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Molecular cloning and nucleotide sequencing of the coat protein gene of citrus tristeza virus

Mary E. Sekiya

Department of Fruit Crops, University of Florida, Gainesville, Florida 32611, U.S.A.

Citrus tristeza virus (CTV) contains approximately 20000 bases of positive-sense ssRNA, encapsidated by a coat protein of approximately 25000 M_r that has previously been reported to consist of at least two size variants, cp1 and cp2. In the present study, a cDNA library of the T36 isolate of CTV was prepared in a protein expression vector and screened with a polyclonal antibody against the coat protein. Five immuno-positive clones produced proteins in Escherichia coli that reacted with monoclonal as well as polyclonal antibodies to the CTV coat protein. Nucleotide sequence analysis of a region common to the five clones revealed the presence of a 669 nucleotide open reading frame flanked by numerous in-frame termination codons. The encoded protein has a predicted M_r of 24909 and an amino acid composition consistent with that previously reported for the CTV coat protein. Comparison of the predicted amino acid sequence of the coat protein with the amino-terminal sequences of cp1 and cp2 indicated that these coat protein species arise from the same primary translation product, as a result of post-translational proteolysis at sites approximately 12 to 15 and 26 amino acids from the amino terminus respectively. These results are the first reported cloning and sequencing of a CTV gene and provide evidence that CTV may be translated using subgenomic RNA.

Present address: Department of Plant Pathology, Cornell University, Ithaca, New York 14853, U.S.A.

Received 1 October 1990; accepted 11 February 1991.