The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove. The propensity of influenza A virus (IAV) to develop resistance to antivirals, as observed in 2009 with oseltamivir (Tamiflu), a neuraminidase inhibitor, calls for the search of new therapeutics. Because of the continuous change in the major viral antigens, vaccine must be renewed each year, and during influenza pandemics, antiviral can provide a first step of protection, at least during the time lapse required for vaccine production. The nucleoprotein (NP) is highly expressed during viral infection and has multiple functions. NP covers the eight single-stranded segments of the genomic RNA and assembles with the three polymerase subunits in a ribonucleoprotein complex (RNP) controlling viral transcription and replication (1). Recent studies unraveled the RNA-free trimeric NP structures of the H1N1 and H5N1 strains of influenza A virus (2-5). NP formed a trimer in the crystal that was stabilized by a swapping loop protruding from one monomer to its neighbor. The overall structure of the nucleoprotein of influenza B virus shared many similarities with its analog of influenza A virus, although NP was tetrameric in the former (6). The oligomerization of NP plays an important role in the maintenance of RNP structure required for function (4,5,(7)(8)(9)(10). Moreover, NP is a highly conserved protein (Ͼ90% ami...

The polyprotein of infectious bursal disease virus (IBDV), an avian birnavirus, is processed by the viral protease, VP4. Previous data obtained on the VP4 of infectious pancreatic necrosis virus (IPNV), a fish birnavirus, and comparative sequence analysis between IBDV and IPNV suggest that VP4 is an unusual eukaryotic serine protease that shares properties with prokaryotic leader peptidases and other bacterial peptidases. IBDV VP4 is predicted to utilize a serine-lysine catalytic dyad. Replacement of the members of the predicted catalytic dyad (Ser-652 and Lys-692) confirmed their indispensability. The two cleavage sites at the pVP2-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing and probed by site-directed mutagenesis. Several additional candidate cleavage sites were identified in the C-terminal domain of pVP2 and tested by cumulative site-directed mutagenesis and expression of the mutant polyproteins. The results suggest that VP4 cleaves multiple (Thr/Ala)-X-Ala Ala motifs. A trans activity of the VP4 protease of IBDV, and also IPNV VP4 protease, was demonstrated by co-expression of VP4 and a polypeptide substrate in Escherichia coli. For both proteases, cleavage specificity was identical in the cis-and trans-activity assays. An attempt was made to determine whether VP4 proteases of IBDV and IPNV were able to cleave heterologous substrates. In each case, no cleavage was observed with heterologous combinations. These results on the IBDV VP4 confirm and extend our previous characterization of the IPNV VP4, delineating the birnavirus protease as a new type of viral serine protease.

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala2(Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.

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The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.