Bioinformatics and Functional Analysis of a New Nuclear Localization Sequence of the Influenza A Virus Nucleoprotein

Abstract

Influenza viruses deliver their genome into the nucleus of infected cells for replication. This process is mediated by the viral nucleoprotein (NP), which contains two nuclear localization sequences (NLSs): NLS1 at the N-terminus and a recently identified NLS2 (212GRKTR216). Through mutagenesis and functional studies, we demonstrated that NP must have both NLSs for an efficient nuclear import. As with other NLSs, there may be variations in the basic residues of NLS2 in different strains of the virus, which may affect the nuclear import of the viral genome. Although all NLS2 variants fused to the GFP mediated nuclear import of GFP, bioinformatics showed that 98.8% of reported NP sequences contained either the wild-type sequence 212GRKTR216 or 212GRRTR216. Bioinformatics analyses used to study the presence of NLS2 variants in other viral and nuclear proteins resulted in very low hits, with only 0.4% of human nuclear proteins containing putative NLS2. From these, we studied the nucleolar protein 14 (NOP14) and found that NLS2 does not play a role in the nuclear import of this protein but in its nucleolar localization. We also discovered a functional NLS at the C-terminus of NOP14. Our findings indicate that NLS2 is a highly conserved influenza A NP sequence.

Contribution of the Nuclear Localization Sequences of Influenza A Nucleoprotein to the Nuclear Import of the Influenza Genome in Infected Cells

“…These results indicate that the NLS2MT virus spread slower and was less infectious than the NLS1MT virus. This difference may be due to the role of NLS2 in the nucleolar localization of NP because, in transfected cells, NLS2 significantly promotes the nuclear import of NP to the same extent as NLS1 [ 18 ]. Hence, a dysfunctional NLS2 affects the infectious cycle of the influenza A virus more dramatically than a mutated NLS1.…”

“…NLS2 was first proposed to be a classical bipartite NLS [ 17 ], but site-directed mutagenesis and crystallographic studies have defined it as monopartite NLS at residues 212–214 ( 212 GRKTR 216 ) that predominantly binds to the importin-α major NLS-binding site [ 13 ]. A recent bioinformatics study found NLS2 to be highly conserved among different influenza A strains, with 67.6% of strains containing 212 GRKTR 216 and 31.2% with the sequence 212 GRRTR 216 [ 18 ]. Performing competition of the nuclear import of NP during infection with chimeric proteins containing NLS1 or NLS2, it was demonstrated that both NLS1 and NLS2 are indispensable for influenza infection [ 13 ].…”

“…Hence, NLS2 has a dual role during influenza A virus infection; first, it serves as a functional NLS for the nuclear import of both incoming vRNPs and newly synthesized NP, and then, it is a nucleolar localization signal of NP. However, the nucleolar localization function of NLS is not exclusive to NP, as it has recently been found that the NLS2 sequence is present in the nucleolar protein 14 (NOP14) and plays a role in the nucleolar accumulation of this protein [ 18 ].…”

“…Similarly, the roles of NLS1 and NLS2 in the nuclear import of NP have been studied using cells transfected with plasmids that express NP or NP with mutations in these NLSs. For example, alanine substitution of basic amino acids of NLS1 impairs the nuclear import NP [ 12 ], and a recent study in cells transfected with plasmids expressing NP-carrying mutations in NLS1 or NLS2 shows that NLS1 and NLS2 contribute equally to the nuclear import of NP [ 18 ].…”