NLSs of Influenza Nucleoprotein as Novel Targets for Antiviral

Background: Influenza A virus poses a serious threat to world public health, particularly the pandemic H1N1 and avian H5N1. Currently, the major anti-influenza strategies are vaccination and antiviral drugs. However, both of these approaches target structural components of the virus which undergo mutation and become resistant to these antiviral approaches. An alternative way to inhibit influenza infection is to interfere with the viral entry into the nucleus, since this process is necessary for viral replication. So far, there are at least two stretches of amino acids sequences located on influenza nucleoprotein (NP) that are know to mediate the nuclear import of the viral ribonucleoproteins (vRNPs) containing the viral genome: NLS1 at the N-terminus of NP, and NLS2 spanning residues 198-216. Sequence allignment shows both NLSs on NP are highly conserved between different strains of influenza A, suggesting that NLSs are ideal candidate for novel antiviral approaches. Methods: To assess this, we are first defining the contribution of the NLSs of NP to nuclear import. The functional role of NLS1 has been very well characterized in previous studies. The specific role of the NLS2 is, however, ill-defined. In our study, we generated chimeric protein by fusing NLS1 or NLS2 to a heterologous protein and characterize the contribution of these NLSs to nuclear import. To analyzed the contribution of the NLS2’s basic amino acids to the nuclear import of the chimera protein, we did site-directed mutagenesis towards NLS2 in chimeric protein. Further analyzation of NLSs's antiviral function was performed by competition assay.  Results:  Our results showed that NLS2 renders a weak nuclear import behavior compared to NLS1. Interestingly, with only one basic amino acid difference (lysine to arginine) the NLS2 of seasonal flu (H1N1 and H5N1) contributes stronger to the nuclear import of the chimera protein than the NLS2 from pandemic H1N1 and avian H5N1. Using site-directed mutagenesis we further analyzed the contribution of the NLS2’s basic amino acids to the nuclear import of the chimera protein. Our results suggest that NLS2 from  pandemic H1N1 and avian H5N1 function as a classical bipartite NLS. However, NLS2 from seasonal flu (H1N1 and H5N1) behaves as a monopartite NLS instead of a bipartite NLS. By performing competition assays using NLS1 and NLS2 chimeras in infected cells, we found that the competing NLSs were able to successfully delayed infection of influenza A virus. Conclusions: NLS2 showed strain-dependent function in nuclear import and contributed less comparing to NLS1. Competition assay results clearly indicate that a good strategy to employ in the development of new influenza antiviral drugs is to interfere with the function of NLSs of influenza NP.