Functional Analysis of a Novel Motif Conserved across Geminivirus Rep Proteins

Abstract

Members of the Geminiviridae have single-stranded DNA genomes that replicate in nuclei of infected plant cells. All geminiviruses encode a conserved protein (Rep) that catalyzes initiation of rolling-circle replication. Earlier studies showed that three conserved motifs-motifs I, II, and III-in the N termini of geminivirus Rep proteins are essential for function. In this study, we identified a fourth sequence, designated GRS (geminivirus Rep sequence), in the Rep N terminus that displays high amino acid sequence conservation across all geminivirus genera. Using the Rep protein of Tomato golden mosaic virus (TGMV AL1), we show that GRS mutants are not infectious in plants and do not support viral genome replication in tobacco protoplasts. GRS mutants are competent for protein-protein interactions and for both double-and single-stranded DNA binding, indicating that the mutations did not impair its global conformation. In contrast, GRS mutants are unable to specifically cleave single-stranded DNA, which is required to initiate rolling-circle replication. Interestingly, the Rep proteins of phytoplasmal and algal plasmids also contain GRS-related sequences. Modeling of the TGMV AL1 N terminus suggested that GRS mutations alter the relative positioning of motif II, which coordinates metal ions, and motif III, which contains the tyrosine involved in DNA cleavage. Together, these results established that the GRS is a conserved, essential motif characteristic of an ancient lineage of rolling-circle initiators and support the idea that geminiviruses may have evolved from plasmids associated with phytoplasma or algae.Geminiviruses are plant viruses with small, circular DNA genomes and twin icosahedral capsids (58). They constitute a large family that is divided into the begomovirus, mastrevirus, curtovirus, and topocuvirus genera based on genome arrangement, insect vector, and host range (68). Geminiviruses amplify their single-stranded (ss) genomes in the nuclei of infected cells using a combination of rolling-circle and recombination-mediated replication (reviewed in references 27, 31, and 35). All geminiviruses encode a conserved protein designated Rep (also known as AL1, AC1, C1, L1, or C1:C2), which mediates initiation of viral replication but does not act as a DNA polymerase (33,42,51). Instead, geminiviruses depend on host replication machinery to copy their genomes (28,31,32).Rep is the only geminivirus protein that is essential for viral replication (18, 56). It is a multifunctional protein that mediates virus-specific recognition of its cognate origin (22) and transcriptional repression (17, 69). Rep initiates and terminates viral DNA synthesis (33, 42, 51) and induces the accumulation of host replication factors in infected cells (48). Rep binds specifically to double-stranded DNA (dsDNA) at a repeated sequence in the 5Ј intergenic region of the viral genome (22, 67), cleaves and ligates DNA within an invariant sequence in a hairpin loop of the plus-strand origin (42, 51), and acts as a DNA helicase to unwind vi...

A novel maize-infecting mastrevirus from La Réunion Island

“…Within the MSRV Rep we identified various amino acid motifs previously identified in other mastreviruses including (1) a likely rolling-circle replication (RCR) motif I sequence (FLTYP [11]), (2) a possible metal ion coordination domain (HLHVLLQ, corresponding to RCR motif II [11]), (3) a possible GRS motif (YHPNIQASR [14]), (4) a possible RCR motif III sequence (YILKSP [11]), (5) a probable dNTP-binding domain sequence (VGX 4 GKTTW 30 DD [8]), (6) a retinoblastoma-related protein binding motif (LHCYE [27]), and (7) a potential oligomerisation domain (SAERLFPTLPSPFV [10]; Supplementary Fig. 2).…”

Identification and characterization of a novel geminivirus with a monopartite genome infecting apple trees

“…3). In addition, the GRS motif, composed of 12 consensus amino acids and possibly involved in rolling circle initiation in several geminiviruses (Nash et al, 2011), was found between aa 77 and 99 of this AGV protein ( Fig. 3), thus indicating that the latter had the typical features of a geminivirus Rep. With regard to the other AGVencoded proteins, Pfam (Finn et al, 2014) search detected conserved domains expected for geminiviral proteins, with the exception of AGV V2 (Table S4).…”

“…BLASTP analysis of the Rep protein (352 aa, encoded by C1 ORF) showed 74% amino acid sequence identity with Rep of East African cassava mosaic virus-Kenya (EACMV-KE, a begomovirus). The motifs FLTYP (motif I), HLH (motif II) and YIEKG (motif III), reported in other geminivirus Rep proteins and required for dsRNA binding, protein conformational changes and viral DNA cleavage (Nash et al, 2011), were identified at aa 18, 60 and 106 of AGV Rep, respectively (Fig. 3).…”

Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease

“…A more detailed analysis showed that Rep proteins encoded by MMDaV contain the motifs reported in other geminivirus Reps (Fig. S3), which include: (i) the rolling-cycle replication (RCR) motif I (FLTFP), required for specific dsDNA binding; (ii) the RCR motif II (HFH), a metal-binding site that may be involved in protein conformation and DNA cleavage; (iii) the RCR motif III (YIQKE), a catalytic site for DNA cleavage (Nash et al, 2011); (iv) the Walker A (GPT-RSGKT) and the Walker B (LYNVIDDI) domains that are crucial components of the nucleotide-binding site (Walker et al, 1982) (Fig. S3).…”