Mutational analysis of the putative nicking motif in the replication-associated protein (AC1) of bean golden mosaic geminivirus

Abstract

Geminiviruses are circular single-stranded DNA viruses that replicate by a rolling circle mechanism involving the viral-encoded AC1 protein. DNA nicking is necessary both for initiating replication of the covalently closed double-stranded DNA templates and for releasing unit-length monomers. The effects of mutations in a putative nicking motif (K101 A Y I D K106; E. V. Koonin and T. V. Ilyina, J. Gen. Virol. 73:2763-2766, 1992) of the AC1-derived protein for bean golden mosaic geminivirus isolate GA (BGMV-GA) were studied. The amino acids equivalent to Y103 and K106 of BGMV-GA are invariant in all whitefly-transmitted geminiviruses. Phaseolus vulgaris plant infectivity assays showed that the mutants K101-->H, K101-->A, and D105-->T produced symptoms, but mutants Y103-->A, Y103-->F, K106-->R, and K106-->H did not. A mutant with a stop codon in the N terminus of the AC4 open reading frame (ORF) produced the same symptoms as the wild-type BGMV-GA. Only those that were infectious replicated in NT-1 tobacco suspension cells. These results indicate that the Y103 and K106 residues are essential for replication, and that this putative DNA-nicking motif of the AC1 ORF may be functional in the rolling circle mechanism of replication for geminiviruses. The potential role of these mutants in the design of antiviral strategies is discussed.