Cloning of the gene for the capsid protein of potato leafroll virus

Abstract

DNA complementary to the RNA of purified potato leafroll virus (PLRV) was synthesized and cloned into the lambda insertion vector NM1149. Overlapping PLRV-specific cDNA clones were isolated that represent some 80% of the viral genome. Sequences coding for the capsid protein were identified by subcloning size-selected cDNAs into the lambda expression vector gt11 and screening with PLRV-specific antisera. The gene for the viral capsid protein was shown to reside in the 3' terminal half of the genomic RNA. Sequence comparisons with the recently published genomes of the beet western yellows virus (BWYV) and the barely yellow dwarf virus (BYDV) reveal some 65% protein sequence homology between the capsid proteins of BWYV and PLRV and some 45% homology between BYDV and PLRV. Furthermore, it is evident that the structural organization of the PLRV genome in the CP gene region is similar to that of BWYV and BYDV.

The location of the 5' end of the potato leafroll luteovirus subgenomic coat protein mRNA

“…This discrepancy may reflect differences between either the length or the sequence of the subgenomic R N A of the two isolates. However, the published sequences of the isolates (Mayo et aL, 1989;Tacke et al, 1989) differ at only five nucleotides in the intergenic region. Moreover, we could find no evidence of a stop 40 nucleotides upstream of the coat protein initiation codon.…”

Ribosomal frameshifting in plants: a novel signal directs the −1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

“…EcoRV digestion of pSP65GUS removed a 230 bp internal GUS fragment (coordinates 584-814 of the wildtype GUS gene; pSP65GUS-EcoRV). A 1235 bp EcoRV fragment of PLRV cDNA clone pCPL3 (Tacke et al, 1989) corresponding to coordinates 1165 -2400 of the PLRV genome (Mayo et al, 1989) and containing the ORF2a/ORF2b overlap region was inserted into pSP65GUS-EcoRV such that both ORF2a and ORF2b were in frame with the flanking GUS sequences (pSFS-WT). Deletion mutants were produced as depicted in Figure LA and contained the frameshift region from coordinates 1165 -2190 (pSFS-mutl), 1735-2397 and 1165-1947.…”

Characterization of a potato leafroll luteovirus subgenomic RNA: differential expression by internal translation initiation and UAG suppression

“…Samples of 10 ~tg of total RNA were glyoxalated, separated by electrophoresis in a 1-2% agarose gel, transferred to nitrocellulose filters and baked for 2 h at 80 °C (Thomas, 1980). Virus-specific hybridization probes were prepared by isolating DNA fragments from the PLRV cDNA clones pCPLI and pCPL2 (Tacke et al, 1989) after restriction with appropriate endonucleases (EcoRl, 2173 bp fragment 1, co-ordinates 640 to 2812 according to the published PLRV sequence of Mayo et al, 1989; AvaI/SacI, 788 bp fragment 2, co-ordinates 2711 to 3498; AvaI/RsaI, 987 bp fragment 3, co-ordinates 2711 to 3697; BssHIl/EcoRI, 1514 bp fragment 4, coordinates 3773 to 5286; see Fig. 1) (Maniatis et al, 1982).…”