Related Experiment Videos
Identification of genes encoding for the cocksfoot mottle virus proteins
Abstract:
Cocksfoot mottle sobemovirus (CfMV) has a monopartite single-stranded positive-sense RNA genome. In wheat germ extract, in vitro translation of CfMV virion RNA resulted in the production of four major proteins of 100, 71, 34 and 12 kDa. In this paper we show the individual genes from which these polypeptides are synthesised. Polyclonal antisera against the proteins encoded by all open reading frames (ORFs) of CfMV were produced. Antibodies were used to immunoprecipitate the in vitro translation products of CfMV genomic RNA as well as the coupled in vitro transcription/translation products of individual viral genes. We demonstrate that the 12, 71, and 100 kDa CfMV proteins are synthesised from the bicistronic genomic RNA of the virus. The CfMV 12 kDa protein is produced from ORF1, the 71 kDa protein from ORF2a (and does not result from the proteolytic cleavage of the polyprotein) and the 100 kDa protein is a polyprotein encoded by ORFs 2a and 2b by a -1 ribosomal frameshift. ORF2b alone does not direct any in vitro protein synthesis. CfMV 34 kDa protein is a coat protein synthesised from the virion-packed subgenomic RNA. The translational strategies of different sobemoviruses are also discussed in this paper.
Insights
Cocksfoot mottle sobemovirus (CfMV) genomic RNA directs the synthesis of viral proteins. Researchers identified the specific open reading frames (ORFs) responsible for producing CfMV's 12, 71, 100, and 34 kDa proteins.
Area of Science:
- Plant virology
- Molecular biology
- RNA virus translation
Background:
- Cocksfoot mottle sobemovirus (CfMV) possesses a single-stranded, positive-sense RNA genome.
- In vitro translation of CfMV RNA yields four major proteins: 100, 71, 34, and 12 kDa.
Purpose of the Study:
- To identify the specific open reading frames (ORFs) responsible for synthesizing the four major CfMV proteins.
- To elucidate the translational strategies employed by CfMV.
Main Methods:
- Production of polyclonal antisera against CfMV-encoded proteins.
- Immunoprecipitation of in vitro translation products using specific antibodies.
- Coupled in vitro transcription/translation of individual viral genes.
Main Results:
- The 12 kDa protein originates from ORF1.
- The 71 kDa protein is synthesized from ORF2a, not via polyprotein cleavage.
- A -1 ribosomal frameshift generates the 100 kDa polyprotein from ORFs 2a and 2b.
- The 34 kDa coat protein is synthesized from a subgenomic RNA.
Conclusions:
- CfMV utilizes distinct translational mechanisms for its genomic and subgenomic RNAs.
- ORF2b alone does not initiate protein synthesis.
- The study clarifies the synthesis pathways of all major CfMV proteins and contributes to understanding sobemovirus translational strategies.