MUTATIONAL ANALYSIS OF THE PUTATIVE NICKING MOTIF IN THE REPLICATION-ASSOCIATED PROTEIN (AC1) OF BEAN GOLDEN MOSAIC GEMINIVIRUS

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 coval ently closed double-stranded DNA templates and for releasing unit-leng th monomers, The effects of mutations in a putative nicking motif (K-1 01 A Y I D K-106; E. V. Koonin and T. V. Ilyina, J. Gen, Virol, 73:276 3-2766, 1992) of the AC1-derived protein for bean golden mosaic gemini virus isolate GA (BGMV-GA) were studied, The amino acids equivalent to Y-103 and K-106 of BGMV-GA are invariant in all whitefly-transmitted geminiviruses. Phaseolus vulgaris plant infectivity assays showed that the mutants K-101 --> H, K-101 --> A, and D-105 --> T produced sympto ms, but mutants Y-103 --> A, Y-103 --> F, K-106 --> R, and K-106 --> 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 wildtype BGMV-G A, Only those that were infectious replicated in NT-1 tobacco suspensi on cells, These results indicate that the Y-103 and K-106 residues are essential for replication, and that this putative DNA-nicking motif o f the AC1 ORF may be functional in the rolling circle mechanism of rep lication for geminiviruses. The potential role of these mutants in the design of antiviral strategies is discussed.