Summary auto-generated
Researchers used deletion mutagenesis to identify the RNA-binding domain of the cucumber mosaic virus (CMV) 3a movement protein. They created a series of in-frame deletion mutants of the 3a gene and expressed these as either inclusion body proteins or glutathione S-transferase (GST) fusion proteins in E. coli. The mutant proteins were purified and tested for their ability to bind single-stranded RNA using UV cross-linking assays with digoxigenin-labeled RNA probes. Analysis of fifteen deletion mutants revealed that an RNA-binding domain is located between amino acids 174 and 233 of the 3a protein. This domain retained RNA-binding activity even when isolated from the full-length protein context, such as when fused to GST. The identified domain is rich in positively charged residues capable of forming ionic interactions with RNA, and is highly conserved across both subgroups of CMV, suggesting its biological importance for viral movement.
Key findings
- The RNA-binding domain of CMV 3a protein is located between amino acids 174 and 233
- This domain can bind single-stranded RNA independently of the rest of the movement protein
- The domain is highly conserved among both CMV subgroups, indicating functional importance
- The domain contains multiple positively charged residues capable of interacting with negatively charged RNA phosphate groups
- The C-terminal region beyond amino acid 233 is not essential for RNA binding but is required for viral infection
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Abstract
A series of in-frame deletion mutants was used to identify a domain within the 3a protein of cucumber mosaic virus (CMV) that is required for RNA-binding activity. Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences were cloned either in pT7-7 or in pGEX-5X3 expression vectors. The mutated 3a proteins or fusions with glutathione S-transferase (GST) were expressed in E. coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxigenin-UTP-labelled RNA probes. Comparative analyses of seven mutated 3a proteins obtained from inclusion bodies and eight GST fusion proteins revealed that there is an RNA-binding domain located between amino acids 174 and 233. This RNA-binding domain is able to bind single- stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV.