Peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells.
The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses.
Lopez-Ochoa
Luisa
L
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.
Ramirez-Prado
Jorge
J
Hanley-Bowdoin
Linda
L
D052158:Aptamers, Peptide / Q000494:pharmacology*; D004279:DNA, Viral / Q000032:analysis; D017791:Geminiviridae / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D019151:Peptide Library; D059828:Plant Cells; D010942:Plant Viruses / Q000187:drug effects*; D010944:Plants / Q000821:virology; D030821:Plants, Genetically Modified; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014779:Virus Replication / Q000187:drug effects*
D052158:Aptamers, Peptide; D004279:DNA, Viral; D019151:Peptide Library; D014764:Viral Proteins; C080288:replication protein AL1, Begomovirus; D013879:Thioredoxins