Computational modeling suggests dimerization of equine infectious anemia virus Rev is required for RNA binding

Abstract

BackgroundThe 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.ResultsThe 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 Å, 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0115-7) contains supplementary material, which is available to authorized users.

Deep sequencing and variant analysis of an Italian pathogenic field strain of equine infectious anaemia virus

“…Furthermore, a majority (McKenna et al., ) of non‐synonymous nucleotide substitutions were located within the intracytoplasmic domain of the molecule with just six present in the surface‐exposed extracellular region. Similarly predicted non‐synonymous SNPs in ORF3 were predominantly restricted to a previously described variable region comprising a coiled‐coil motif located between the central region that is responsible for dimerization along with binding to the viral Rev responsive element and the second or C‐terminal arginine‐rich motif (ARM2) responsible (in conjunction with ARM1) for RNA binding (Umunnakwe et al., ). However, SA and F2 contained lysine (K) instead of the more prevalent arginine (R) residue at amino acid position 46 within the first ARM (ARM1) while F2 possessed cysteine (C) instead of tryptophan (W) at position 13 within the nuclear export signal (NES) and negatively charged glutamic acid (E) instead of non‐polar alanine (A) at position 34.…”

A Novel, Fully Spliced, Accessory Gene in Equine Lentivirus with Distinct Rev-Responsive Element

“…2D , the levels of Mat expression significantly decreased in the presence of LMB. In addition, a Rev mutant (Rev L95D ), that disrupted nuclear export activity ( 33 , 34 ), was unable to mediate the expression of Mat to the same extent as the wild-type Rev ( Fig. 2E ).…”

A novel fully-spliced accessory gene in equine lentivirus with distinct Rev-responsive element

“…As shown in Fig 2D, the levels of Mat expression significantly decreased in the presence of LMB. In addition, a Rev mutant (RevL95D), that disrupted nuclear export activity [26, 27], was unable to mediate the expression of Mat to the same extent as the wild-type Rev (Fig 2E). These results indicated that Rev-mediated Mat expression depends on the nuclear export activity of Rev through the CRM1-pathway.…”

“…Thus, Rev binding and subsequent multimerization are necessary for Rev-RRE complex function. Previous studies have identified the RNA binding domain and multimerization of EIAV Rev [24][25][26][27]. Considering that Rev-mediated RNA export is essential for lentiviral replication, the Rev-RRE complex is a potential therapeutic target in lentiviruses.…”