How Are Antivirals used in Influenza Virus Infection?

By Yuchen Lin

Influenza virus infection, commonly known as the flu, is a widespread respiratory disease. There are four types of influenza viruses: Type A, B, C, and D. The first three types have the ability of human-infection, whilst Type D viral infection has not yet been detected in humans but is well known to infect cattle and pigs. Influenza A viruses are of particular concern as they are the only type to evolve into new strains (Isabelle Marois et al., 2014) and cause seasonal pandemics (World Health Organization, 2018). 

In 2009, an outbreak of a new type of influenza, A/H1N1, that contained a novel form of the influenza gene (Chan, 2009) led to the swine flu pandemic that was responsible for around 575400 deaths worldwide. After the pandemic had ended, the virus continued to circulate as a seasonal flu virus and caused deaths every year (Centers for Disease Control and Prevention, 2019). Although large outbreaks of influenza are uncommon, the circulating virus can still result in three to five million cases of serious illness and up to 650000 deaths yearly (Hartl, 2017). Vaccination seems to be the best possible strategy to minimise the cases of serious illness and deaths from influenza, but creating lasting effective vaccines is difficult; vaccines only contain antibodies that recognize a specific viral antigen, so if the virus mutates, the vaccine is no longer effective. 

Antivirals that target conserved viral pathways may then be a better approach. Antivirals and antibiotics are all antimicrobials, but unlike antibiotics which destroy the target pathogen, antivirals only suppress and inhibit the replication and reproduction processes of viruses (Shiel, n.d.). Antivirals are relatively harmless for hosts, and they result in a lower rate of complications and hospitalizations from influenza (Harvard Health Publishing, 2015). There were two main classes of antivirals used for Influenza A infection of the swine flu pandemic: adamantane, that targets the matrix protein 2 (M2) proton-channel, and viral neuraminidase (NA) inhibitors (Isabelle Marois et al., 2014).

Adamantane blocks proton transport by binding to the M2-proton-channel. The M2 homotetramer protein in Influenza A viruses is abundant on the surface of infected cells. It maintains an acidic microenvironment to trigger viral ribonucleoprotein complex (RNP) uncoating. The uncoating process is vital in the early and late stages of viral replication. Adamantanes prevent RNP uncoating, which blocks the release of the virus into the cytoplasm. However, adamantane resistance exists in nearly all circulating Influenza A viruses. Mutations in the virus lower the binding affinity of adamantane to M2 and allow proton influx in the presence of adamantane, reducing the effectiveness of the antiviral (Pielak & Chou, 2010).

Viral glycoprotein NA, a receptor-destroying enzyme, allows for the release and spread of budding virions by cleaving the interaction between sialic acid residues on the surface of infected cells and the virus. Inhibitors, such as oseltamivir and zanamivir, bind to NA to occlude its cleavage, so the release of virions is prohibited – this is one such antiviral strategy. But viruses can generate substitutions at the binding site of NA to reduce the binding affinity for inhibitors (Joseph S. Bresee et al., 2018). Both of these antiviral strategies have limitations; in severe influenza infection, if they are administered 48 hours after symptoms first appeared, the influenza A virus may have mutated to develop resistance (Davide Corti et al., 2017). Therefore, novel therapeutics against new targets are needed to circumvent antiviral resistance. 

The major glycoprotein on the surface of an influenza A virus, hemagglutinin (HA), is one potential target. It is a highly conserved region for viral attachment. HA contains a fusion peptide, a receptor binding site, a metastable structural motif, and transmembrane domain (Luo, 2012). Endosomal acidification will induce irreversible conformational changes in HA, exposing the fusion peptide to enable viral attachment. Then, interactions between the fusion peptide and endosomal membrane release RNP into the cytoplasm for replication (Davide Corti et al., 2017). Other targets, such as calcium-dependent proteins that participate in virus replication cycles have potential due to their chemical concentration requirements – for example, intracellular Ca2+ concentration is essential for promoting the endocytic pathway and viral RNA transcription, but if disrupted, could effectively prevent the spread of viral infection.

HA trimer has a head and a stem domain, which are linked by one disulfide bond. Three helices from the stem domain together with the short C- and N-terminal loops from the head domain form the binding pocket with an inner hydrophobic core and a highly charged periphery (Kadam & Wilson, 2016). Arbidol, the broad-spectrum antiviral (Qiang Liu et al., 2013) that targets the HA fusion machinery, binds at the fusion peptide above the stem region (see Fig 1.). It functions as a molecular glue to stabilize HA in the prefusion conformation.

Fig 1. HA binding site with Arbidol bound. HA is shown in grey cartoons. Residues from helix-A and helix-C of protomer1 are shown in purple-blue sticks. Residues from helix-C’ of protomer2 are in hotpink sticks. Residues from HA1 are shown as yellow sticks. The colour of the residue name represents the domain it is in. Arbidol is displayed as orange sticks. 

On the HA of H7N9 influenza virus, four internal salt bridges at the binding site pose steric clash when Arbidol binds, so conformational changes are induced. Some salt bridges are broken while new ones are formed. Specifically, an Arginine residue at the binding cavity is reoriented to be solvent-exposed. This change opens the cavity for Arbidol to bind. Arbidol makes hydrophobic interactions with the cavity at one of the three binding sites of HA trimer. Due to the lack of polar residues, Arbidol cannot form direct H-bonds with the cavity. Thus, its bulky thiophenol group occupies the space. Meanwhile, intramolecular H-bonds are built between Arbidol and one helix of HA. As a result, Arbidol can be constrained within the cavity space to prevent the binding of HA to cells inside our body (Kadam & Wilson, 2016). In contrast, the salt bridges on HA of H3N2 influenza virus do not pose steric hindrances when Arbidol binds. Therefore, the HA accommodates Arbidol easier without major conformational changes in its binding site (Kadam & Wilson, 2016). 

Upon Arbidol binding, it clamps the two protomers of HA together. Thus, even at a low pH, the acid-induced conformational rearrangement that leads to viral entry is halted (Damian C Ekiert et al., 2009). As a result, influenza virus infection can be impeded. However, through experiments (Rupert J Russell et al., 2008), researchers discovered that Arbidol prefers to bind to only one domain of HA trimer, and the same binding position located in the other domain is blocked by an extra turn of helix. Therefore, viral fusion may still occur, or there exists a novel binding site for fusion inhibitors which has not been detected yet. Moreover, the binding indentation of HA is shallow, so the binding affinity and efficacy of Arbidol need improvements. Strengthening the hydrophobic interaction or polar intramolecular interactions between HA and Arbidol may help (Kadam & Wilson, 2016). There are other promising therapeutics for pre-and post-viral infection. For example, the lysosomotropic alkalinizing agents (LAA) block viral plaque formation, and the calcium modulators (CMs) inhibit calcium flow in viruses. Those non-covalent antivirals are much more ideal than peptidomimetics or covalent inhibitors because they minimize the potential toxicity caused by non-specific reactions with hosts. Sometimes, combinations of antivirals give better results on inhibiting viral infection with reduced side effects (Isabelle Marois et al., 2014).

Influenza is linked to severe complications such as asthma or heart failure, and due to the rapid evolution of influenza A virus circulating strains, antiviral drugs that target broad viral pathways are urgently needed. Antivirals that inhibit viral fusion, transcription, replication, and reproduction are considered to be ideal in dealing with influenza. However, they still have shortcomings; further research is required in investigating antivirals with higher efficacy to protect vulnerable populations from potentially deadly influenza virus infections.

References:

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Marois, I. et al. (2014) ‘Inhibition of influenza Virus Replication by Targeting Broad Host Cell Pathways’, PLOS ONE, 9(10):e110631. doi: 10.1371/journal.pone.0110631. 

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