- Volume 70, Issue 4, 1989

Chicory yellow mottle virus (CYMV) is a nepovirus isolated from chicory in southern Italy. The strain CYMV-T, besides the genomic RNAs, contains additional encapsidated RNA components, the most prominent of which have estimated M r values of 500000 (500K) and 170000 (170K). Northern blot hybridization experiments confirmed that the 170K molecule is a satellite RNA and indicated that the 500K RNA is neither a subgenomic RNA nor a trimeric form of the 170K RNA, but is another satellite RNA. The two satellite RNAs also differ significantly at their termini. The 170K RNA has a free hydroxyl group at the 5′ terminus, where the 500K RNA is blocked. At the 3′ termini, the 170K RNA has a cyclic 2′,3′-phosphate whereas the 500K RNA has a poly(A) tail. Thus CYMV-T seems capable of supporting two satellites with structural characteristics that resemble two types of satellite previously found in association only with two different nepoviruses.

The nucleotide sequence of cDNA copies of grapevine fanleaf virus (strain F13) satellite RNA has been determined. The primary structure obtained was 1114 nucleotides in length, excluding the poly(A) tail, and contained only one long open reading frame encoding a 341 residue, highly hydrophilic polypeptide of M r 37 275. The coding sequence was bordered by a leader of 14 nucleotides and a 3′-terminal non-coding region of 74 nucleotides. No homology has been found with small satellite RNAs associated with other nepoviruses. Two limited homologies of eight nucleotides have been detected between the satellite RNA in grapevine fanleaf virus and those in tomato black ring virus, and a consensus sequence U. G/UGAAAAU/AU/AU/A at the 5′ end of nepovirus RNAs is reported. A less extended consensus exists in this region in comovirus and picornavirus RNA.

A near full-length cDNA clone of tobacco rattle virus (TRV) RNA-1 was constructed by joining together nine overlapping cDNA clones using restriction sites in the regions of overlap. At the 5′ end of the cDNA, oligonucleotide mutagenesis was used to insert nucleotides which were missing from the cDNA placing the construct immediately on the 3′ side of the Pr promoter of phage λ to create pTR7116. Extraneous non-viral nucleotides had been deleted from the 3′ end of the TRV cDNA to create a unique SmaI site in pTR7116 in which the nucleotides CCC were provided by the viral cDNA, and GGG by the vector. As a result, pTR7116 could be linearized with SmaI and transcribed in vitro to yield RNA molecules with 5′ and 3′ termini identical to those of natural TRV RNA-1. These transcripts were infectious when inoculated onto leaves of tobacco and produced the subgenomic RNA species typical of an infection with TRV RNA-1.

Reactions of virions of the potyvirus wheat streak mosaic virus (WSMV) with homologous antiserum in leaf-dips depended on the age of infection of the leaf. Virions from young and recently expanded leaves were completely or partially covered with immunoglobulin whereas virions from leaves infected earlier were not decorated. However, in ultrathin sections virions could be immunolabelled in leaves of all ages. These results suggest that the virions can be partially degraded upon extraction, particularly those from older leaves. Thus, a negative reaction with leaf-dip serology of a suspected WSMV infection does not in itself constitute evidence that the plant is not infected with WSMV. The same principle has implications for the routine serological identification of potyviruses in leaf-dips.