Open Collections

Transcriptional and translational regulation of a subgenomic mRNA of cucumber necrosis virus

University of British Columbia

Cucumber necrosis virus (CNV) is a spherical virus which encapsidates a small messenger sense RN A genome. During infection, CN V generates a 0.9 kb subgenomic mRN A which directs the synthesis of two distinct proteins, p20 and p21, from different nested open reading frames (ORFs). Sequences comprising the core promoter for the synthesis of the CN V 0.9 kb subgenomic mRN A were determined using deletion analysis and site-directed mutagenesis. The results indicated that the CN V 0.9 kb subgenomic mRN A core promoter lies within a region located 20 nucleotides upstream and 6 nucleotides downstream of the transcription initiation site and that nucleotides immediately surrounding the initiation site also regulate promoter activity. Comparison of sequences within the core promoter region with the corresponding region in other tombusviruses revealed that the tombusvirus promoter shares a region of near complete identity in 14 of the 26 core promoter nucleotides. Similarities to other well studied plant and animal virus promoters or to other putative CNV promoters were not apparent. Expression of both CNV p20 and p21 from the 0.9 kb subgenomic mRN A represents one of the rare cases of production of two proteins from the same coding region of a single mRNA . In vitro translation of synthetic transcripts corresponding to the 0.9 kb subgenomic mRN A but containing point substitutions in the AU G codons for either p20 or p21 indicated that these proteins are indeed separately initiated from different nested ORFs. The regulation of the synthesis of these proteins was investigated through examining the effects of codon context and leader length on the efficiency of translation. Nucleotide substitutions introduced into the -3 and +4 positions of the p21 AU G codon verified that purines in these positions are favored and demonstrated the similar contribution of the -3 and +4 positions to the efficiency of initiation codon selection in plants. Further analyses also indicated that the codon context of the upstream p21 AU G codon affects expression from the downstream p20 AU G codon and that an increase in the length of the subgenomic mRN A leader decreases expression from the downstream site. These observations are in accordance with the "Kozak rules" for accession of internal AU G codons by leaky ribosomal scanning and provide the first example of an effect of leader length on the efficiency of translation initiation in a plant (viral) mRNA.

Thesis/Dissertation

application/pdf

2009-02-19

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/4819

Microbiology and Immunology

University of British Columbia

UBCV

DSpace