Emerging antiviral strategies to interfere with influenza virus entry

Abstract

Influenza A and B viruses are highly contagious respiratory pathogens with a considerable medical and socioeconomical burden and known pandemic potential. Current influenza vaccines require annual updating and provide only partial protection in some risk groups. Due to the global spread of viruses with resistance to the M2 proton channel inhibitor amantadine or the neuraminidase inhibitor oseltamivir, novel antiviral agents with an original mode of action are urgently needed. We here focus on emerging options to interfere with the influenza virus entry process, which consists of the following steps: attachment of the viral hemagglutinin to the sialylated host cell receptors, endocytosis, M2-mediated uncoating, low pH-induced membrane fusion, and, finally, import of the viral ribonucleoprotein into the nucleus. We review the current functional and structural insights in the viral and cellular components of this entry process, and the diverse antiviral strategies that are being explored. This encompasses small molecule inhibitors as well as macromolecules such as therapeutic antibodies. There is optimism that at least some of these innovative concepts to block influenza virus entry will proceed from the proof of concept to a more advanced stage. Special attention is therefore given to the challenging issues of influenza virus (sub)type-dependent activity or potential drug resistance.

Keywords: M2 channel; antiviral; hemagglutinin; influenza virus; nucleoprotein.

Figure 1

Overview of the influenza virus entry and replication process. In the inset on the right, the different virion components are specified. (a) After binding of the viral HA to sialylated glycans on the host cell surface, the virus is internalized by endocytosis. (b) Acidification of the endosome leads to activation of the M2 proton channel and virion acidification, resulting in virus uncoating (i.e., dissociation of the vRNPs from the M1 capsid protein). The low pH inside the endosome also triggers a conformational change in the HA, leading to fusion of the viral and endosomal membranes. After vRNP release in the cytoplasm and dissociation of residual M1, nuclear localization signals in NP direct the transport of the vRNPs into the nucleus. (c) In the nucleus, the viral polymerase starts mRNA synthesis by cleaving off 5′‐capped RNA fragments from host cell pre‐mRNAs. Then, viral mRNA transcription is initiated from the 3′ end of the cleaved RNA cap. (d) Viral mRNAs are transported to the cytoplasm for translation into viral proteins. HA, M2, and NA are processed in the endoplasmic reticulum and the Golgi apparatus, and subsequently transported to the cell membrane. (e) Besides viral mRNA synthesis, the viral polymerase performs the unprimed replication of vRNAs. The vRNAs are first transcribed into positive‐stranded cRNAs, which then function as the template for the synthesis of new vRNAs. During their synthesis, vRNAs and cRNAs are encapsidated by NPs. Export of the newly formed vRNPs into the cytoplasm is mediated by an M1‐NS2 complex that is bound to the vRNPs. (f) As they reach the cell membrane, the vRNPs associate with viral glycoproteins at the plasma membrane from which new virions bud off. Finally, the NA cleaves the sialic acid termini on viral and cell membrane glycoproteins, thereby releasing the progeny virions from the host cell.

Figure 2

Structure and classification of influenza A HAs. (A) Structure of the viral hemagglutinin, showing the binding site for sialic acid (violet) in the globular head domain (blue ribbon structure), as well as the binding pockets in the HA stem structure for fusion inhibitors reported to prevent the HA conformational change, that is, the small‐molecule inhibitor TBHQ (orange) and the broad‐acting antibodies F10 (pink) and CR6261 (yellow). Two HA subunits are represented by their combined molecular surface, while the third one is shown in a ribbon diagram. [Reprinted by permission from Macmillan Publishers Ltd: Nature Structural & Molecular Biology Ref. Das et al.10 © (2010).] (B) Phylogenetic tree of influenza A HAs. Group 1 (cyan) can be subdivided into three clades (H8, H9, and H12; H1, H2, H5, and H6; H11, H13, and H16). Group 2 (green) is subdivided in two clades (H3, H4, and H14; H7, H10, and H15). The newly identified H17 is classified in the H1 clade of group 1.35 [Taken from Russell et al.,55 Copyright (2008) National Academy of Sciences, USA.] (C) Detail of the HA RBS indicating the binding mode of the CDR‐H3 loop (heavy‐chain complementarity determining region 3) of antibody CH65, which acts as a sialic acid mimic. The HA RBS is colored pink and the CDR‐H3 loop is shown in blue. The residues relevant for the antibody‐HA interaction are labeled; some of these are conserved HA1 residues involved in sialic acid binding (Ser136₁, Trp153₁, and Leu194₁). [Taken, with permission, from Whittle et al.79] (D) Cartoon of the structural changes in HA during the HA‐mediated membrane fusion process. [a] The HA RBS binds to the sialylated cell receptor (in green). [b] The acidic pH in the endosome induces HA refolding, which leads to the exposure of the fusion peptide (in red) and its insertion in the endosomal membrane. [c] As a result of further conformational changes in HA, the viral and endosomal membranes are pulled together. [d] Mixing of the outer membrane leaflets generates the prefusion stalk intermediate. The dashed lines separate the inner and outer membrane leaflets. [Taken from Hamilton et al., 251 with permission]

Figure 3

Chemical structures of diverse antiviral agents reported to inhibit the entry of, among others, influenza viruses. The sulfated sialyl lipid NMSO₃ may act upon influenza virus binding107; glycyrrhizin may reduce membrane fluidity125, 126; and dextran sulfate184 and arbidol197 probably interfere with the low pH‐induced fusion process (see the text for all details).

Figure 4

Solid‐state NMR structure of amantadine‐bound A/M2 proton channel in lipid bilayers. Side view showing the luminal site. His37 and Trp41 function as pH sensor and gate, respectively, while Val27 acts as a gatekeeper controlling the entrance of protons. The amantadine binding pocket is formed by Val27, Ala30, Ser31, and Gly34. Substitution of these residues causes amantadine resistance. [Reprinted by permission from Macmillan Publishers Ltd: Nature Ref. Cady et al.155 © (2010).]

Figure 5

Chemical structures of amantadine, rimantadine, and a selection of published analogues. The codes shown are those used in the original reports. The spiro‐adamantane compound 9161 possesses activity against mutant A/M2 ion channels. The imine compound 8e166 and spiro compound 4b167 are both ∼200‐fold more potent than amantadine. Compounds 8168 and 24168 are ring‐contracted and ring‐expanded polycyclic analogues, respectively.

Figure 6

Chemical structures of small‐molecule inhibitors of the HA conformational change. For each compound, the subtype specificity, as far as tested, is given in brackets. See the text for references on individual compounds.

Figure 7

Chemical structure and NP‐binding site of nucleozin. (A) Chemical structure of nucleozin (R = H),236 3061 (R = Cl),238 and compound 3 (R = OMe).239 (B) X‐ray structure of the oligomeric complex of compound 3 with influenza virus NP. Six molecules of compound 3 bridge two NP trimers (NP trimer A and NP trimer B) to form a hexamer. [Taken from Gerritz et al.,239 with permission.] Critical interactions made by compound 3 include a hydrogen bond with Ser376 and a π‐stacking interaction with Tyr289.