Conserved Structural Motif Identified in Peptides That Bind to Geminivirus Replication Protein Rep.

The geminivirus replication protein, Rep, has long been recognized as a high-value target for control of geminivirus infections as this protein is highly conserved and essential for viral replication and proliferation. In addition, inhibition of viral replication has been pursued through various antiviral strategies with varying degrees of success, including inhibitory peptides that target Rep. While much effort has centered around sequence characterization of the Rep protein and inhibitory peptides, detailed structural analysis has been missing. This study computationally investigated the presence of common structural features within these inhibitory peptides and if these features could inform if a particular peptide will bind Rep and/or interfere with viral replication. Molecular dynamics simulations of the inhibitory peptide library showed that simply possessing stable structural features does not inform interference of viral replication regardless of the binding of Rep. Additionally, nearly all known Rep inhibitory peptides sample a conserved ß-sheet structural motif, possibly informing structure-function relationships in binding Rep. In particular, two peptides (A22 and A64) characterized by this structural motif were computationally docked against a wide variety of geminivirus Rep proteins to determine a mechanism of action. Computational docking revealed these peptides utilize a common Rep protein sequence motif for binding, HHN-x1/2-Q. The results identified residues in both Rep and the inhibitory peptides that play a significant role in the interaction, establishing the foundation for a rational structure-based design approach for the construction of both broadly reactive and geminivirus species-specific inhibitors.

Peptide aptamers that bind to geminivirus replication proteins confer a resistance phenotype to tomato yellow leaf curl virus and tomato mottle virus infection in tomato.

Geminiviruses constitute a large family of single-stranded DNA viruses that cause serious losses in important crops worldwide. They often exist in disease complexes and have high recombination and mutation rates, allowing them to adapt rapidly to new hosts and environments. Thus, an effective resistance strategy must be general in character and able to target multiple viruses. The geminivirus replication protein (Rep) is a good target for broad-based disease control because it is highly conserved and required for viral replication. In an earlier study, we identified a set of peptide aptamers that bind to Rep and reduce viral replication in cultured plant cells. In this study, we selected 16 of the peptide aptamers for further analysis in yeast two-hybrid assays. The results of these experiments showed that all 16 peptide aptamers interact with all or most of the Rep proteins from nine viruses representing the three major Geminiviridae genera and identified two peptide aptamers (A22 and A64) that interact strongly with different regions in the Rep N terminus. Transgenic tomato lines expressing A22 or A64 and inoculated with Tomato yellow leaf curl virus or Tomato mottle virus exhibited delayed viral DNA accumulation and often contained lower levels of viral DNA. Strikingly, the effect on symptoms was stronger, with many of the plants showing no symptoms or strongly attenuated symptoms. Together, these results established the efficacy of using Rep-binding peptide aptamers to develop crops that are resistant to diverse geminiviruses.

Functional analysis of a novel motif conserved across geminivirus Rep proteins.

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.

High-frequency reversion of geminivirus replication protein mutants during infection.

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.