Identification of Influenza Virus Inhibitors Targeting NS1A Utilizing Fluorescence Polarization–Based High-Throughput Assay

This article describes the development of a simple and robust fluorescence polarization (FP)–based binding assay and adaptation to high-throughput identification of small molecules blocking dsRNA binding to NS1A protein (nonstructural protein 1 from type A influenza strains). This homogeneous assay employs fluorescein-labeled 16-mer dsRNA and full-length NS1A protein tagged with glutathione S-transferase to monitor the changes in FP and fluorescence intensity simultaneously. The assay was optimized for high-throughput screening in a 384-well format and achieved a z′ score greater than 0.7. Its feasibility for high-throughput screening was demonstrated using the National Institutes of Health clinical collection. Six of 446 small molecules were identified as possible ligands in an initial screening. A series of validation tests confirmed epigallocatechine gallate (EGCG) to be active in the submicromolar range. A mechanism of EGCG inhibition involving interaction with the dsRNA-binding motif of NS1A, including Arg38, was proposed. This structural information is anticipated to provide a useful basis for the modeling of antiflu therapeutic reagents. Overall, the FP-based binding assay demonstrated its superior capability for simple, rapid, inexpensive, and robust identification of NS1A inhibitors and validation of their activity targeting NS1A.

ISG15 conjugation system targets the viral NS1 protein in influenza A virus–infected cells

ISG15 is an IFN-α/β–induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a “loss of function” mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.

Influenza A Virus Polymerase Is an Integral Component of the CPSF30-NS1A Protein Complex in Infected Cells

ABSTRACT The NS1A protein of influenza A virus binds the cellular CPSF30 protein, thereby inhibiting the 3′-end processing of all cellular pre-mRNAs, including beta interferon pre-mRNA. X-ray crystallography identified the CPSF30-binding pocket on the influenza virus A/Udorn/72 (Ud) NS1A protein and the critical role of two hydrophobic NS1A amino acids outside the pocket, F103 and M106, in stabilizing the CPSF30-NS1A complex. Although the NS1A protein of the 1997 H5N1 influenza A/Hong Kong/483/97 (HK97) virus contains L (not F) at position 103 and I (not M) at position 106, it binds CPSF30 in vivo to a significant extent because cognate (HK97) internal proteins stabilize the CPSF30-NS1A complex in infected cells. Here we show that the cognate HK97 polymerase complex, containing the viral polymerase proteins (PB1, PB2, and PA) and the nucleocapsid protein (NP), is responsible for this stabilization. The noncognate Ud polymerase complex cannot carry out this stabilization, but it can stabilize CPSF30 binding to a mutated (F103L M106I) cognate Ud NS1A protein. These results suggested that the viral polymerase complex is an integral component of the CPSF30-NS1A protein complex in infected cells even when the cognate NS1A protein contains F103 and M106, and we show that this is indeed the case. Finally, we show that cognate PA protein and NP, but not cognate PB1 and PB2 proteins, are required for stabilizing the CPSF30-NS1A complex, indicating that the NS1A protein interacts primarily with its cognate PA protein and NP in a complex that includes the cellular CPSF30 protein.

The H5N1 Influenza Virus NS Genes Selected after 1998 Enhance Virus Replication in Mammalian Cells

ABSTRACT The NS1A proteins of human influenza A viruses bind CPSF30, a cellular factor required for the processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including beta interferon mRNA. Here we show that the NS1A protein of the pathogenic H5N1 influenza A/Hong Kong/483/97 (HK97) virus isolated from humans has an intrinsic defect in CPSF30 binding. It does not bind CPSF30 in vitro and causes high beta interferon mRNA production and reduced virus replication in MDCK cells when expressed in a recombinant virus in which the other viral proteins are encoded by influenza A/Udorn/72. We traced this defect to the identities of amino acids 103 and 106 in the HK97 NS1A protein, which differ from the consensus amino acids, F and M, respectively, found in the NS1A proteins of almost all human influenza A virus strains. X-ray crystallography has shown that F103 and M106, which are not part of the CPSF30 binding pocket of the NS1A protein, stabilize the NS1A-CPSF30 complex. In contrast to the HK97 NS1A protein, the NS1A proteins of H5N1 viruses isolated from humans after 1998 contain F103 and M106 and hence bind CPSF30 in vitro and do not attenuate virus replication. The HK97 NS1A protein is less attenuating when expressed in a virus that also encodes the other internal HK97 proteins and under these conditions binds to CPSF30 to a substantial extent in vivo. Consequently, these internal HK97 proteins largely compensate for the absence of F103 and M106, presumably by stabilizing the NS1A-CPSF30 complex.

Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein: Striking Differences between Different Virus Subtypes

ABSTRACT Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin α isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin α/β pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.

The CPSF30 Binding Site on the NS1A Protein of Influenza A Virus Is a Potential Antiviral Target

ABSTRACT The emergence of influenza A viruses resistant to the two existing classes of antiviral drugs highlights the need for additional antiviral drugs, particularly considering the potential threat of a pandemic of H5N1 influenza A viruses. Here, we determine whether influenza A virus replication can be selectively inhibited by blocking the ability of its NS1A protein to inhibit the 3′-end processing of cellular pre-mRNAs, including beta interferon (IFN-β) pre-mRNA. Pre-mRNA processing is inhibited via the binding of the NS1A protein to the cellular CPSF30 protein, and mutational inactivation of this NS1A binding site causes severe attenuation of the virus. We demonstrate that binding of CPSF30 is mediated by two of its zinc fingers, F2F3, and that the CPSF30/F2F3 binding site on the NS1A protein extends from amino acid 144 to amino acid 186. We generated MDCK cells that constitutively express epitope-tagged F2F3 in the nucleus, although at only approximately one-eighth the level of the NS1A protein produced during virus infection. Influenza A virus replication was inhibited in this cell line, whereas no inhibition was observed with influenza B virus, whose NS1B protein lacks a binding site for CPSF30. Influenza A virus, but not influenza B virus, induced increased production of IFN-β mRNA in the F2F3-expressing cells. These results, which indicate that F2F3 inhibits influenza A virus replication by blocking the binding of endogenous CPSF30 to the NS1A protein, point to this NS1A binding site as a potential target for the development of antivirals directed against influenza A virus.