DNA Helicase Activity Is Associated with the Replication Initiator Protein Rep of Tomato Yellow Leaf Curl Geminivirus

Reyes

et al. 2011

109

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...

mentioning

confidence: 99%

Functional Analysis of a Novel Motif Conserved across Geminivirus Rep Proteins

Nash

Reyes

et al. 2011

109

“…AL1 (also called AC1, C1, and Rep) is essential for replication (23), while AL3 (also called AC3, C3, and REn) enhances viral DNA accumulation (86). AL1 is a multifunctional protein that mediates the virusspecific recognition of its cognate origin (28), is required for the initiation and termination of viral DNA synthesis (28,49,70), and acts as a DNA helicase (18,19). A variety of protein interactions have been demonstrated for TGMV AL1 and other geminivirus replication proteins, including the formation of homomultimers (72) and interactions with AL3/REn (83,84) and coat protein (CP) (56).…”

mentioning

confidence: 99%

Interaction between Geminivirus Replication Protein and the SUMO-Conjugating Enzyme Is Required for Viral Infection

Sánchez-Durán

Ascencio-Ibáñez

et al. 2011

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells by using plant DNA polymerases. These viruses encode a protein designated AL1, Rep, or AC1 that is essential for viral replication. AL1 is an oligomeric protein that binds to double-stranded DNA, catalyzes the cleavage and ligation of single-stranded DNA, and induces the accumulation of host replication machinery. It also interacts with several host proteins, including the cell cycle regulator retinoblastoma-related protein (RBR), the DNA replication protein PCNA (proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme [SCE1]). The SCE1-binding motif was mapped by deletion to a region encompassing AL1 amino acids 85 to 114. Alanine mutagenesis of lysine residues in the binding region either reduced or eliminated the interaction with SCE1, but no defects were observed for other AL1 functions, such as oligomerization, DNA binding, DNA cleavage, and interaction with AL3 or RBR. The lysine mutations reduced or abolished virus infectivity in plants and viral DNA accumulation in transientreplication assays, suggesting that the AL1-SCE1 interaction is required for viral DNA replication. Ectopic AL1 expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected, indicating that AL1 modifies the sumoylation state of selected host proteins. These results established the importance of AL1-SCE1 interactions during geminivirus infection of plants and suggested that AL1 alters the sumoylation of selected host factors to create an environment suitable for viral infection.Geminiviruses constitute a large family of plant viruses with circular, single-stranded DNA (ssDNA) genomes packaged within geminate particles (77, 82). They infect a broad range of plants and cause devastating crop diseases (57, 63). The family Geminiviridae is classified into four genera, Begomovirus, Curtovirus, Topocuvirus, and Mastrevirus, based on their genome organizations, host ranges, and insect vectors (25,26). The largest genus corresponds to the begomoviruses, which can have bipartite genomes (A and B components), like Tomato golden mosaic virus (TGMV), or monopartite genomes, like Tomato yellow leaf curl Sardinia virus (TYLCSV).Geminiviruses replicate through double-stranded DNA (dsDNA) intermediates (34). Begomoviruses encode two proteins involved in viral replication. AL1 (also called AC1, C1, and Rep) is essential for replication (23), while AL3 (also called AC3, C3, and REn) enhances viral DNA accumulation (86). AL1 is a multifunctional protein that mediates the virusspecific recognition of its cognate origin (28), is required for the initiation and termination of viral DNA synthesis (28,49,70), and acts as a DNA helicase (18,19). A variety of protein interactions have been demonstrated for TGMV AL1 and other geminivirus replication proteins, including the formation of homomultimers (72) and interactions with AL3/REn (83, 84) a...

“…Both viruses encode a replication protein designated AL1 (also named AC1, C1, or Rep), which is required for the initiation and termination of viral DNA synthesis (20,32,45) and acts as a DNA helicase (11,12). The AL1 protein also reprograms mature plant cells to create a permissive environment for viral replication through interactions with the host retinoblastoma-related protein (RBR), which regulates cell division and differentiation in plants (14,17,31).…”

mentioning

confidence: 99%

High-Frequency Reversion of Geminivirus Replication Protein Mutants during Infection

Argüello‐Astorga

Ascencio-Ibáñez

et al. 2007

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.