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. 2000 Oct;74(20):9766-70.
doi: 10.1128/jvi.74.20.9766-9770.2000.

Leader proteinase of the beet yellows closterovirus: mutation analysis of the function in genome amplification

C W Peng  1 , V V Dolja
Affiliations

Leader proteinase of the beet yellows closterovirus: mutation analysis of the function in genome amplification

C W Peng et al. J Virol. 2000 Oct.

Abstract

The beet yellows closterovirus leader proteinase (L-Pro) possesses a C-terminal proteinase domain and a nonproteolytic N-terminal domain. It was found that although L-Pro is not essential for basal-level replication, deletion of its N-terminal domain resulted in a 1, 000-fold reduction in RNA accumulation. Mutagenic analysis of the N-terminal domain revealed its structural flexibility except for the 54-codon-long, 5'-terminal element in the corresponding open reading frame that is critical for efficient RNA amplification at both RNA and protein levels.

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Figures

FIG. 1
FIG. 1
Genomic map of BYV (top) and diagram of the cDNA clone of the mini-BYV genome, pBYV-GUS-p21, tagged by insertion of the GUS gene (bottom). Boxes represent BYV ORFs 1a to 8 encoding L-Pro, replication-associated proteins possessing putative methyltransferase (MET), RNA helicase (HEL), and RNA polymerase (POL) domains, 6-kDa protein (p6), HSP70 homolog (HSP70h), 64-kDa protein (p64), minor capsid protein (CPm), major capsid protein (CP), 20-kDa protein (p20), and 21-kDa protein (p21). Each curved arrow designates the self-processing site for the BYV L-Pro. Arrows marked CP and p21 on the pBYV-GUS-p21 map show approximate positions of the 5′ termini of the subgenomic RNAs expressing GUS and p21 and driven by the CP and p21 promoters, respectively. fs, the frameshift mutation inactivating BYV replicase (26). Selected restriction endonuclease sites are shown below the pBYV-GUS-p21 diagram. Arrows marked SP6 and T7 show positions and orientations of the corresponding RNA polymerase promoters.
FIG. 2
FIG. 2
Mutagenic analysis of the function of the N-terminal and proteinase domains of L-Pro in BYV genome amplification. The 5′-terminal part of the BYV genome including the noncoding leader region (L), L-Pro coding region (box), and part of the methyltransferase domain (Met) is shown in the middle. Asterisks, translation start codons; G-G, scissile glycine-glycine bond cleaved by L-Pro. Vertical lines mark sites of the alanine-scanning mutations A1 to A12. N-ATG, mutant in which the original start codon was replaced with ACA and an artificial start codon was introduced at the indicated position; S-1 and DELL, mutants in which in-frame deletions were introduced in the L-Pro coding region; N-ATG-ΔN and N-ATG-ΔALL, mutants in which deletions were introduced into the L-Pro coding region possessing an artificial start codon.
FIG. 3
FIG. 3
Northern analysis of the RNA accumulation in protoplasts transfected with parental and mutant BYV variants. Lane GUS-p21, parental BYV-GUS-p21 variant. Other lanes represent the mutants marked at the top and mock-transfected protoplasts (lane Mock). Arrows mark the positions of genomic RNA and subgenomic RNAs (sgRNAs) encoding GUS and p21, as well as of background bands corresponding to plant rRNAs (26). The membrane was overexposed to visualize low levels of BYV RNA accumulation detected in N-ATG and A1 mutants.
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