Identification of a high affinity nucleocapsid protein binding element from the bovine leukemia virus genome

Abstract

Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5'-untranslated region (5'-UTR) of the genome. Recent studies suggest that a major packaging determinant of bovine leukemia virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5'-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (K(d)=136±21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369-U399, K(d)=67±8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism.

Figure 1

(A) Representation of the Bovine Leukemia Virus (BLV) RNA genome showing relative locations of the splice donor site (SD) and RNA packaging signal. Nucleotides are numbered using the first residue after the 5′-cap as position number 1. The 18 nucleotide Primer Binding Site was indicated by numbers nt322 and nt339. Secondary structure of a segment of the BLV (B) 5′UTR (shown in red is the 10-nucleotide linker connecting the SL_V and SL_VI stem loops) (C) 5′gag. (D) Denaturing PAGE results (12 %(w/v) polyacrylamide) showing relative electrophoretic migration and sample purity for the RNA constructs used in these studies. Lane numbers 1, 2, 3, 4, 5 correspond to RNA constructs SL_V-VI, SL_1-2^gag, SL_V, SL_VI respectively. To increase the transcription yields, non-native GG residues shown in blue were added at the 5′ termini of SL_V-VI. 5′ termini GG of SL_VI are native (E) Amino acid sequence and the zinc-binding mode of the BLV NC protein. Non-native residues derived from the PreScission protease cleavage site (used to cleave GST during purification) are shown in blue.

Figure 2

NATIVE-PAGE (12%) data showing titration of BLV NC with RNA segments (A) from 5′gag, SL_1-2^gag (lanes 1–4) and from 5′UTR, SL_V-VI (lanes 5–8) ([RNA]=30μM; lanes 1–4 and 5–8 correspond to addition of 0.0, 0.5, 1.0, 1.25 equivalents of NC to SL_1-2^gag and SL_V-VI, respectively). The presence of smearing RNA bands with NC increments for NC- SL_1-2^gag titration indicates that NC binds SL_1-2gag via nonspecific-weak interactions, whereas formation of a well defined, electrophoretic band shift upon addition of NC into SL_V-VI indicates that high affinity NC binding site resides within the 5′UTR. Representative ITC data obtained for NC binding to (B) SL_1-2^gag and (C) SL_V-VI under different ionic conditions (shown in the inset). Top panel: raw data with each peak corresponding to the heat produced upon addition of NC ([NC] = 75 μM, 10μl per injection) to RNA, ([RNA]=5 μM, 1.41 ml). Bottom panel: binding isotherms obtained after peak integration.

Figure 3

Representative ITC data obtained upon titration of BLV NC with (A) 10nt SL_V-VI Linker RNA (5′-CAACUCUCUG-3′) (B) SL_V hairpin RNA. Different colors denote the different conditions employed (shown in the inset). Under low ionic strength (magenta panels) NC binds to SL_V via very weak interactions whereas to Linker via tight interactions (Equilibrium dissociation constant for NC-Linker complex, K_d = 92 ± 16 nM). However, under conditions of physiological-like ionic strength (green panels), NC did not bind to either the Linker or SL_V RNAs.

Figure 4

(A) Native-PAGE (14%) data showing results of titrating SL_VI with NC (5mM NaCl, 1 mM MgCl₂). Under the conditions employed, the SL_VI RNA migrates as monomeric (major, M) and dimeric (minor, D) species. Addition of NC leads to formation of a specific 1:1 NC-RNA complex that migrates faster than the dimeric RNA and 2:1 NC: SL_VI complex that migrates slower than the dimeric RNA. (B) Representative ITC data obtained for titration of SL_VI (5 μM, 1.41ml) with NC (75 μM, 10 μl per injection) under conditions of 5mM NaCl (magenta panel) and 5mM NaCl+1mM MgCl₂ (cyan panel). Under low ionic strength (5mM NaCl), SL_VI exhibits two NC binding sites (Equilibrium dissociation constants for the first site and the second site are K_d1 < 1nM and K_d2 = 93 ± 37nM respectively). Weaker binding site is inhibited upon addition of 1mM MgCl₂. (C) Representative ITC data obtained for titration of SL_VI in the presence of varying concentrations of NaCl. Different colors denote the different NaCl concentrations employed (shown in the inset). The equilibrium dissociation constants were measured as 67 ± 8 nM ([NaCl] = 150 mM), 181 ± 49 nM ([NaCl] = 200 mM) and 932 ± 245 nM ([NaCl] = 300 mM). The Plot of the log (K_d, nM) versus −log ([NaCl], M) (correlation coefficient=0.9982) (see the inset), which afforded an extrapolated dissociation constant of 2 ± 0.6 pM at [NaCl] =10mM. (D) ITC data obtained upon titration of SL_VI with NC under physiological-like ionic conditions (140 mM KCl, 1 mM MgCl₂) showing that replacement of Na+ with K+ does not affect the binding affinity (K_d= 69 ± 16nM).