Nuclear localized Influenza nucleoprotein N-terminal deletion mutant is deficient in functional vRNP formation

Abstract

Background: The influenza RNA dependent RNA polymerase synthesizes viral RNA in the nucleus as functional viral ribonucleoprotein (vRNP) complexes with RNA and nucleoprotein (NP). The N-terminus of NP contains an unconventional nuclear localization signal (NLS) important for initial vRNP nuclear localization but which also interacts with various host factors. Methods. To study the role of the N-terminus of NP aside from NLS function, we generated an N-terminal NP deletion mutant, del20NLS-NP, encoding the conventional SV40 T-antigen NLS in place of the first 20 amino acids of NP. We characterized expression, location, and activity of del20NLS-NP compared to wild type NP using reconstituted vRNP assays, cellular fractionation, Western blotting, and reverse transcription-PCR. We assessed NP nucleotide binding with gel-shift assays and analyzed NP complexes using 1D blue native gel electrophoresis. Results: del20NLS-NP is expressed, localized in the nucleus and cytoplasm, and maintains ability to bind nucleic acids. Despite this, del20NLS-NP exhibits a defect in viral RNA expression exacerbated by increasing vRNA template length. We find diminished del20NLS-NP high molecular weight complexes in protein extracts; evidence the defect is with functional vRNP formation. Interestingly, the shortest template, NS vRNA, exhibits a limited defect. However, this is not due to short template size, but rather activity of the NS protein(s). Expression of NS1 rescues the gene expression defect primarily at the protein level, a finding consistent with the known role of NS1 as a viral mRNA translational enhancer. NS1 mutant analysis confirms NS1-RNA binding is not required for the translational enhancement and reveals the NS1-CPSF30 interaction surface is essential. Conclusions: del20NLS-NP is a nuclear localized NP mutant able to bind nucleic acids but inefficient for assembly of functional vRNPs inside the host cell. Our results add to growing evidence the N-terminus of NP plays important roles aside from vRNP nuclear localization. We demonstrate the utility of this partially functional NP mutant to characterize the influence of additional proteins on viral gene expression. Our studies reveal the NS1-CPSF30 interaction surface is required for the ability of NS1 to enhance viral protein translation, supporting a function for this NS1 domain in the cytoplasm.