MUTATIONAL ANALYSIS OF A PUTATIVE NTP-BINDING DOMAIN IN THE REPLICATION-ASSOCIATED PROTEIN (AC1) OF BEAN GOLDEN MOSAIC GEMINIVIRUS

Bean golden mosaic virus (BGMV) is a whitefly-transmitted, ssDNA gemin ivirus with a bipartite genome. AC1 is the only ORF required for gemin iviral replication. A putative NTP-binding motif, EGX(4)GKTX(32)DD, wa s present in the derived amino acid sequence of the replication-associ ated protein from the AC1 ORF for 13 geminiviruses including BGMV-GA ( Guatemalan isolate, amino acids 221-263). We analyzed the phenotypes o f mutations within this domain using a rapid and sensitive PCR-based a ssay for geminiviral replication developed for these studies. Replicat ion in tobacco cells (NT-1 suspension cells) and infection of beans we re abolished when codons were changed from E228 to H or D262 to R with in the NTP-binding site. A temperature-sensitive replication phenotype was conferred by changing E221 to R within the putative NTP-binding d omain. Replication was unaffected by changing a nonconserved codon nea r the putative NTP-binding domain from l190 to R. Our results demonstr ate that the putative NTP-binding domain is required for geminiviral r eplication. The role of NTP hydrolysis and the possible value of these mutants in a trans-dominant interference scheme for virus-derived res istance are discussed. (C) 1995 Academic Press, Inc.