Related Experiment Videos
An intragenic revertant of a poliovirus 2C mutant has an uncoating defect
1Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142.
Abstract:
A revertant was isolated from a temperature-sensitive poliovirus 2C mutant, 2C-31, which is defective in viral RNA synthesis. This revertant, called 2C-31R1, grew well at 39 degrees C and was not defective in RNA synthesis. However, in contrast to its parental mutant, 2C-31R1 was cold sensitive and could hardly grow at all at 32 degrees C. Analysis of a single-cycle growth revealed that 2C-31R1 was defective in virion uncoating at 32 degrees C, and a substantial amount (more than 30%) of input viruses could be recovered as infectious particles from an infected cell lysate up to 6 h postinfection. The uncoating defect and the inability to grow at cold temperatures could be overcome by a brief incubation at the permissive temperature (39 degrees C) before the infection was continued at 32 degrees C. cDNA cloning and mix-and-match recombination experiments indicated that the defect in uncoating was the result of two secondary point mutations, seven nucleotides apart, in the 2C-coding sequence downstream of the inserted linker which is the original mutation in the parental 2C-31 genome. Another revertant, 2C-31R3, isolated from the same 2C-31 stock, was not defective in uncoating and appeared to be a secondary revertant that contained an intragenic suppressor for the uncoating defect. The uncoating defect of 2C-31R1 could be complemented by type 2 poliovirus. These results suggested that protein 2C, in addition to its role in viral RNA synthesis, has a function in determining virion structure.
Insights
Poliovirus protein 2C, essential for RNA synthesis, also plays a role in virion structure. A temperature-sensitive mutant revealed a cold-sensitive defect in uncoating, linked to secondary mutations in the 2C gene.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Poliovirus protein 2C is known to be essential for viral RNA synthesis.
- Temperature-sensitive mutants are valuable tools for dissecting viral protein functions.
- Understanding poliovirus replication is crucial for developing antiviral strategies.
Purpose of the Study:
- To investigate the function of poliovirus protein 2C beyond its role in RNA synthesis.
- To characterize the genetic basis of a temperature-sensitive poliovirus mutant and its revertants.
- To elucidate the role of protein 2C in poliovirus virion structure and uncoating.
Main Methods:
- Isolation and characterization of temperature-sensitive poliovirus mutants and their revertants.
- Single-cycle growth analysis to assess viral replication and defects.
- cDNA cloning and mix-and-match recombination experiments to identify mutation sites.
- Complementation studies using different poliovirus types.
Main Results:
- A temperature-sensitive mutant (2C-31) defective in RNA synthesis yielded a revertant (2C-31R1) with a cold-sensitive defect in virion uncoating at 32°C.
- The uncoating defect in 2C-31R1 was attributed to two secondary point mutations in the 2C-coding sequence.
- Another revertant (2C-31R3) suppressed the uncoating defect, indicating intragenic suppression.
- The uncoating defect was complementable by type 2 poliovirus.
Conclusions:
- Poliovirus protein 2C possesses a dual function, involved in both viral RNA synthesis and virion structure.
- Specific mutations in the 2C protein can lead to defects in virion uncoating, independent of RNA synthesis.
- These findings provide new insights into the structural role of 2C protein in the poliovirus life cycle.