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Structural studies of turnip crinkle virus have been extended to include the identification of high-affinity coat protein binding sites on the RNA genome. Virus was dissociated at elevated pH and ionic strength, and a ribonucleoprotein complex (rp-complex) was isolated by chromatography on Sephacryl S-200. Genomic RNA fragments in the rp-complex, resistant to RNase A and RNase T1 digestion and associated with tightly bound coat protein subunits, were isolated using coat-protein-specific antibodies. The identity of the protected fragments was determined by direct RNA sequencing. These approaches allowed us to study the specific RNA-protein interactions in the rp-complex obtained from dissociated virus particles. The location of one protected fragment downstream from the amber terminator codon in the first and largest of the three viral open reading frames suggests that the coat protein may play a role in the regulation of the expression of the polymerase gene. We have also identified an additional cluster of T1-protected fragments in the region of the coat protein gene that may represent further high-affinity sites involved in assembly recognition.

Genome-length cDNA clones of turnip crinkle virus (TCV) were constructed with SmaI and XbaI restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of modified lambda and T7 phage promoters such that in vitro transcription resulted in RNAs with 5 extra nucleotides at the 3' end, and 1, 2, or 14 extra nucleotides at the 5' end depending on the construction. Transcripts with 14 extraviral 5' nucleotides were not infectious, while transcripts with 1 or 2 additional 5' nucleotides, with or without 5'-cap analog included in transcription reactions, were biologically active. These were approximately an order of magnitude less infectious than RNA extracted from TCV virions. The additional 5' nucleotides were not maintained in progeny viral RNAs isolated from plants.

The nucleotide sequence of the gene immediately following the nucleocapsid protein gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, is presented. Serological reactions of SYNV proteins with antibodies elicited by a fusion protein constructed from the sequenced gene indicate that this gene encodes an SYNV structural protein designated M2. The 5' end of the M2 protein mRNA appears to correspond to position 1700 relative to the 3' end of SYNV RNA, because an extension product that maps to this position was synthesized by reverse transcription of polyadenylated [poly(A)+] RNA from infected tobacco that had been primed with an SYNV-specific oligodeoxyribonucleotide. The 3' end of the gene encoding the M2 protein is defined by a recombinant DNA plasmid derived from poly(A)+ RNA from SYNV-infected plants. This plasmid contains an insert with a 3'-terminal region corresponding to a uridylate-rich sequence present at positions 2832 to 2836 on SYNV genomic (g) RNA. These data thus suggest that the M2 protein mRNA is 1132 nucleotides (NT) long, excluding the poly(A) tail, and consists of a 50-NT untranslated 5' region, a 1035-NT open reading frame (ORF), and a 47-NT untranslated 3'region. The ORF is capable of encoding a 345-amino acid protein with a calculated molecular weight of 38,332. A small region of the M2 protein appears to have some similarity to the phosphoproteins of other rhabdoviruses. An identical 14-NT region occurs at the two sequenced gene junctions on SYNV gRNA and shares homology with regions separating the genes of some animal rhabdoviruses.

Defective interfering (DI) RNAs have been isolated from a broad spectrum of animal viruses and have recently been identified in plant virus infections. Because of their ubiquitous nature, DIs are thought to play an important role in virus replication and yields. DI RNAs have now been found in association with a natural isolate of turnip crinkle virus (TCV-B) and are generated de novo after inoculation of turnip with virus derived from cloned transcripts. DI RNA G, naturally found in the TCV-B isolate, is a mosaic molecule with 5' and 3' viral segments and a repeat of 36 nucleotides at the beginning of the 3' segment. The 5'-terminal 21 nucleotides of DI RNA G were not similar to genomic TCV sequences but did resemble sequences found at the 5' end of other small RNAs associated with TCV (satellite RNAs). DI RNA G interferes with the accumulation of TCV genomic RNA and, unlike other DI RNAs, intensifies the symptoms of its helper virus. Infection of turnip with virus derived from cloned transcripts of TCV-B resulted in de novo generation of a DI RNA, DI1 RNA. DI1 RNA differed from DI RNA G by containing exact 5' and 3' ends of TCV as well as an internal virus segment.

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3' terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5' terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.