Influenza A viruses cause a highly contagious respiratory infection, and both seasonal and pandemic strains remain a persistent health issue for humans. Influenza pandemics occurred three times in the 20th century (1918, 1957 and 1968) and in 2009 the novel H1N1 influenza (swine flu) became the first influenza pandemic of the 21st century. Equally troubling are the continued human infections with the highly pathogenic H5N1 avian influenza (bird flu) in Asia, Europe, and Africa. This subtype has not yet gained the ability to readily transmit from human to human, but research has demonstrated this virus is able to evolve increased transmissibility in the ferret model [1, 2]. The lack of preparation for the rapidly spreading novel H1N1 and continued threats of a possible avian influenza outbreak make it painfully evident that more knowledge of the basic molecular activities required for influenza replication is needed so that new antiviral targets can be identified and therapies developed.
Influenza is a negative strand RNA virus with eight vRNA segments. The influenza RNA dependent RNA polymerase (RdRP) is a three-subunit complex comprised of PA, PB1, and PB2, which transcribes and replicates viral RNA in the nucleus. The viral RdRP binds an RNA panhandle formed by complimentary interaction between the 5’ and 3’ ends of the viral genome [3, 4]. NP encapsidates each vRNA template and also makes direct protein contacts with the viral RdRP . Together, the viral RdRP, NP and each vRNA segment form functional viral ribonucleoprotein complexes (vRNPs) responsible for transcription and replication of the viral genome in the nucleus of the host cell.
The mechanisms of transcription by the viral RdRP are well characterized. Influenza viral mRNAs are similar to host messages as they contain both a 5’ cap and 3’ polyA tail, but these modifications are not acquired through host cellular processing. The 5’ cap is stolen from nascent cellular mRNAs via a ‘cap-snatching’ mechanism . The influenza RdRP interacts with host RNA polymerase II C-terminal domain , placing the viral RdRP within the environment of nascent cellular mRNAs, which acquire the 5’ cap modification early during transcription. The PB2 subunit of the influenza RdRP recognizes and binds cellular capped mRNAs  while the PA subunit uses its intrinsic endonuclease activity to cleave the 5’ capped RNA [9, 10]. The resulting cellular capped RNA fragment is used by the PB1 subunit of the influenza RdRP as primer for polymerization and viral mRNA synthesis. Polyadenylation of the 3’ end of the viral mRNA occurs by a ‘stuttering’ mechanism, wherein the viral RdRP slips along five to seven uracil residues approximately 17 to 22 nucleotides from the 5' end of each vRNA template, resulting in the polymerization of a poly A tail [11, 12]. Influenza gene expression further requires the splicing of two viral mRNAs and the nuclear export of spliced mRNAs (NS2 and M2), intron containing mRNAs (NS1 and M1), and intronless mRNAs (NP,PA, PB1, PB2, HA, and NA). Evidence suggests a model wherein the resident RdRP of the vRNP is responsible for transcription, while a soluble RdRP is responsible for replication from the vRNA template .
NP does not directly participate in the transcriptional activities described above, but NP is essential for efficient transcription in the host cell as part of the functional vRNP. Replication actively requires NP as NP encapsidates both the vRNA and cRNA replication products, and NP is required for synthesis of template sized RNA in vitro
. However, NP is more than a structural RNA binding protein. NP is intricately involved in promoting viral RNA replication: NP is required for anti-termination at the polyA addition site during vRNA to cRNA replication , NP and the RdRP stabilize nascent cRNA and vRNA replication products , and although NP is not essential for RNA replication on short templates , NP protein interaction with the viral RdRP enhances unprimed RNA initiation in vitro
. The cRNA is then used as template to produce more vRNA. Viral RNA replication leads to increased vRNA templates available fo transcription, amplifying production of viral mRNAs. Thus, in addition to coating the RNA template, NP plays an integral role in viral RNA replication and proper viral gene expression.
NP is a multi-functional protein that interacts with a number of viral and host proteins at various times during infection (for review see ). The N-terminus of NP contains an unconventional nuclear localization signal at amino acids 3-13 (nNLS- SxGTKRSYxxM) critical for both NP and vRNP nuclear localization [20, 21]. Recombinant virus encoding mutations within the unconventional NLS were attenuated with NP localized primarily to the cytoplasm, resulting in decreased viral gene expression . The N-terminus also interacts with host RNA processing factor UAP56, as NP amino acids 1-20 were sufficient to bind UAP56 . UAP56 is a member of the DEAD-box family of RNA dependent ATPases and RNA helicases , and is involved in cellular mRNA remodeling during mRNA processing and nuclear export . Experiments with purified NP and UAP56 proteins in vitro indicate that UAP56 functions as a chaperone to promote free NP binding to nascent viral RNA replication products resulting in enhanced viral RNA synthesis [23, 26]. The goal of this study was to address the role of N-terminal NP interactions in the context of the host cell. To accomplish this, we designed a plasmid encoding the conventional NLS from SV40 T-antigen (PKKKRKV) in place of the N-terminal 20 amino acids of NP (del20NLS-NP).
We report here the characterization of del20NLS-NP using transfection to express reconstituted vRNPs in human embryonic kidney cell line (293 T). We find that del20NLS-NP is expressed, localized, and binds nucleotides as wild type NP (WT-NP). However, RNA expression from influenza vRNA templates in the presence of del20NLS-NP is significantly decreased compared to WT-NP. Increasing vRNA template length exacerbates the RNA expression defect. To assess vRNP formation we analyzed cell protein extracts for NP containing high molecular weight complexes using 1D blue native gel electrophoresis and found substantial decrease in high molecular weight complexes containing del20NLS-NP compared to WT-NP. These results contribute evidence that aside from the importance for nuclear localization, the N-terminus of NP is required for efficient formation of vRNPs.