Rec (formerly Corf) function requires interaction with a complex, folded RNA structure within its responsive element rather than binding to a discrete specific binding site - PubMed (original) (raw)
Rec (formerly Corf) function requires interaction with a complex, folded RNA structure within its responsive element rather than binding to a discrete specific binding site
C Magin-Lachmann et al. J Virol. 2001 Nov.
Abstract
It was recently reported that the human endogenous retrovirus HTDV/HERV-K encodes the regulatory protein Rec (formerly designated Corf), which is functionally equivalent to the nuclear export adapter proteins Rev of human immunodeficiency virus and Rex of human T-cell leukemia virus. We have demonstrated that the Rec protein interacts with a characteristic 429-nucleotide RNA element, the Rec-responsive element (RcRE), present in the 3' long terminal repeat of HTDV/HERV-K transcripts. In analogy to the Rev and Rex proteins, which have distinct RNA binding sites in their responsive elements, we have proposed that Rec may also have a defined binding site in the RcRE. In this report, we demonstrate that not every HTDV/HERV-K copy present in the human genome contains an active RcRE, and we characterize mutations that abrogate Rec function. In addition, we demonstrate that Rec function requires binding to a complex, folded RNA structure rather than binding to a discrete specific binding site, in contrast to Rev and Rex and their homologous responsive elements. We define four stem-loop structures in the RcRE that are essential for Rec function. Finally, we demonstrate that both Rev and Rex can mediate nuclear export through the RcRE but that their binding sites are different from each other and from that of Rec.
Figures
FIG. 1
Alignment of RcRE sequences of different genomic LTRs. Mutational clusters M0 to M5 are depicted in bold letters. cons, consensus.
FIG. 2
Biological activity of RcRE sequences from HTDV/HERV-K genomes. The HIV Gag precursor in cell lysates was determined with an HIV p24 capture assay after cotransfection of the indicated pHIVgagRcRE reporter vectors into HLtat cells with the effector plasmid pRec or p(−) (A), pRev (B), or pRex (C). Expression of pHIVgagRcRE pcK30 in the presence of pRec was set as 100%; all other data are relative to this value. Error bars indicate average deviations.
FIG. 3
Activity of RcRE hybrids containing pcK30 and LTR21 sequences. (A) Schematic representation of hybrid sequences. pcK30 sequences are depicted as hatched bars; LTR21 sequences are depicted as white bars; 0 to 5 indicate the positions of mutations or consensus sequences. (B to D) Determination of the HIV Gag precursor in cell lysates after cotransfection of the indicated pHIVgagHyRcRE vectors into HLtat cells with the effector plasmid pRec or p(−) (B), pRev (C), or pRex (D). Error bars indicate average deviations.
FIG. 3
Activity of RcRE hybrids containing pcK30 and LTR21 sequences. (A) Schematic representation of hybrid sequences. pcK30 sequences are depicted as hatched bars; LTR21 sequences are depicted as white bars; 0 to 5 indicate the positions of mutations or consensus sequences. (B to D) Determination of the HIV Gag precursor in cell lysates after cotransfection of the indicated pHIVgagHyRcRE vectors into HLtat cells with the effector plasmid pRec or p(−) (B), pRev (C), or pRex (D). Error bars indicate average deviations.
FIG. 4
Predicted folding structures of active and inactive RcREs. (A) pcK30. (B) LTR21. The structures were predicted with DNASIS software using algorithms defined by Zuker (33).
FIG. 5
Activity of RcRE deletion mutants of pcK30. The HIV Gag precursor in cell lysates was determined after cotransfection of the indicated pHIVgagdel vectors (for exact positions of deletions, see Materials and Methods) into HLtat cells with the effector plasmid pRec or p(−) (A), pRev (B), or pRex (C). Error bars indicate average deviations.
FIG. 6
In vitro binding of recombinant purified Rec to RcRE sequences transcribed in vitro. RNA gel shift assays were performed as described in Materials and Methods. (A) Titration of Rec on biologically active pcK30 RcRE and on inactive RcRE LTR21 (six lanes at left). Competition for Rec binding to pcK30 RcRE with active (pcK30, Hy3, Hy7, and Hy10) and inactive (LTR21, Hy0, Hy1, and Hy2) RcREs (eight lanes at right). (B) Titration of Rec on active pcK30 RcRE and on inactive RcREdel2 (six lanes at left). Competition for Rec binding to pcK30 RcRE with active (pcK30 and del14) and inactive (LTR21 and del2) RcREs (four lanes at right). (C) Summary of the competition experiments.
FIG. 7
Predicted folding structures of single stem-loop formations in HyRcRE8 and RxRE. The structures were predicted with DNASIS, the RxRE secondary structure being identical to that described by Van Brussel et al. (30). The hypothetical Rex binding site in HyRcRE8 and the defined Rex binding site in the RxRE are indicated by the parallel lines.
References
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