Regulation of closterovirus gene expression examined by insertion of a self-processing reporter and by northern hybridization

Abstract

A reporter open reading frame (ORF) coding for a fusion of bacterial beta-glucuronidase (GUS) with a proteinase domain (Pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (BYV). Insertion of this reporter ORF between the first and second codons of the BYV ORFs encoding the HSP70 homolog (HSP70h), a major capsid protein (CP), and a 20-kDa protein (p20) resulted in the expression of the processed GUS-Pro reporter from corresponding subgenomic RNAs. The high sensitivity of GUS assays permitted temporal analysis of reporter accumulation, revealing early expression from the HSP70h promoter, followed by the CP promoter and later the p20 promoter. The kinetics of transcription of the remaining BYV genes encoding a 64-kDa protein (p64), a minor capsid protein (CPm), and a 21-kDa protein (p21) were examined via Northern blot analysis. Taken together, the data indicated that the temporal regulation of BYV gene expression includes early (HSP70h, CPm, CP, and p21 promoters) and late (p64 and p20 promoters) phases. It was also demonstrated that the deletion of six viral genes that are nonessential for RNA amplification resulted in a dramatic increase in the level of transcription from one of the two remaining subgenomic promoters. Comparison with other positive-strand RNA viruses producing multiple subgenomic RNAs showed the uniqueness of the pattern of closterovirus transcriptional regulation.

FIG. 1

Tagging of BYV by insertion of the self-processing reporter. (A) Diagram of the BYV genome with ORFs 1a to 8 encoding leader proteinase (L-Pro), replication-associated proteins harboring putative methyltransferase (MET), RNA helicase (HEL), and RNA polymerase (POL) domains; p6; HSP70h; p64; CPm; CP; p20; and p21. The bottom part of panel A depicts a reporter GUS-Pro ORF encoding a fusion of GUS with Pro derived from TEV. Large vertical arrows indicate the sites of reporter insertion into BYV cDNA clone and the names of the resulting plasmids. Smaller rounded arrows designate the self-processing sites for the BYV L-Pro and reporter GUS-Pro. (B) Diagram of the deletion variant pBYV-GUS-p21. The designations are the same as those for panel A, except for the arrows marked CP and p21, which show the approximate positions of the 5′ termini of the sgRNAs driven by the CP and p21 promoters.

FIG. 2

Kinetics of GUS activity accumulation in protoplasts transfected by the three tagged BYV variants. The activity is expressed as a percentage of the absolute maximum for each variant. The means and standard deviations from four independent transfections were used to generate the graph.

FIG. 3

Kinetics of accumulation of the genomic RNA and three sgRNAs encoding p64, CPm, and p21 in protoplasts transfected by wild-type BYV transcripts. The RNA levels are expressed as percentages of the absolute maximum for each variant. Northern hybridization and a ³²P-labeled RNA probe of negative polarity were used to detect and quantify RNA species. The means and standard deviations from four independent transfections were used to generate the graph.

FIG. 4

Immunoblot analysis of the CP accumulation in protoplasts transfected by the wild-type BYV transcripts. M, sample obtained from mock-inoculated protoplasts at 5 d.p.t.; V, capsid protein standard derived from purified and dissociated BYV virions.

FIG. 5

Relative levels of the genomic RNA and sgRNA species at their respective maxima. The protoplasts were transfected by the wild-type RNA transcripts. The sgRNAs are designated in accord with their coding specificity and presented in order from 5′ to 3′ (see Fig. 1A). The levels of genomic RNA and sgRNAs encoding HSP70h, p64, CPm, and CP were determined at 5 d.p.t., whereas that of p21 sgRNA was measured at 2 d.p.t. Black bars show standard deviations. ND, not determined. Although sgRNA encoding p20 is visible on Northern blots, its quantitation is impractical due to a high background.

FIG. 6

Northern hybridization analysis of the RNAs derived from protoplasts at 86 h posttransfection. The types of the RNA transcripts used are shown on the top. Four lanes for each variant correspond to independent transfections. The RNA probe was the same as that described in the legend for Fig. 3. Positions of the genomic (g) RNAs and sgRNAs encoding HSP70h (hsp), p64, CPm (cpm), CP (cp), p20, and p21 corresponding to the wild-type transfection are shown at the left. WT, wild-type transcripts derived from pBYV-NA clone; int, intermediate-sized, probably defective RNA (6, 31). Asterisks designate the background bands corresponding to plant rRNAs (31). Note that the bands of the CP sgRNAs for the wild-type and HSP70hGUS variants overlap the band of smaller rRNA. The designation ←cp→ shows the position of the sgRNA derived from the CP promoter for the CPGUS and p20GUS variants. The designations on the right indicate positions of the genomic RNA and sgRNAs for the GUS-p21 variant; ‘cp’ corresponds to the GUS-encoding sgRNA derived from the CP promoter. The genomic RNAs and sgRNAs derived from the CP and p21 promoters are quantified in Table 1. The sizes of the genomic RNAs are 15.5 kb for the wild type, 17.8 kb for HSP70hGUS, CPGUS, and p20GUS variants, and 12.6 kb for the GUS-p21 variant. The estimated sizes of the sgRNAs in the wild-type BYV (16) are as follows: 6.1 kb (hsp), 4.4 kb (p64), 2.6 kb (cpm), 1.8 kb (cp), 1.2 kb (p20), and 0.8 kb (p21). The size of the CP sgRNA in CPGUS and p20GUS variants is 4.1 kb, whereas the size of the sgRNA expressing GUS under the control of the CP promoter (‘cp’) is 2.8 kb.