PMID- 25533001 OWN - NLM STAT- MEDLINE DCOM- 20151012 LR - 20181113 IS - 1742-4690 (Electronic) IS - 1742-4690 (Linking) VI - 11 DP - 2014 Dec 23 TI - Computational modeling suggests dimerization of equine infectious anemia virus Rev is required for RNA binding. PG - 115 LID - 10.1186/s12977-014-0115-7 [doi] LID - 115 AB - BACKGROUND: The lentiviral Rev protein mediates nuclear export of intron-containing viral RNAs that encode structural proteins or serve as the viral genome. Following translation, HIV-1 Rev localizes to the nucleus and binds its cognate sequence, termed the Rev-responsive element (RRE), in incompletely spliced viral RNA. Rev subsequently multimerizes along the viral RNA and associates with the cellular Crm1 export machinery to translocate the RNA-protein complex to the cytoplasm. Equine infectious anemia virus (EIAV) Rev is functionally homologous to HIV-1 Rev, but shares very little sequence similarity and differs in domain organization. EIAV Rev also contains a bipartite RNA binding domain comprising two short arginine-rich motifs (designated ARM-1 and ARM-2) spaced 79 residues apart in the amino acid sequence. To gain insight into the topology of the bipartite RNA binding domain, a computational approach was used to model the tertiary structure of EIAV Rev. RESULTS: The tertiary structure of EIAV Rev was modeled using several protein structure prediction and model quality assessment servers. Two types of structures were predicted: an elongated structure with an extended central alpha helix, and a globular structure with a central bundle of helices. Assessment of models on the basis of biophysical properties indicated they were of average quality. In almost all models, ARM-1 and ARM-2 were spatially separated by >15 A, suggesting that they do not form a single RNA binding interface on the monomer. A highly conserved canonical coiled-coil motif was identified in the central region of EIAV Rev, suggesting that an RNA binding interface could be formed through dimerization of Rev and juxtaposition of ARM-1 and ARM-2. In support of this, purified Rev protein migrated as a dimer in Blue native gels, and mutation of a residue predicted to form a key coiled-coil contact disrupted dimerization and abrogated RNA binding. In contrast, mutation of residues outside the predicted coiled-coil interface had no effect on dimerization or RNA binding. CONCLUSIONS: Our results suggest that EIAV Rev binding to the RRE requires dimerization via a coiled-coil motif to juxtapose two RNA binding motifs, ARM-1 and ARM-2. FAU - Umunnakwe, Chijioke N AU - Umunnakwe CN AD - Department of Animal Science, Iowa State University, Ames, IA, 50011, USA. nkemjika@iastate.edu. AD - Program in Bioinformatics and Computational Biology, Iowa State University, Ames, IA, 50011, USA. nkemjika@iastate.edu. FAU - Loyd, Hyelee AU - Loyd H AD - Department of Animal Science, Iowa State University, Ames, IA, 50011, USA. heri1008@iastate.edu. FAU - Cornick, Kinsey AU - Cornick K AD - Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, 50011, USA. kcornick@iastate.edu. FAU - Chavez, Jerald R AU - Chavez JR AD - Department of Animal Science, Iowa State University, Ames, IA, 50011, USA. jrchavez@iastate.edu. FAU - Dobbs, Drena AU - Dobbs D AD - Department of Genetics, Developmental and Cell Biology, Iowa State University, Ames, IA, 50011, USA. ddobbs@iastate.edu. AD - Program in Bioinformatics and Computational Biology, Iowa State University, Ames, IA, 50011, USA. ddobbs@iastate.edu. FAU - Carpenter, Susan AU - Carpenter S AD - Department of Animal Science, Iowa State University, Ames, IA, 50011, USA. scarp@iastate.edu. LA - eng GR - R01 CA128568/CA/NCI NIH HHS/United States GR - CA128568/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20141223 PL - England TA - Retrovirology JT - Retrovirology JID - 101216893 RN - 0 (Gene Products, rev) RN - 0 (RNA, Viral) SB - IM MH - Gene Products, rev/*chemistry/*metabolism MH - Infectious Anemia Virus, Equine/*physiology MH - Models, Molecular MH - Protein Binding MH - Protein Conformation MH - *Protein Multimerization MH - RNA, Viral/*metabolism PMC - PMC4299382 EDAT- 2014/12/24 06:00 MHDA- 2015/10/13 06:00 PMCR- 2014/12/23 CRDT- 2014/12/24 06:00 PHST- 2014/08/26 00:00 [received] PHST- 2014/11/27 00:00 [accepted] PHST- 2014/12/24 06:00 [entrez] PHST- 2014/12/24 06:00 [pubmed] PHST- 2015/10/13 06:00 [medline] PHST- 2014/12/23 00:00 [pmc-release] AID - s12977-014-0115-7 [pii] AID - 115 [pii] AID - 10.1186/s12977-014-0115-7 [doi] PST - epublish SO - Retrovirology. 2014 Dec 23;11:115. doi: 10.1186/s12977-014-0115-7.