Detection of potato mop-top virus capsid readthrough protein in virus particles

Potato mop-top furovirus (PMTV) RNA 3 encodes a 20 kDa coat protein and a 67 kDa readthrough protein produced by suppression of the amber stop codon terminating the coat protein gene. Researchers cloned a C-terminal fragment of the readthrough protein, expressed it as a glutathione S-transferase fusion protein in E. coli, and generated antiserum against the purified fusion protein. Using this antiserum, they detected the readthrough protein in extracts from PMTV-infected leaves via ELISA and immunosorbent electron microscopy. Immunogold labeling studies revealed that the readthrough protein was located near one extremity of some virus particles, with labeling predominantly at the concave end where particles uncoil. Although only a small percentage of particles (approximately 1%) were gold-labeled, the labeling was highly specific with minimal background, suggesting the readthrough protein associates with only a subset of virions. These findings provide the first direct evidence that the readthrough domain is expressed and remains attached to virus particles, and may play a role in fungal vector transmission analogous to readthrough proteins in related furoviruses like beet necrotic yellow vein virus.

Key findings

• PMTV readthrough protein (67 kDa) is expressed in infected plants at approximately 17% suppression frequency of the coat protein stop codon
• Immunogold electron microscopy showed readthrough protein located specifically at one extremity (concave end) of virus particles, with only ~1% of particles labeled
• The readthrough protein remains attached to a subset of virions rather than existing as free protein in the plant, as evidenced by specific trapping and labeling patterns
• Antiserum against the readthrough protein C-terminal domain successfully detected the protein in infected leaf extracts using ELISA