Genes required for replication of the 15.5-kilobase RNA genome of a plant closterovirus

Abstract

A full-length cDNA clone of beet yellows closterovirus (BYV) was engineered and used to map functions involved in the replication of the viral RNA genome and subgenomic RNA formation. Among 10 open reading frames (ORFs) present in BYV, ORFs 1a and 1b suffice for RNA replication and transcription. The proteins encoded in these ORFs harbor putative methyltransferase, RNA helicase, and RNA polymerase domains common to Sindbis virus-like viruses and a large interdomain region that is unique to closteroviruses. The papain-like leader proteinase (L-Pro) encoded in the 5'-proximal region of ORF 1a was found to have a dual function in genome amplification. First, the autocatalytic cleavage between L-Pro and the remainder of the ORF 1a product was essential for replication of RNA. Second, an additional L-Pro function that was separable from proteolytic activity was required for efficient RNA accumulation. The deletion of a large, approximately 5.6-kb, 3'-terminal region coding for a 6-kDa hydrophobic protein, an HSP70 homolog, a 64-kDa protein, minor and major capsid proteins, a 20-kDa protein, and a 21-kDa protein (p21) resulted in replication-competent RNA. However, examination of mutants with replacements of start codons in each of these seven 3'-terminal ORFs revealed that p21 functions as an enhancer of genome amplification. The intriguing analogies between the genome organization and replicational requirements of plant closteroviruses and animal coronavirus-like viruses are discussed.

FIG. 1

(A) Gene organization of BYV and summary of the introduced mutations. L-Pro, papain-like leader proteinase (the arrow shows the site of L-Pro-mediated cleavage); MET and HEL, putative methyltransferase and RNA helicase domains in the ORF 1a product; POL, putative RNA polymerase; HSP70h, an HSP70 homolog; CPm and CP, the minor and major capsid proteins, respectively; p6, p64, p20, and p21, the 6-, 64-, 20-, and 21-kDa products of ORFs 2, 4, 7, and 8, respectively. Asterisks denote replacement of the start codons in each of ORFs 2 to 8; fs, frameshift mutation. AN, SB, and SS, expanded deletions in the 3′-terminal part of the BYV genome. (B) Diagrammatic representation of the full-length BYV-Cal cDNA flanked by the SP6 RNA polymerase promoter in pBYV-NA. The most 5′-terminal and 3′-terminal trinucleotides in the BYV cDNA are shown. The SmaI restriction endonuclease site engineered downstream from the BYV cDNA (boldface) and selected sites used for cloning and mutagenesis are indicated above the diagram, together with their positions in the genome (kilobases). Four BYV cDNA fragments used to generate pBYV-NA are shown as double lines above the diagram. The boundaries of the five cDNA subclones engineered for site-directed mutagenesis are marked below the diagram.

FIG. 2

Northern hybridization analysis of the plus-strand (A) and minus-strand (B) BYV-specific RNAs in transfected tobacco protoplasts. The RNA transcripts used for protoplast transfections are indicated at the top of each lane. (−), no RNA added; WT, wild-type transcript; fs, frameshift mutation in ORF 1a; AN, SB, and SS, expanded deletions in the 3′-terminal part of the BYV genome illustrated in Fig. 1A; g, genomic RNA; int, intermediate-size RNA; numbers, sgRNAs that express corresponding BYV ORFs 2 through 8. The estimated sizes of BYV-Cal sgRNAs are as follows: 2/3, 6.1 kb; 4, 4.4 kb; 5, 2.6 kb; 6, 1.8 kb; 7, 1.2 kb; 8, 0.8 kb (22). The asterisks beside panel B show the positions of background bands.

FIG. 3

Mutagenic analysis of L-Pro function in BYV RNA replication. (A) L-Pro coding region with a summary of the introduced deletions and the BglII site used to generate the 1-B and B-4 mutants. (B) Northern analysis of plus-strand RNA. (C) Northern analysis of minus-strand RNA. 1DL to 4DL, four relatively small (72 to 96 nucleotides) in-frame deletions introduced into the L-Pro-encoding region; dcl, deletion of nine codons, including two glycine codons that specify an L-Pro scissile bond; 1-4, 1-B, and B-4, expanded in-frame deletions in the L-Pro-encoding region outside of the proteinase domain; WT, wild type; fs, frameshift mutation.

FIG. 4

Effects of the mutations in the L-Pro coding region on the levels of RNA replication and proteolytic activity of L-Pro. The levels of plus or minus strands of genomic RNA in transfected protoplasts were quantified and compared to the level of proteolytic maturation of L-Pro in vitro for each mutant. Values expressed as percentages of the wild type (wt) are means and standard deviations from at least four independent experiments.

FIG. 5

Characterization of mutants with replaced start codons in ORFs 2 through 8. (A) Northern analysis of plus-strand RNA. (B) Northern analysis of minus-strand RNA. WT and fs, wild-type and frameshift mutant transcripts used for protoplast transfection, respectively. The numbers at the top correspond to mutant ORFs (cf. Fig. 1A). For designations of BYV-specific and background RNA bands marked by asterisks, see the legend to Fig. 2.