeficiency virus type 1 (HIV-1)1 encodes the regulatory protein Rev, which is absolutely required for viral replication. Rev promotes the nuclear export of incompletely spliced HIV-1 mRNA species, which are specified by the presence of a complex RNA structure, the Rev response element (RRE). Rev interacts directly with a purine-rich stem-loop called IIB within the RRE, and through oligomerization additional Rev molecules bind to lower affinity sites throughout the RRE (reviewed in Ref. 1). Rev is a 116-amino acids protein that has been shown to continuously shuttle between the nucleus and the cytoplasm (2, 3). Nuclear import of Rev is mediated by an arginine-rich nuclear localization signal (NLS) embedded in the RNA binding domain of the Rev protein (amino acids 34 -50) (4 -6). The NLS of Rev has been reported to associate with importin ␤, the large subunit of the conventional importin ␣/importin ␤ NLS receptor heterodimer, as well as with B23, a mammalian protein involved in the nuclear import of ribosomal proteins (7-9). Nuclear export of Rev is mediated by the nuclear export signal (NES), which contains a conserved stretch of characteristically spaced leucine residues (10, 11). Mutation of any of three leucines (Leu 78 , Leu 81 , or Leu 83 ) in the NES-core domain generates nonfunctional Rev proteins that loose their ability to exit the nucleus (2-4,12-16). The fact that these mutants remain active for specific binding and oligomerization on the RRE in vitro, has prompted the suggestion that they are unable to interact with a functionally important cellular export factor(s) (4). This model has gaine...

show abstract

“…The plasmids for Escherichia coli expression were generated by PCR amplification of either or both DRBMs in the pcDNA3 constructs, adding EcoRI and BamHI restriction sites by which the fragments were inserted into the pET H-His vector (Jensen et al 1997), yielding the d1 (ADAR2 amino acids 69-152), d2 (ADAR2 amino acids 223-307), and d1d2 (ADAR2 amino acids 69-307) with C-terminal 6xHis-tags.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

Poulsen¹,

Jørgensen²,

Heding³

et al. 2006

RNA

View full text Add to dashboard Cite

Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.

show abstract

Probing the structure of HIV‐1 Rev by protein footprinting of multiple monoclonal antibody‐binding sites

Cited by 22 publications

References 28 publications

Persistence of attenuated HIV-1 rev alleles in an epidemiologically linked cohort of long-term survivors infected with nef-deleted virus

Persistence of attenuated HIV-1 rev alleles in an epidemiologically linked cohort of long-term survivors infected with nef-deleted virus

The Specificity of the CRM1-Rev Nuclear Export Signal Interaction Is Mediated by RanGTP

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

Contact Info

Product

Resources

About