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, EGX4GKTX32DD, 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 K228 to H or D262 to R within the putative 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 1190 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.
Hanson
S F
SF
Department of Plant Pathology, University of Wisconsin-Madison 53706, USA.
Hoogstraten
R A
RA
Ahlquist
P
P
Gilbertson
R L
RL
Russell
D R
DR
Maxwell
D P
DP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D017931:DNA Primers; D007887:Fabaceae / Q000821:virology; D017791:Geminiviridae / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
C095136:AC1 protein, geminivirus; D017931:DNA Primers; D011994:Recombinant Proteins; D012265:Ribonucleotides; D014764:Viral Proteins