Identification of the Coat Protein Gene of Tomato Golden Mosaic Virus

Abstract

SUMMARYAn open reading frame, designated AR1, on DNA component A of tomato golden mosaic virus has been identified as the virus coat protein gene on the basis of the molecular weight and amino acid composition of the virus capsid polypeptide, the mass spectra and N-terminal sequences of peptides produced by cyanogen bromide cleavage of the capsid polypeptide and by the binding of antibodies, induced by immunization of a rabbit with a fl-galactosidase-AR1 fusion protein, to the capsid polypeptide in a Western blot.Tomato golden mosaic virus (TGMV) belongs to the Geminivirus group of plant viruses, members of which are characterized by double-icosahedral particles and genomes of circular ssDNA (Hamilton et al., 1981 ;Matthews, 1982). Nucleotide sequence analysis of the infectious cloned DNA components of TGMV, DNA A and DNA B, has identified six open reading frames (ORFs) with the potential to code for proteins with 100 amino acids or more (Hamilton et al., 1984). Four of these, designated AR1, ALl, AL2 and AL3, are located on DNA A, and the other two, designated BR1 and BLI, are on DNA B, where R and L indicate a rightward or leftward orientation with respect to the "common regions', intergenic regions of about 200 bp in each DNA and having an almost identical sequence. None of the protein products of these ORFs has been directly identified. We now present evidence which proves that ORF AR1 codes for the coat protein.TGMV was isolated from systemically infected Nicotiana benthamiana plants and purified by sucrose density gradient centrifugation as described by Stein et al. (1983). To ensure that the preparation was completely free from host proteins, virus was further purified by centrifugation for 20 h at 115 000 g in a caesium sulphate density gradient in a Beckman SW50.1 rotor. The virus formed a single band with a density of 1.34 g/ml. The ultraviolet absorbance spectrum of the virus had a maximum at 258 nm and a minimum at 235 nm with an A260 :A280 ratio of 1.56 (not corrected for light scattering). When the virus was disrupted by heating in 1 ~o SDS containing 1 ~ 2-mercaptoethanol for 3 rain at 100 °C and analysed by SDS-PAGE (Laemmli, 1970), followed by staining with Coomassie Brilliant Blue R250, a single protein band was detected.The Mr of the capsid polypeptide species was determined by mixing with protein standards (ovalbumin, 45K; deoxyribonuclease, 31K; trypsinogen, 24K; tobacco mosaic virus capsid polypeptide, 17-5K; cytochrome c, 12K), subjecting the mixture to SDS-PAGE in 12~, 14~, 16 ~o, 18 ~o and 20 ~ polyacrylamide gels and measuring the relative mobility (RF) of each protein t Present address:

Chlorotic Curly Stunt: A Severe Begomovirus Disease of Bottle Gourd in Northern India

“…The DNA A genome contains four major genes encoding coat protein (CP), replication initiator protein (Rep), transcriptional transactivator protein and replication enhancer protein [11,31,32]. The DNA B contains two genes encoding proteins required for movement of virus, host range and pathogenicity [10,28].…”

Mutagenesis of the AC3 open reading frame of African cassava mosaic virus DNA A reduces DNA B replication and ameliorates disease symptoms

“…The virus coat protein genes of ACMV and TGMV have been identified as virus-sense ORFs (AV1 and AR1) on DNA A Kallender et al, 1988). Mutational analyses have shown that a functional coat protein is not required for systemic spread or symptom development of either virus (Gardiner et al, 1988;Morris et al, 1988;.…”

DNA sequences essential for replication of the B genome component of tomato golden mosaic virus.

“…Vira1 ORFs are defined according to genome component (A versus 6) and orientation relative to the common region (Hamilton et al, 1984). Rightward transcription spans ORF AR1, which encodes the 28.7-kD viral capsid protein (Kallender et al, 1988). Leftward transcription spans ORFs ALI (40.3 kD), AL2 ' Current address: Department of Botany and Microbiology, Auburn University, Auburn, AL 36849.…”