Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellows virus and comparison with the corresponding geminivirus and circovirus structures

Abstract

Nanoviruses are a family of plant viruses that possess a genome of multiple circular single-stranded DNA (ssDNA) components and are strikingly similar in their replication mode to the plant geminiviruses and to the circoviruses that infect birds or mammals. These viruses multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyze the initiation of replication on a double-stranded DNA (dsDNA) intermediate and the resolution of the ssDNA into circles. Here we report the solution NMR three-dimensional structure of the endonuclease domain from the master Rep (M-Rep) protein of faba bean necrotic yellows virus (FBNYV), a representative of the nanoviruses. The domain comprises amino acids 2-95 (M-Rep2-95), and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domains, consisting of a central 5-stranded antiparallel beta-sheet covered on one side by an alpha-helix and irregular loops and on the other, more open side of the domain, by an alpha-helix containing the catalytic tyrosine residue (the catalytic helix). Longer domain constructs extending to amino acids 117 and 124 were also characterized. They contain an additional alpha-helix, are monomeric, and exhibit catalytic activity indistinguishable from that of M-Rep2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central beta-sheet. A comparison with the previously determined Rep endonuclease domain structures of tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus, and that of porcine circovirus type 2 (PCV2) Rep allows the identification of a positively charged surface that is most likely involved in dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.

FIGURE 1

(A) Endonuclease activity of FBNYV M-Rep protein domains. Oligonucleotide F12 (TAGTATT^ACCCC), containing the conserved nonamer sequence of the nanovirus replication origin (in italics) with five nucleotides after the scissile bond (^) was used as substrate. The reaction was monitored by SDS-PAGE (12%), followed by Coomassie staining. The change in electrophoretic mobility compared to the free proteins (−) after covalent adduct formation between the protein and the penta-nucleotide is easily seen (+). (B) Effect of divalent metals on the cleavage of oligonucleotide F12 by M-Rep_2-95. Lanes are labelled by the added metal. Mg/n indicates addition of both Mg²⁺ and Mn²⁺; E indicates excess addition of EDTA to the Mn²⁺ containing reaction before addition of oligonucleotide F12.

FIGURE 2

2D ¹H-¹⁵N HSQC spectrum of FBNYV M-Rep_2-95 (0.6 mM in 20 mM sodium phosphate, pH 6.6, 0.6 M NaCl, 1 mM DTT, 8% D₂O). Backbone amide resonances are labeled according to residue position in the protein sequence and the side chain NH₂ signals are connected and labeled by residue type and number. Likewise, the side chain NH resonances of Trp and Arg residues are labeled by residue type and number, and position in the side chain. Folded signals in the ¹⁵N dimension are marked with asterisks.

FIGURE 3

Three-dimensional structure of FBNYV M-Rep_2-95. (A) Stereoview representation displaying best-fit superpositions of 30 conformers in the final ensemble (residues 5 to 95). Backbone heavy atoms are shown in black while side chain heavy atoms are displayed in blue (H,K,R), red (D,E), brown (F,Y,W), green (A,I,L,P,V), yellow (C,M,S,T), and magenta (N,Q). (B) Ribbon representations of M-Rep_2-95. The central 5-stranded β-sheet is shown in cyan, the helix α₁ in green, and the helix α₂ carrying the catalytic tyrosine is colored in yellow. The strands and helices are numbered and the N- and C-termini labeled. Loop residues exhibiting substantial flexibility (¹⁵N{¹H} NOE < 0.7) or non-detected NH resonances are colored in orange and magenta, respectively. Proline residues are colored in black.

FIGURE 4

Heteronuclear ¹⁵N{¹H} NOE values of the backbone amides of FBNYV M-Rep2-95. Errors are indicated as vertical lines for each point. Secondary structure elements are indicated by grey boxes.

FIGURE 5

Region of the 2D ¹ H-¹⁵N HSQC spectra of FBNYV M-Rep₂-₉₅ illustrating the decrease in signal intensity in the free protein (A), upon addition of 0.4 (B), 0.8 (C), and 1.6 (D) molar equivalents of MnCl₂. Intensities (contour levels) were normalized and resonances are labeled by residue number in the polypeptide sequence.

FIGURE 6

Localization of amide sites affected by manganese binding in the structure of FBNYV M-Rep_2-95. The strongly affected sites are represented as red spheres, less affected ones in orange, and borderline ones in grey (see text). Perturbed NH₂ side chain sites are shown as sticks in red or orange with the NH₂ atoms displayed as spheres.

FIGURE 7

Structure-based alignment of the Rep endonuclease domain sequences from the nanoviruses faba bean necrotic yellows virus (FBNYV, accession number O39828), milk vetch dwarf virus (MVDV, NP_619769), subterranean clover stunt virus (SCSV, NP_620694), and banana bunchy top virus (BBTV, NP_604483)), the circovirus porcine circovirus type 2 (PCV2, AAQ94098)), and the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSV, CAA43466). The secondary structure elements present in FBNYV M-Rep_2-95 (identical color coding as in Figure 3B), as well as those in the extension Tag²¹-M-Rep_1-.117 (indicated with “…” and highlighted in pale blue and orange), together with those of the previously determined structures of the Rep endonuclease domains of PCV2 (20) and TYLCSV (13) are displayed below the corresponding sequences. The conserved RCR initiator protein sequence motifs 1, 2, and 3 are labeled above the alignment by 1111, *2*, and 33333, respectively. Putative divalent metal binding residues are boxed and labeled above the alignment by an asterisk. The pale yellow bars flanking helix α₂ in FBNYV indicate regions adopting a 3₁₀ helix conformation (see text). Sequence identities (Iden.) and similarities (Siml.) of the different sequences compared to the FBNYV Rep sequence are provided at the end of the alignment.

FIGURE 8

Comparison of the three dimensional structures of the viral RCR initiator endonuclease domains from the geminivirus TYLCSV (PDB 1L2M, left), the nanovirus FBNYV (PDB 2HWT, center) and the circovirus PCV2 (PDB 2HWO, right). Ribbon representation (A): β-strands in the central β-sheet are depicted in cyan and α-helices in yellow, red and green. Active site regions of TYLCSV (B), FBNYV (C), and PCV2 (D) endonuclease domains, including residues in the conserved sequence motifs I (blue), II (green), and III (red), as well as putative metal binding residues not present in these motifs (black). Electrostatic surface representation (E) of TYLCSV (left), FBNYV (center) and PCV2 (right). Negative and positive potentials are colored red and blue, respectively, with neutral regions in white. Secondary structure elements are visible through the partially transparent surface and color coded as in (A) (generated with Swiss-PdbViewer 3.7 (33)).