Nucleotide sequence of a Singapore isolate of zucchini yellow mosaic virus coat protein gene revealed an altered DAG Motif

Abstract

A cDNA clone of zucchini yellow mosaic virus (ZYMV) RNA was mapped to the 3' terminal region. The nucleotide sequence revealed a single open reading frame of 1035 nucleotides followed by a 3' noncoding region of 215 nucleotides. The putative protease cleavage site for the release of coat protein (CP) was deduced to be between Glu-Ser (at amino acid position 66- 67), which would result in a protein of 279 amino acids. This non-aphid- transmissible Singapore isolate of ZYMV showed a change of DAG to GAG triplet near the N-terminal of the CP. The CP gene was expressed as a protein fused to the β-galactosidase in Escherichia coli and as an unfused protein in Saccharomyces cerevisiae. Zucchini yellow mosaic virus (ZYMV) is a potyvirus with characteristic flexuous rod-like particles. The genome consists of a positive-sense, single-stranded RNA of about 10 kb that encodes 7-8 viral specific proteins in a single open reading frame (ORF). The viral genes are translated as a large precursor polyprotein that undergoes proteolytic processing by virus-encoded proteinases (1) to yield mature viral proteins. The coat protein (CP) of potyviruses has been mapped to the C-terminal region of this polyprotein (2). Mosaic disease caused by ZYMV was first observed in Singapore in 1989 (3). This unique Singapore isolate of ZYMV, designated ZYMV-S, was found to be nontransmissible by Myzus persicae Sulzer and Aphis gossypii Glover (unpublished). Its symptom development in pumpkin (Curcurbita pepo L. 'elite') also differs from the ZYMV of Connecticut and Florida strains. Expression of viral CP in transgenic plants can confer a degree of resistance to the virus from which the CP gene was obtained (4). In order to study CP-mediated protection, we have determined the nucleotide sequence of the ZYMV-S CP gene and expressed it in bacteria and yeast. The ZYMV-S was purified according to Chng (5). Viral RNA was extracted and a complementary DNA (cDNA) library was constructed in pBluescript SK + (pSK +, Stratagene). The clone pZY-22 contains a 4.2 kb insert. Partial sequencing showed that this clone began with a poly (A) tail, which mapped it to the 3' terminal of the viral RNA. The plasmid sequencing was carried out on both strands of the clone pZY-22 by the method of Sanger et al. (6). For expression of the CP gene in E. coli and S. cerevisiae, two synthetic oligonucleotide primers were made to the 3' and 5' ends of the CP gene. The primer sequences were 5'- CGCGGATCCAGGCTTGCAAACGGAGTCTAA-3' (made to nucleotides 1228-1249) and 5'- CGGGATCCGATGTCAGACACTCAAACAAGAGAGGCGGG-3' (made to nucleotides 199-225; beginning with the first Ser residue of ZYMV-S CP). An ATG codon was introduced into the 5' primer for ribosome recognition in the yeast system. The complete CP gene was amplified by polymerase chain reaction (PCR) and cloned into the BamHl site of pSK+, where the CP gene is placed in-frame to the lac Z gene. The resulting recombinant pZYCP was transformed into E. coli XL1-Blue. When the PCR fragment was cloned into the yeast shuttle vector pCG112 (7), it is placed immediately downstream of a regulatable promoter from the yeast GALI gene (P(GALI)). The resulting plasmid, designated YEpZYCP-P(GALI), was transformed into yeast strain DBY745. The total cellular proteins extracted from E. coli harboring pZYCP and yeast cells harboring YEpZYCP-P(GALI) were electrophoresed in 10% NaDodSO4-PAGE. They were electroblotted onto nitrocellulose filters and probed with ZYMV-S CP antibody, followed by goat-anti-rabbit globulin conjugated with horseradish peroxidase. The CP specific proteins were detected by the enhanced chemillumination method (ECL, Amersham). The nucleotide sequence elucidated from the cDNA clones encompassed the 3' terminal 1250 nucleotides of ZYMV-S, terminating with a poly (A) tract. This sequence showed significant homology with the 3' terminal regions of other potyviruses, which are known to encode the potyviral CP. Alignment with three recently reported ZYMV isolates (8- 10) revealed 87% homology, while alignment with other potyviruses revealed homology of only 64.1% (PVY) to 67.3% (WMV-2). The positive strand of the 3' terminal sequence of ZYMV-S opens into a single ORF, which ends at nucleotide position 1035 (Fig. 1) with an ochre codon (TAA). This ORF is potentially capable of encoding a protein of 345 amino acids, followed by a 3' untranslated region of 215 nucleotides. A consensus sequence UAUGU found 90 nucleotides upstream of the poly (A) tail may be involved in polyadenylation of potyvirus RNA (11), as demonstrated in some yeast genes (12).