Defective RNA replication by poliovirus mutants deficient in 2A protease cleavage activity

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Poliovirus 2A protease (2Apro) is crucial for viral polyprotein processing.
• 2Apro also cleaves eukaryotic initiation factor 4F (p220), inhibiting host cell protein synthesis.
• The necessity of 2Apro's host shutoff function for poliovirus replication is unclear.

Purpose of the Study:

• To investigate the role of 2Apro's trans-cleavage activity (host cell shutoff) in poliovirus replication.
• To determine if 2Apro's cis-cleavage activity is sufficient for viral replication.
• To correlate specific 2Apro mutations with poliovirus RNA replication efficiency.

Main Methods:

• Site-directed mutagenesis was used to create poliovirus 2Apro mutants with altered cis and trans cleavage functions.
• In vitro transcription/translation assessed polyprotein processing and p220 cleavage.
• Northern blot and RT-PCR analyzed viral RNA replication in transfected COS-7 and HeLa cells.

Main Results:

• Mutants with impaired trans-cleavage (D38E, Y89L) showed no detectable RNA replication.
• Mutants with partial loss of trans-cleavage (Y88S, Y88L) exhibited very low RNA replication levels.
• Mutations affecting only cis-cleavage or with wild-type phenotype did not impair replication.

Conclusions:

• Poliovirus 2Apro's ability to cleave host factors (trans function) correlates with efficient viral RNA replication in vivo.
• This correlation appears independent of 2Apro's role in polyprotein processing (cis function).
• The host cell shutoff activity of 2Apro may be essential, not just facilitative, for productive poliovirus replication.