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 geminivirus with a bipartite genome. AC1 is the only ORF required for geminiviral replication. A putative NTP-binding motif, EGX(4)GKTX(32)DD, was present in the derived amino acid sequence of the replication-associated protein from the AC1 ORF for 13 geminiviruses including BGMV-GA (Guatemalan isolate, amino acids 221-263). We analyzed the phenotypes of mutations within this domain using a rapid and sensitive PCR-based assay for geminiviral replication developed for these studies. Replication in tobacco cells (NT-1 suspension cells) and infection of beans were abolished when codons were changed from E228 to H or D262 to R within the NTP-binding site. A temperature-sensitive replication phenotype was conferred by changing E221 to R within the putative NTP-binding domain. Replication was unaffected by changing a nonconserved codon near the putative NTP-binding domain from l190 to R. Our results demonstrate that the putative NTP-binding domain is required for geminiviral replication. The role of NTP hydrolysis and the possible value of these mutants in a trans-dominant interference scheme for virus-derived resistance are discussed. (C) 1995 Academic Press, Inc.