Characterization of PA-N terminal domain of Influenza A polymerase reveals sequence specific RNA cleavage

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Influenza virus employs a unique "cap-snatching" mechanism to initiate viral mRNA synthesis.
• This process involves hijacking and cleaving host cell pre-mRNAs to generate primers.
• The PA subunit of the influenza polymerase complex possesses the endonuclease activity responsible for this cleavage.

Purpose of the Study:

• To investigate the sequence selectivity and mechanism of the influenza virus PA endonuclease.
• To determine if the PA subunit exhibits intrinsic sequence specificity in RNA cleavage.
• To elucidate the interplay between PA endonuclease activity and the PB2 subunit's cap-binding in determining RNA cleavage sites.

Main Methods:

• Biochemical assays were used to analyze the endonuclease activity of the PA subunit.
• Experiments were conducted using both isolated PA endonuclease domains and the full influenza polymerase complex.
• RNA cleavage products were analyzed to determine sequence preference and site selection.

Main Results:

• The PA subunit was identified as a sequence-selective endonuclease with a strong preference for cleaving RNA at the 3' end of guanine (G) bases.
• This guanine specificity was observed in both isolated PA domains and the native influenza polymerase complex.
• While cap binding by the PB2 subunit generally directs cleavage downstream of the cap, the presence of a guanine within nucleotides 10-13 preferentially directs cleavage to that guanine.

Conclusions:

• The influenza virus PA endonuclease possesses intrinsic sequence selectivity, preferentially targeting guanine residues.
• This guanine specificity, combined with cap-binding by PB2, fine-tunes the site of host mRNA cleavage during influenza virus replication.
• These findings provide the first biochemical evidence for the sequence-selective endonuclease activity of the influenza polymerase PA subunit.