A cDNA clone to clover yellow vein potyvirus genome is highly infectious

Abstract

We obtained a highly infectious cDNA clone of clover yellow vein virus (CIYVV). The cDNA fragments, from which a full-length cDNA clone was constructed, were sequenced, and the complete nucleotide sequence of C1YVV RNA was determined. The viral genome is 9584 nucleotides (nt) in length excluding the poly(A) tail and contains one open reading frame (ORF) encoding a large polyprotein of 3072 amino acids. The non-coding region preceding the ORF is 190 nt long. The termination codon is followed by a 175-nt sequence. Seven potential protease NIa, one HC-pro and one P1 protease recognition sites were found in the C1YVV polyprotein by searching for cleavage consensus sequences among the potyvirus group. The cleavage dipeptides of C1YVV NIa protease are Q(E)/S(A,G). The F is conserved at the -2 position from the cleavage site except for at the P3/6K1 junction, and the V conserved at the -4 position among many potyviruses is not present at all. The genome organization of C1YVV was determined, and the amino acid sequence was compared with that of other potyviruses. The full-length cDNA clone of C1YVV was constructed by combining cDNA fragments and placed it under the control of the cauliflower mosaic virus 35S promoter. The full-length cDNA was constructed so that no extra nucleotide was present at the transcription initiation site and only 10 adenine residues were present at the 3' end of the C1YVV cDNA clone. Mechanical inoculation of a circular-formed plasmid DNA onto broad bean seedlings led to systemic infection, and the symptoms were similar to those caused by the wild-type virus but rather mild. Plasmid diluted as low as 500 pg/microl was able to induce symptoms, demonstrating that this full-length C1YVV cDNA is more infectious than any other infectious cDNAs so far reported. Filamentous particles reacting with the antiserum to C1YVV were observed in the crude sap of infected plants by immunoelectron microscopy, and genome replication was demonstrated by RT-PCR of 3' non-coding regions of C1YVV genome in total plant RNAs.