@article{xiong_kim_kendall_lommel_1993, title={SYNTHESIS OF THE PUTATIVE RED-CLOVER NECROTIC MOSAIC-VIRUS RNA-POLYMERASE BY RIBOSOMAL FRAMESHIFTING INVITRO}, volume={193}, ISSN={["0042-6822"]}, DOI={10.1006/viro.1993.1117}, abstractNote={
 Abstract
 
 The red clover necrotic mosaic virus (RCNMV) genome is split between two single-stranded RNA species termed RNA-1 and RNA-2. RNA-1 directs the synthesis of 88-kDa (p88), 57-kDa (p57), 37-kDa (p37), and 27-kDa (p27) polypeptides and RNA-2 a 35-kDa (p35) polypeptide in vitro. The coding order of the RNA-1 products was determined to be 5′-p27-p57-p37-3′. Antibodies to synthetic peptides representing the carboxyl terminal portions of p27 and p57 immunoprecipitated their respective polypeptides in addition to p88, suggesting that p88 is a fusion protein. A frameshift heptanucleotide sequence element has been identified in RCNMV RNA-1. In addition, a stable stem-loop secondary structure adjacent to the heptanucleotide sequence is predicted. Together, these sequence elements suggest that a ribosomal frameshifting event occurs which allows translational readthrough of the p27 open reading frame into the p57 open reading frame, generating the observed p88 product. An RNA-1 expression construct fusing the p57 and the CP open reading frame was engineered to investigate the ribosomal frameshifting event. CP antibodies immunoprecipitated a fusion protein of the predicted size containing the carboxyl portion of CP. Site-directed mutagenesis of the frameshift element indicates that in vitro, p88 can also be expressed alternatively by suppression of an amber termination codon. Based on these data, we propose that the putative RCNMV RNA polymerase is an 88-kDa polypeptide expressed by a ribosomal frameshifting mechanism similar to those utilized by retroviruses.
 
}, number={1}, journal={VIROLOGY}, author={XIONG, Z and KIM, KH and KENDALL, TL and LOMMEL, SA}, year={1993}, month={Mar}, pages={213–221} }
 @article{xiong_lommel_1991, title={RED-CLOVER NECROTIC MOSAIC-VIRUS INFECTIOUS TRANSCRIPTS SYNTHESIZED INVITRO}, volume={182}, ISSN={["0042-6822"]}, DOI={10.1016/0042-6822(91)90687-7}, abstractNote={The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two essentially nonhomologous ssRNAs of 3.9 kb (RNA-1) and 1.45 kb (RNA-2) which are each capped at the 5' terminus with m7GpppA. cDNA clones short of full length by several nucleotides at both termini have been generated to both RNAs. Oligonucleotide-directed mutagenesis was employed to generate a series of RNA-1 and -2 transcription vectors in which the bacteriophage T7 RNA polymerase promoter was fused to full-length cDNA clones. Yields of in vitro transcripts initiating with wild-type viral 5'-terminal adenosine were extremely low. Efficient transcription was achieved only when one, or alternatively two, nonviral guanosines were engineered 5' to the authentic viral sequence at the transcription start site. m7GpppG-capped or -uncapped RCNMV RNA-1 and RNA-2 transcripts were infectious and induced symptoms identical to those of wild-type virus infection when coinoculated on the systemic hosts Nicotiana benthamiana and N. clevelandii, and on the local lesion host Chenopodium amaranticolor. Uncapped in vitro transcripts were somewhat less infectious. Progeny virus derived from infectious transcript inoculum was as infectious as wild-type virus. Primer extension analysis indicated that the 5'-terminal nonviral guanosine residues were not maintained in the progeny virus.}, number={1}, journal={VIROLOGY}, author={XIONG, ZG and LOMMEL, SA}, year={1991}, month={May}, pages={388–392} }