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Viruses 2013, 5(1), 352-373; doi:10.3390/v5010352
Published: 22 January 2013
Abstract: Influenza A virus (IAV) has caused seasonal influenza epidemics and influenza pandemics, which resulted in serious threat to public health and socioeconomic impacts. Until now, only 5 drugs belong to two categories are used for prophylaxis and treatment of IAV infection. Hemagglutinin (HA), the envelope glycoprotein of IAV, plays a critical role in viral binding, fusion and entry. Therefore, HA is an attractive target for developing anti‑IAV drugs to block the entry step of IAV infection. Here we reviewed the recent progress in the study of conformational changes of HA during viral fusion process and the development of HA-based IAV entry inhibitors, which may provide a new choice for controlling future influenza pandemics.
Main Influenza A viruses (IAV) cause acute respiratory diseases in humans, birds, and other mammals, representing one of the major threats to public health. Wild birds are the reservoir of influenza A viruses. An avian strain can adapt to the human host and attain human-to-human transmission capability through acquired mutations. An unexpected human adaptation of an influenza subtype or strain rather than currently circulating influenza viruses may cause pandemic flu. The pandemics of 1918 H1N1 (Spanish flu), 1957 H2N2 (Asian flu), 1968 H3N2 (Hong Kong flu) and 2009 H1N1 (swine flu) symbolize the devastating public health and socioeconomic impacts of pandemic flu and keep us alert to any such outbreak. Additionally, seasonal flu is responsible for about 50,000 deaths per year. The H5N1 type IAV, which infected 18 patients in Hong Kong and caused 6 death in 1997, is a potentially serious threat to human health in the near future because of its high mortality (about 60%) and potential human-to-human transmission.
Influenza viruses mutate frequently because of their segmented RNA genome, making it almost impossible to produce a timely and sufficiently effective vaccine to prevent the potential oseltamivir-resistant H5N1 influenza A viruses epidemic outbreaks. Therefore, it is the only way to use anti-influenza agents for treatment and prevention at the beginning of pandemic outbreak of a virulent influenza strain, which gives time for the development and widespread dissemination of an effective vaccine. There are two classes of anti-influenza drugs up to now available in the clinic, which targeting the M2 ion channel and neuraminidase (NA) expressed on the virus envelope, respectively. Adamantanes block the ion channel formed by the M2 protein, which is critical in the release of viral ribonucleoprotein complexes (vRNPs) into the cytoplasm . Although ion channel inhibitors can be effective against influenza virus infection, they have been reported to cause central nervous system (CNS) side effects. Also, currently circulating IAV strains are mostly resistant to adamantanes . Thus adamantanes are not recommended for a general and uncontrolled use.
Two neuraminidase inhibitors, oseltamivir and zanamivir, were both approved in 1999 for treatment and prevention for acute uncomplicated flu caused by influenza A and B. Neuraminidase inhibitors interfere with the enzymatic activity of the NA protein, which is critical for the efficient release of newly synthesized viruses from infected cells. However, resistant virus strains are constantly emerging, especially to oseltamivir . Different from the oral administration oseltamivir, zanamivir can only be inhaled due to its low bioavailability, which makes the limited use of this drug. In 2009, a new NA inhibitor, peramivir, was authorized for the emergent treatment of certain hospitalized patients with known or suspected 2009 H1N1 influenza.
It seems quite pressing to seek for new anti-influenza medications. Up to now, the life cycle of influenza virus has been well understood, allowing for the validation of several therapeutic targets. Among them, hemagglutinin (HA) is one of the most appealing ones. Till now 16 subtypes of HA have been identified and can be further subdivided into 5 clades and 2 groups (Figure 1). This malleable nature of HA imposes a great difficulty to conduct rational drug design. Furthermore, the variety of HA may be even strengthened by antigenic drift and antigenic shift [4,5]. Here, we described the functional and structural studies leading to the discovery of HA as a new anti-influenza target, and also how structural information is facilitating the rational design of new IAV entry inhibitors targeting HA.
2. The Virus Entry Process and the Function of Hemagglutinin
Hemagglutinin is encoded by the fourth negative-stranded RNA segment of influenza A viral genomes. This RNA segment encodes HA0 with 566 residues. HA0 is post-translationally glycosylated and trimerized with chaperon in endoplasmic reticulum (ER) in infected cell. Subsequently, HA0 undergoes an extra- or intra- cellular cleavage process into HA1 and HA2, which is a critical step for the maturation of influenza virus progenies to acquire their infectivity . Most HA subtypes (H1, H2 and H3) of the circulating influenza viruses in human, have a conserved cleavage site with a sole basic amino acid residue R343 in specified sequence Q/E-X-R, which is only recognized by extracellular tissue-restrict trypsin-like proteases . However, for highly pathogenic avian influenza (HPAI) viruses, the multi-basic HA0 cleavage sites (R-X-R/K-R) of H5- and H7- subtyped HA are recognized by ubiquitously expressed intracellular proteases, facilitating systemic virus spread and greater pathogenicity .
After proper proteolytic cleavage and glycosylation of HA0, disulfide bound subunits HA1 (about 327 residues) and HA2 (about 222 residues) form the fusogenic homotrimer, whose structure resembles a “mushroom” planted in the viral envelope (Figure 2). The globular head of the “mushroom” is mainly constructed with HA1 subdomains, including receptor binding subdomain, vestigial esterase subdomain and antigenic epitope . The receptor binding subdomain of HA1 recognizes α-2,3 or α-2,6 linked terminal sialic acids (SAs) in membrane glycoprotein receptor of host cell, therefore can prime the virus-cell adsorption and endocytosis for virus entry . The stem of the “mushroom” is mainly composed of the inner trimeric HA2 ectodomain subunits, embraced by the N- and C-terminal segments of HA1. The first 23 residues of N-terminal of HA2 is the functional fusion peptide (FP). FP is accommodated in a hydrophobic pocket formed partially by the fusion domain of HA1 . The acidification induced rearrangment of HA1 is a prerequisite for the exposure and release of FP subdomain of HA2 from inner pocket . Subsequently, low pH-triggered HA2 reconformation results in the fusion of the viral envelope with endosome membranes (Figure 2) . The fusion allows the release of viral ribonucleocapsid (vRNP) into cytoplasm of an infected cell, leading to the completion of the entry step.
5. Inhibitors Targeting the Cleavage of the HA0 to HA1 and HA2
As the H3-subtype avian flu virus X31 HA crystal structure and H1, H5, H9, H7-subtype avian influenza virus HA crystal structure are unfolded, people get a better understanding of haemagglutinin. The crystal structure show that HA is trimer forms. Each monomer of HA can be hydrolyzed into subunits HA1 and HA2 by host protease. The two hydrolyzed subunits are then connected by disulfide (Figure 2). The structure of HA precursor (HA0) and the structure of the hydrolysis are very alike. Only after six residues of the C-terminal of HA1 and 12 residues of the N-terminal of HA2 subunit distributing can they fuse. Especially the 12 residues of HA2 cover polar amino acids in HA0 which is exposed to liquid environment. This makes the HA vulnerable to hydrogen ion excitation and its structure changes .
The hydrolysis of HA0 into HA1 and HA2 is a necessary step for avian influenza virus to be contagious. Therefore, if any molecule can prevent HA0 from being hydrolyzed into the subunits of HA1 and HA2, it possesses the ability to fight against viral infection. Some serine protease inhibitors, such as anti-protease peptide constituted of 58 amino acids , e-aminocaproic acid , Nafamostat , pulmonary surfactant (a kind of surface active lipoprotein complex)  and mucous protease inhibitors , can reduce the hydrolysis of HA0 and the infection of influenza viruses in both cell models and animal models (Figure 5). As a kind of injections to treat the symptoms of bleeding, cow pancreas inhibitor, protease peptide (Trasylol bayer), has been applied to the clinical practice. However, it was withdrawn from the clinical application with the study finding that the drug could increase mortality in 2008 .
There are some FDA approval pulmonary surfactants, such as Exosurf, Curosurf, Infasurf and Survanta, all of which can enhance lung compliance, and prevent the baby respiratory distress syndrome. Quasi peptide inhibitor (dec-RVKR-CMK) resisting alkaline amino acid protease can inhibit the replication of H7 avian flu with high pathogenicity . Recently, some researchers reported the MPSL/TMPRSS13 can hydrolyze H5 and H7 subtypes . Therefore, natural inhibitors in the human body against MPSL/TMPRSS13 can serve as a lead compound to develop antiviral drugs which targeting haemagglutinin. It was found that the quasi peptide inhibitor also can restrain MPSL/TMPRSS13 and other insulin proteases such as plasmin, which may be the mechanism of action for this inhibitory peptide against influenza virus infection.
6. HA-Targeting Natural Products
In addition to above inhibitors, some natural molecules also possess good inhibitory activity against influenza A virus infection (Figure 6). For example, catechins isolated from green tea, like EGCG, were found to exhibit mild anti-influenza effect . Further modifications of catechins derived a set of better inhibitors. To be better anti-influenza drug candidates, catechin derivatives possess broad spectrum anti-influenza activity. Besides, curcumin, the widely used spice and coloring agent in Indian food, was proved to be a good virus entry inhibitor targeting HA with EC50 value of 0.47 μM . Modification of curcumin may produce a series of novel HA targeting inhibitors. Another kind of small molecule inhibitor is derived from andrographolide, like AL-1. AL-1 showed significant activity against avian influenza A (H9N2 and H5N1) and human influenza A H1N1 viruses in vitro . AL-1 is capable of direct interfering with viral HA to block viral binding to cellular receptors as was demonstrated by its inhibitory activity on viral adsorption to red blood cells. In 2009, Li’s group in China reported the first three small saponins molecule which inhibit HA. These three compounds can potently inhibit the entry of a H5N1 virus (A/Viet Nam/1203/2004) with IC50at low μM level. Further modifications revealed that the reduction of R1 to a disaccharide chain would abolish the inhibitory activity of these inhibitors . Therefore, anti-influenza agents from natural products, especially those from Traditional Chinese Medicine (TCM), are promising lead compounds. Some lead compounds from TCM are extensively and intensively reviewed by Xu et al. in 2010 .
HA, the major surface protein of IAV, mediates the viral binding, membrane fusion and viral entry. Determination of the crystal structures of HA0 provides important information for understanding the pH-induced conformational changes of HA at the pre-fusion, intermediate and post-fusion states for development of anti-influenza drugs. Although a series of anti-influenza drugs targeting the NA and M2 ion channel are currently available, the emergency of drug-resistance viruses has raised the great concern on their ineffectiveness against the newly emerging IAVs and the HPAI viruses. Thus, it is essential to develop novel anti-IAV drugs with new targets. A number of protein-based or small molecule anti-IAV agents have been shown to interfere with the HA-mediated membrane fusion by targeting the receptor binding, blocking the cleavage of HA0, or by inhibiting the low pH-mediated conformation changes of HA. It seems not easy to find or design small compounds targeting the binding event of influenza virus, but the conformational change of HA2 which mediates membrane fusion, is a promising target for developing anti-influenza drugs. Novel influenza virus entry inhibitors may provide more selections for combination therapy with NA inhibitors and M2 ion channel blockers for treating and preventing influenza virus infection and potential pandemic outbreak.
This work was financially supported by the National Nature Science Foundation of China (No. 30772602, 81102792). The statements in this paper reflect the reviews of the authors and we apologize for any unintended missed reference in this review.
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