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<sup><a href="#FN2" rid="FN2" class=" fn">†</a></sup><sup><a href="#FN3" rid="FN3" class=" fn">‡</a></sup>

Susana Vega-Rocha

Bruno Gronenborn

Angela Gronenborn

Ramón Campos-Olivas

^{Structural and Computational Biology Program. Spanish National Cancer Center (CNIO). Madrid 28029. Spain}

^{Institut des Sciences du Vegetal. Centre National de la Recherche Scientifique. 91198 Gif-sur-Yvette Cedex, France}

^{Department of Structural Biology. University of Pittsburgh School of Medicine, BST3, 3501 5th Avenue, Pittsburgh, PA 15261. USA}

Corresponding author: Ramón Campos-Olivas, Structural and Computational Biology Program., Spanish National Cancer Center (CNIO)., C. Melchor Fernandez Almagro, 3, Madrid 28029. Spain, Tel: +34-912246900, Fax: +34-912246976, E-mail: se.oinc@sopmacr

Abstract

Nanoviruses are a family of plant viruses that posses 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-Rep_2-95) and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domain, consisting of a central 5-stranded antiparallel β-sheet covered on one side by an α-helix and irregular loops and on the other, more open side of the domain, by an α-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 α-helix, are monomeric and exhibit catalytic activity indistinguishable from that of M-Rep_2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central β-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 dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.

Abstract

Nanoviruses possess the smallest known virions and the smallest individual genome components of all plant viruses (1) and are causative disease agents for a variety of important food and fodder legumes (2). The nanovirus genome is multipartite and consists of 6 to 8 molecules of circular ssDNA, ranging in size from 977 to 1111 nucleotides, individually encapsidated in small icosahedral virions of 17–20 nm in diameter. Each component encodes, in virion (+) sense polarity, a single protein and contains a common region that is part of the viral origin of replication (3). This region is partially conserved among all genomic components of a given nanovirus and contains sequence elements (including a common nonanucleotide sequence flanked by inverted repeats that may form a hairpin-loop structure) functionally equivalent to those found in the intergenic regions of plant geminiviruses and animal circoviruses (4, 5), that possess only one or two genomic components. The replication of the multiple nanovirus genomic DNAs is initiated by a single replication initiator protein, the so-called master Rep protein (M-Rep) (6), that cleaves a specific phosphodiester bond in the conserved nonanucleotide sequence at the viral replication origin. Despite the occurrence of multiple additional Rep proteins encoded by other DNA molecules associated with the nanoviruses, solely the M-Rep is required and sufficient to catalyze replication initiation of the nanovirus genomic DNAs (3, 7, 8). M-Rep is a multifunctional protein of ~33 kDa involved in both initiation and termination of rolling circle replication. It is related in sequence and biological function to the rolling circle replication initiator proteins of gemini- and circoviruses.

The distinct biochemical activities of Rep proteins from gemini-, circo- and nanoviruses are associated with different parts of the protein: (1) Sequence specific recognition of the replication origin and endonuclease activity, i.e. DNA cleavage and joining at the replication origin, reside in the N-terminal region (9–13). (2) Oligomerization of geminivirus Rep has been assigned to the central region, comprising amino acids 119-180 (12), although a corresponding domain has not yet been identified for nanovirus or circovirus Rep proteins. (3) The ATPase activity, essential for virus replication (14), resides in the C-terminal part of Rep from around amino acid 180 onwards (15), and very recently, helicase activity of Rep or its ATPase domain oligomers have been demonstrated for two different geminivirus Rep proteins (16, 17).

Amino acids involved in the different activities of the replication initiator proteins constitute characteristic motifs conserved throughout large families of genetic entities that multiply by rolling circle replication, including gemini-, circo- and nanoviruses (18, 19). In particular, three conserved sequence motifs of rolling circle replication initiator proteins (19) are localized in the N-terminal endonuclease domain of Rep. The recently determined structures of the endonuclease domains of the Rep proteins from the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSV) (13) and from porcine circovirus type 2 (PCV2) (20) revealed structural and biochemical features corroborating the functional roles of these sequence motives (see below) and further rationalized their conservation in the RCR initiator proteins. Since nanovirus replication initiator proteins, including the M-Rep, are smaller than those of gemini- and circoviruses, and since their amino acid motifs are slightly different, it is instructive to determine and compare the structure of the endonuclease domain of a nanovirus M-Rep protein with those of the corresponding gemini- and circoviruses. Interestingly, infectivity of cloned viral DNAs has only been achieved for a single nanovirus to date, namely faba bean necrotic yellows virus (FBNYV), an expanding pathogen of legume crops, rendering it especially important and amenable to reverse genetics (21). Origin-specific DNA cleavage and nucleotidyl transfer activities have been demonstrated in vitro for the complete FBNYV M-Rep protein, with tyrosine 79 identified as the catalytic residue (3). In addition, divalent Mg^{or Mn}^{cations were found to be essential for DNA cleavage by two different nanovirus M-Rep proteins (}3, 22).

Here we report the three-dimensional solution NMR structure of the catalytic domain of the M-Rep protein of FBNYV and further characterize its in vitro endonuclease activity. In addition, we investigated the metal dependence of the DNA cleavage reaction, and identified residues involved in metal binding. Finally, a comparative analysis of the present structure with those of previously determined representative Rep endonuclease domains of a geminivirus (13) and a circovirus (20) is presented.

Footnotes

^{This work was supported in part by}Plan Nacional de Biotecnologia grant BIO2001–2287 from the Spanish Ministry of Science and Technology (to R.C.O) and the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the National Institute of Health (to A.M.G.).

^{BMRB and PDB accession codes: The assignment of NMR signals for the proteins M-Rep}_2-95 and Tag^-M-Rep_1-117 have been deposited at the BMRB with access numbers 7112 and 7218, respectively. The 30-conformer ensemble, as well as the average minimized structure, have been deposited in the PDB with access code 2HWT.

Footnotes

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