Nucleotide sequences of the two ORFs upstream to the coat protein gene of cymbidium mosaic virus.

Abstract

We report here the nucleotide sequences of the two ORFs upstream to the coat protein gene cymbidium mosaic virus (CyMV), elucidated from a cDNA clone raised to CyMV RNA. This clone (pCYM 46 in BlueScript phagemid), which encompassed the 3' region of the CyMV genome, was sequenced in both directions by the chaintermination method of Sanger et al. [8]. The two ORFs sequenced form part of the 'triple gene block' found in potexand carla-viruses [3, 5, 7]. In CyMV, a potexvirus, the ORF immediately upstream to the coat protein gene codes for a putative protein of 10 kDa, while the next ORF upstream to the 10K ORF codes for one of 14 kDa. The 10 kDa and 14 kDa putative proteins respectively show homologies to the 7 kDa and 13 kDa proteins from the triple gene blocks of other potexviruses. These proteins contain conserved blocks of hydrophobic amino acids which possibly constitute membrane-spanning domains [1, 6, 9]. The nucleotide sequences and the derived amino acid sequences of the two ORFs are shown in Fig. 1. The organization of the two ORFs on the genome shows similarities with other potexviruses [ 1, 6]: the triple gene block is located upstream to the coat protein gene but there is no overlap between them. In CyMV the coat protein gene is separated from the triple gene block by two non-coding nucleotides. Within the triple gene block, the 5' end of the 10K ORF overlaps with the 3' end of the 14K ORF. Figure 2 shows the regions of conserved amino acids between the CyMV 10 kDa and 14 kDa proteins and their counterparts in a few potexand carla-viruses. Putative membrane-spanning domains of hydrophobic amino acids are indicated by bold print. In vitro translation experiments using the wheat germ extract have illustrated that the 10 kDa and the 14 kDa proteins were not efficiently translated from the full-length genomic RNA of CyMV. The proteins could however be translated from shorter in vitro transcripts generated from the cDNA clone pCYM46 (unpublished results). This cDNA clone contained the coat protein gene of CyMV (24 kDa), the 10K and the 14K ORFs, and some upstream sequences to the 14K ORF. In vitro translation yielded only three polypep-