Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit

Summary auto-generated

This study identifies the nuclear localization signals (NLS) of the influenza virus PA polymerase subunit using deletion analysis and reporter protein assays in COS-1 cells expressing recombinant simian virus 40 constructs. The researchers found that the PA protein contains a complex nuclear targeting structure involving two independent NLS regions: region I (amino acids 124-139), which contains a nucleoplasmin-like NLS, and region II (amino acids 186-247), which lacks a consensus NLS motif but contributes to nuclear transport. A point mutation at position 154 completely abolished nuclear localization. Small deletions within these regions prevented nuclear transport, while large deletions removing entire regions still permitted nuclear targeting of truncated proteins. PA amino-terminal sequences (residues 1-280) directed the reporter protein β-galactosidase partially into the nucleus, but sequences 1-186 alone did not. Critically, cytoplasmic PA mutant proteins could not be rescued by superinfection with influenza virus, demonstrating that functional NLS sequences are required during viral infection. These findings reveal an unusually complex NLS organization, suggesting the two NLS regions exist in separate protein domains with precise spatial orientation requirements.

Key findings

The PA polymerase subunit contains two independent nuclear localization signals: a nucleoplasmin-like NLS at amino acids 124-139 and a non-canonical NLS within region II (186-247)
Position 154 acts as a critical structural element; a point mutation (Glu154Gly) completely eliminated nuclear transport despite proximity to an NLS
Both NLS regions are required for efficient wild-type nuclear transport, but either region alone can partially direct transport of truncated PA proteins to the nucleus
PA protein sequences direct reporter protein translocation to the nucleus only when both regions I and II are included, indicating the domains function cooperatively
Cytoplasmic PA mutants cannot be rescued by influenza virus superinfection, confirming that functional NLS sequences are essential during viral infection

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Abstract

^¹ Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, Cantoblanco, 28049 Madrid
and^² Centro Nacional de Microbiología, Inmunología y Virología Sanitarias, Instituto Carlos III, Majadahonda, 28220 Madrid, Spain

The protein regions involved in the nuclear translocation of the influenza virus PA polymerase subunit have been identified by deletion analysis of the protein expressed from a recombinant simian virus 40. Two regions seem to play a role in the process: region I (amino acids 124 to 139) and region II (amino acids 186 to 247). A nucleoplasmin-like nuclear translocation signal (NLS) has been identified in region I and an additional NLS appears to be present in region II, although no consensus targeting sequence can be detected. Alteration in any of the regions identified by short deletions completely prevented nuclear transport, whereas elimination of the regions I or II by large amino- or carboxy-terminal deletions did not prevent nuclear targeting of the truncated protein. In addition, a point mutation at position 154 completely eliminated nuclear transport. A -galactosidase fusion protein containing the 280 amino acid terminal region of the PA protein was partially transported to the nucleus and mutant PA proteins with a cytoplasmic phenotype could not be rescued by superinfection with influenza virus. These results suggest that the PA protein contains a functional nuclear targeting region which is required in influenza virus infection, with two independent NLSs, one in region I and the other in region II.