3-3-1. Binding to negatively charged natural molecules of influenza A viruses
3-3-2. Binding to a protein complex, major histocompatibility complex (MHC) class II, of H17-H18 HAs
Nongluk Sriwilaijaroen
Associate Professor, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University
Nongluk Sriwilaijaroen was a visiting research student at the Medical Research Service, Department of Veteran’s Affairs Medical Center and Oregon Health and Science University, Portland, Oregon, USA in 2002-2003 and obtained her Ph.D. from the Department of Biochemistry, Faculty of Science, Mahidol University, Thailand in 2004. She started working as a lecturer in Graduate Studies, Faculty of Medicine, Thammasat University in 2005 and was a lecturer in the Department of Preclinical Sciences in the same faculty in 2008. She is currently an associate professor in the Department of Preclinical Sciences. In 2007, she began research on influenza under the supervision of Prof. Dr. Yasuo Suzuki in College of Life and Health Sciences, Chubu University, Japan. Her present research focuses on viral receptors, especially sialyl glycans, and new drug discovery and development.
Yasuo Suzuki
Visiting Professor of Chubu University and Professor Emeritus of the University of Shizuoka
Yasuo Suzuki was a full professor of University of Shizuoka School of Pharmaceutical Sciences (1989-2006) and served as dean of the Graduate School (1996-1998) and School of Pharmaceutical Sciences, University of Shizuoka (1998-2002). He published his first influenza work on “new ganglioside molecules carrying different molecular species of sialic acid which bind to human influenza viruses” in J. Biol. Chem. in 1985. After that work, he developed a new scientific field, glycovirology. He moved to Chubu University in 2006 as professor and director of Health Science Hills of the university. His recent work is focused on the key words (1) sialyl sugar chains, (2) influenza viruses, and (3) viral drug discovery. He was awarded The Pharmaceutical Society of Japan Award 2004 and Chunichi Cultural Award 2004.
Influenza caused by influenza A virus is one of the most widely distributed zoonotic diseases and occasionally leads to a pandemic (pdm). An influenza virus that has caused a pandemic usually becomes a seasonal influenza; however, the currently remaining seasonal viruses in human circulation are only the 1968pdm-derived H3N2 and 2009pdm-derived H1N1 variants. Influenza type A viruses have two major spike glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and the viruses are further classified into subtypes that have so far been identified: H1-H18 HA subtypes and N1-N11 NA subtypes. In contrast to H17N10 and H18N11 subtypes, which have so far been found only in bats and for which sialyl sugar chains are not used for their infection1, H1-H16 HA and N1-N9 NA subtypes have been found in wild waterfowls and some have been found in a variety of animals2. H1-H16 HAs are responsible for the binding to sialyl sugar chains on host receptors and entry of the viral particles into host cells, whereas NAs are responsible for the hydrolysis of sialic acids (Sias) on host decoy/actual receptors to release the virus from the traps/infected cells3. Sialyl sugar chains are widely distributed in animals and their chemical structures vary among animal species and tissues. Sialyl receptor binding specificity of HAs has long been studied extensively and it was found to be related to the dominant sialyl linkage type on the host target sites of influenza A viruses4-10. The use of recently developed chemical and biological technologies and instruments has revealed the diversity of sialyl sugar receptors and/or nonsialyl receptors that are selectively used by H1-H16 HAs and that are selectively used by H17-H18 HAs. Here we review recent progress in research on receptor binding specificity of influenza A virus HAs.
Sialic acid (Sia) has two molecular lineages in nature: Neu (neuraminic acid, 5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid) and KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). H1-H16 influenza A virus HAs bind to Neu derivatives, mainly to 5-N-acetylneuaminic acid (Neu5Ac) and 5-N-glycolylneuraminic acid (Neu5Gc). However, no influenza virus that binds to KDN derivatives has been reported. While single terminal Sia is necessary for virus binding, a tandem repeat of Sias, such as Siaα2-8Sia, and a Sia linked to an internal galactose (Gal) in a sugar chain, such as that on GM1 and GM2 gangliosides, are not recognized by influenza A viruses11, 12. The viruses appear to be able to bind to non-reducing terminal Sia linked to a penultimate sugar residue, Gal, N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), via an α2-3 linkage, except for GlcNAc, or via an α2-6 linkage13. Nonetheless, only Siaα2-3Gal (preferred by avian viruses) and Siaα2-6Gal (preferred by human and classical swine viruses) are commonly found in human11,14,15 and porcine16 respiratory tissues and in avian intestinal tissues17. Dominant sugar chains bound by the viruses are monosialo-lactosamine (LacNAc) type I (Galβ1-3GlcNAc) and type II (Galβ1-4GlcNAc), which are Siaα2-3(6)Galβ1-3(4)GlcNAcβ1- chains18; however, type 2 structures are expressed ubiquitously in animal tissues, whereas type 1 structures are expressed mainly in the gastrointestinal tract11.
It has been widely recognized that human H3N2 variants, which have been circulating in humans since 1968, have acquired reduced binding preference to a short human-type receptor, Siaα2-6Galβ1-4GlcNAcβ1- (6′Sialyl LacNAc)19,20, and have acquired strong binding preference to a long human-type receptor, 6′Sialyl polyLacNAc, over time during epidemic evolution 20,21. Using influenza A viruses isolated from different host species and in different periods of circulation, including the pandemic viruses A/Narita/1/09 (H1N1) and A/California/04/09 (H1N1), the seasonal viruses A/Aichi/28/04 (H1N1), A/Kitakyushu/10/06 (H1N1), A/Yamaguchi/20/06 (H1N1), A/Aichi/75/08 (H3N2) and A/Aichi/102/08 (H3N2), the avian viruses A/duck/Hokkaido/Vac-1/04 (H5N1), A/duck/Tsukuba/394/05 (H5N3) and A/mallard/Hokkaido/24/09 (H5N1), and the swine virus A/swine/Tochigi/1/08 (H1N2), we analyzed the effects of the length of sialyl LacNAc sugar chains on binding specificity by using synthetic 3′and 6′sialylglycopolymers with one and three LacNAc (LN) repeats, Neu5Acα2-3(6)(Galβ 1-4GlcNAc)1,3 β-pAP linked to a poly-α-L-glutamic acid (PGA)11. Based on the results we obtained, we classified influenza A viruses into two groups: group 1 viruses that bind nonselectively to both short α2-3Neu5Ac-LN and long α2-3Neu5Ac-LN-LN-LN sugar chains, i.e., all avian influenza A viruses tested, and group 2 viruses that bind preferentially to terminal α2-6Neu5Ac human-type receptor. Group 2 viruses can be further divided into subgroup 1 viruses including swine (H1N2/08) and pandemic (H1N1/09) viruses, which bind nonselectively to both short α2-6Neu5Ac-LN and long α2-6Neu5Ac-LN-LN-LN sugar chains, and subgroup 2 viruses including seasonal (H1N1/04, H1N1/06 and H3N2/08) viruses, which bind selectively to long α2-6Neu5Ac-LN-LN-LN sugar chains. Interestingly, swine (H1N2/08) virus and pandemic (H1N1/09) virus, the H1pdm of which was derived from a North American swine virus, in subgroup 1 carry only one N-glycan on each head monomer of their HAs. Each head monomer of the HAs of seasonal H1N1/04 and H1N1/06 viruses derived from re-emerged H1N1/77 virus has 4 glycosylation sites and that of seasonal H3N2/08 virus derived from H3N2/68pdm have 6 glycosylation sites11. As well as an increase in the number of glycosylation sites around the receptor binding site (RBS), viruses with long-term circulation (40 years for H3N2/08, 27 years for H1N1/04 and 29 years for H1N1/06) have acquired RBS amino acid substitutions over time that could affect the virus receptor binding specificity. Lin et al. 19 showed that site-specific mutagenesis of the HA D225N substitution into the H3N2/04 HA is a key mutation observed in H3N2 viruses circulating in 2005 that causes a loss of hydrogen-bond formation with the 2-Gal residue, resulting in reduction of short receptor binding. Although N225D reversion with other RBS amino acid substitutions, K158N, F159Y and N189K, since 2014 needs to be further investigated11, it is possible that the binding specificity of influenza A viruses to the length of sialyl sugar chains depends on the number of HA head glycosylation sites and RBS amino acids that are a consequence of host immune escape for continuing to cause seasonal epidemics.
We identified the sialyl N-glycans in normal human alveoli and found abundant short 3′Sia-LN (22.32 mol%) and 6′Sia-LN (16.10 mol%) sugar chains but only one longer Neu5Ac-LN-LN structure (0.15 mol%)11. Sialyl polyLN structures with 1-10 LN units were detected in the human upper respiratory tract14,15. Differential sialyl glycan structures found along the human respiratory tract could be a key factor explaining why infection with a pandemic (H1N1) 2009 virus, which binds both short 6′Sia-LN and long 6′Sia-LN-LN-LN, is more associated with influenza pneumonia22 than is infection with a long-term circulating seasonal virus, which selectively binds to long 6′Sia-polyLN. This finding indicated that the receptor binding specificity of influenza A viruses depending on the length of sialyl sugar chains contributes to the determination of viral tropism and pathogenicity, and the long sialyl sugar chains that are efficiently bound by all viruses should be taken into consideration when designing a vaccine and drugs targeting the HA RBS.
Binding of H1-H16 viral HAs to specific sialyl sugar chains on an endocytosis site is required for successful infection. In 2018, Fujioka and coworkers23 showed that the voltage-dependent Ca2+channel Cav1.2, a cellular protein that is linked to specific sialyl sugar chains, is an endocytosis site and that influx of Ca2+ to the inside of the host cells is important for internalization and infection. They found by a membrane binding assay that HAs of influenza A/Puerto Rico/8/34 (H1N1) virus bind to Cav1.2 and that sialidase treatment, but not treatment with the Ca2+ chelator EGTA, partially inhibits the HA- Cav1.2 binding. It was suggested that HAs bind to sialyl and nonsialyl regions on Cav1.2 and that binding to Ca2+ of the Cav1.2 is not necessary for HA binding. The use of a membrane from cells expressing truncation mutants of Cav1.2 indicated that segment IV of Cav1.2 is a binding site of HAs. Using Cav1.2 with mutations in potential sialylated asparagine residues, N1436Q, N1487Q, and N1436Q + N1487Q, in segment IV showed reduction of binding to HAs in comparison with using the wild-type Cav1.2, suggesting that sialyl sugar chains on the N1436 and N1487 residues of Cav1.2 segment IV are receptors of HAs.
All of the tested Ca2+ channel blockers (CCBs), including amlodipine, verapamil and diltiazem, and the intracellular Ca2+ chelator BAPTA-AM inhibited PR8 infection in a dose-dependent manner, and all of them except for verapamil significantly inhibited influenza A/Aichi/2/68 (H3N2) infection. Among the tested compounds, diltiazem and BAPTA-AM were the most effective inhibitors of virus infection. Using the fluorescent resonance energy transfer-based Ca2+ sensor Yellow Cameleon (YC3.60), diltiazem was shown to inhibit Ca2+ oscillations induced by both PR8 and Aichi virus induction in two different host Cos-1 and A549 cells and to inhibit virus internalization, suggesting that voltage-dependent Ca2+ channels (VDCCs) are critical for virus entry. Among the various types of VDCCs, L-type channels are found ubiquitously, and among the L-VDCC subtypes, Cav1.2 is found in a broad range of tissues and is more highly expressed than other subtypes in human cell lines. siRNA knockdown of Cav1.2 in A549 and 293T cells inhibited PR8 induction of Ca2+ oscillations, virus entry and infection, indicating that Cav1.2 is required for virus infection.
By immunohistochemistry, Cav proteins were shown to be present with different amounts along the murine respiratory system. Intranasal treatment of anesthetized mice with diltiazem 1 day before or after PR8 virus administration significantly reduced the amount of virus in nasal lavage fluid, indicating that diltiazem can be used as a prophylactic and therapeutic agent against influenza virus infection. Pretreatment of the human bronchial epithelial cells (BEAS-2B cells), which had been cultured on and in Matrigel to generate spheroid forms mimicking alveoli and an epithelial monolayer mimicking the bronchial epithelium, respectively, with diltiazem prior to PR8 virus exposure reduced virus infection of host cells, indicating that diltiazem can work effectively in the human respiratory system.
3-3-1. Binding to negatively charged natural molecules of influenza A viruses
Recent studies24 on binding of influenza A virus to natural molecules from the N-glycome of human lung tissue by using a human lung–shotgun N-glycan microarray (HL-SGM) led to the unexpected finding that all tested avian (H1N9 and H6N1), swine (H1N1, H1N2 and H3N2) and human (H1N1 and H3N2) strains can bind on the HL-SGM char IDs that were not bound by a α2-6Sia-binding lectin (SNA) and a α2-3Sia-binding lectin (MAL-I), indicating that all viruses can bind N-glycans without a sialic acid. Further details of the virus binding were studied by using the seasonal influenza A/Pennsylvania/08/2008 (H1N1) virus. Several experiments confirmed that phosphorylated glycans are non-sialylated glycans bound by the virus. For example, binding of the virus on the HL-SGM treated with bovine alkaline phosphatase was on chart IDs that were different from chart IDs of binding of the virus on the HL-SGM treated with Arthrobacter ureafaciens neuraminidase. Binding of the virus on the HL-SGM treated with neuraminidase was competitively inhibited in the presence of an antibody fragment of the single-chain variable domain M6P-1 (Fv M6P-1), which binds specifically to mannose-6-phosphate glycans, and it was completely abolished when the HL-SGM used for competitive binding of the virus with Fv M6P-1 was also treated with phosphatase following neuraminidase treatment. Binding of the virus to sialylated glycans on the untreated HL-SGM was not affected in the presence of Man6P, whereas binding of the virus to the neuraminidase-treated HL-SGM was reduced in the presence of Man6P, suggesting that the virus binds to phosphorylated glycans at a site different from the Sia receptor binding site. The use of other phosphorylated or sulfated sugars, such as mannose-6-sulfate and fructose-6-phosphate, induced much less inhibition of virus binding than that induced by Man6P, indicating that phosphorylated high mannose-type N-glycans should be binding substrates of influenza A viruses. The authors suggested that phosphorylated glycans may be cofactors or facilitators in enhanced virus entry into a host cell. However, the binding site on influenza viruses and the role of phosphorylated glycans on host epithelial cells in virus infection should be further clarified.
An earlier study25 showed by TLC immunostaining assays that a negatively charged phosphatidylinositol with 14-methyloctadecanoic acid and palmitic acid from the bacterium Rhodococcus equi strain S420 bound to all of the tested human (H1, H2 and H3), duck (H1-H7 and H9-H12) and swine (H1 and H3) influenza A viruses and to purified HAs (which were cleaved off from A/Aichi/2/68(H3N2) viruses with bromelain) as well as to human influenza B viruses. The results of that study suggested that binding of this compound to the viruses is different from binding of sialyl sugar chains to the viruses in that the phosphatidylinositol-virus binding is virus strain-independent. This compound was shown to inhibit virus hemagglutination, virus-induced hemolysis and infection of human H1, H2 and H3 in MDCK cells. Phosphatidylinositols (PIs) from the bovine liver (carrying stearic acid and arachidonic acid) and soybeans (carrying palmitic acid and linoleic acid), but not the smaller molecule L-α-glycerophospho-D-myo-inositol from soybeans lacking a fatty acid moiety, also showed inhibitory activities against virus hemagglutination, virus-mediated hemolysis and virus infection of influenza A/Aichi/2/68 (H3N2) even though they have lower activities than those of PI with 14-methyloctadecanoic acid and palmitic acid. In addition, negatively charged phosphatidylserine (PS) from the bovine brain, but not neutral phospholipids including phosphatidylethanolamine from the bovine brain, phosphatidylcholine from the bovine liver and sphingomyelin from the bovine brain displayed both virus-binding activity and inhibitory activities against virus hemagglutination and virus-induced hemolysis at tested concentrations. However, PS has lower binding and inhibitory activities than all of the tested PIs. It was assumed that an acidic phospholipid may adhere near the viral HA fusion loop and its fatty acid tails may be inserted into an interior HA portion where an HA fusion peptide is located, resulting in prevention of HA-induced membrane fusion. The advantage of using acidic phospholipids as influenza virus inhibitors is that they are not substrates of influenza virus neuraminidase.
3-3-2. Binding to a protein complex, major histocompatibility complex (MHC) class II, of H17-H18 HAs
Recently, new influenza-like virus genome sequences (H17N10 and H18N11 subtypes) were discovered in bats26-28. Surprisingly, HA molecules of these viruses do not bind to canonical Sia-containing avian and human type receptors, and NA molecules do not have sialidase activities, i. e., do not cleave the glycosidic linkage between Sia and penultimate sugar of sugar chains. Therefore, it was believed that the bat-derived influenza viruses may use a different entry mechanism than that used by H1-H16 influenza A viruses. In 2019, Karakus et al. 29 and Giotis et al. 30 found that a major histocompatibility complex class II (MHC-II) human leukocyte antigen (HLA) DR isotype is required for the entry of pseudotyped viruses bearing H17 or H18 HAs and thus acts as a determinant of cell susceptibility to infection by H17N10 and H18N11 viruses. They identified genes encoding membrane proteins that were expressed in virus-susceptible cells but not expressed in non-susceptible cells and tested the effect of down-regulation of expression of the gene candidates by gene knockout and/or RNA interference and the effect of blockage of the membrane protein function by specific monoclonal antibodies on H17- and H18-pseudotyped virus infection. The results suggested that MHC-II HLA-DR is crucial for the entry of bat-derived influenza A (H17N10 and H18N11) viruses. The pseudotyped viruses had the capacity to enter human HLA-DR+ cells, indicating that the bat viruses have zoonotic potential. SARS, Ebola and Nipah viruses have already been transmitted from bats to humans, indicating that there is an urgent need for extensive molecular studies on the roles and interactions of host and bat viral components needed for viral multiplication including the function of viral NA, typically a target of successful licensed anti-influenza N1-N9 viruses, and surveillance of the spread of the bat viruses to other animals and humans.
Since interactions between influenza virus HAs and receptors are key determinants of host tropism and pathogenesis, several research groups have published important findings. We collected and analyzed those well-known and recently published data for a better understanding of virus attachment and entry into a host cell. Currently, it is clear that H1-H16 viruses bind specifically to Sia species and linkage types on their target epithelial cells and that they undergo changes in binding preference to a new Sia species and linkage type when they cross a species barrier to a new host. Long-term circulation of both seasonal H1 and H3 viruses in humans appears to change their binding specificity from binding to both short and long sialyl sugar chains to binding to only long sialyl sugar chains. These variants have presumably been selected from a mutation conferring a balance between escaping host immunity and infecting the host cell. Recent findings that binding of virus HAs to the VDCC Cav1.2 channel induces Ca2+ influx and virus entry into the host cell and that virus entry can be inhibited by CCBs or by knocking down VDCC Cav1.2 indicated that sialyl sugar chains on VDCC Cav1.2 are necessary Sia receptors as endocytosis sites for virus infection23. However, experiments on binding of virus HAs to glycosylation-deficient mutants of Cav1.2 and to sialidase-treated Cav1.2 showing incomplete inhibition in comparison with that of wild-type Cav1.2 indicated that Cav1.2 contains both sialyl and nonsialyl regions interacting with virus HAs. Determination of nonsialyl region interactions between Cav1.2 and the virus HA is needed for the development of antiviral drugs and for determining whether this nonsialyl-binding region on the virus HA changes according to the virus host.
It has also been found that all of the tested influenza A viruses can bind to negatively charged nonsialyl molecules, phosphorylated glycans. However, both the binding site and the role of the binding need to be further investigated. There are several possibilities: the binding might be for landing of the virus on the host cell surface and allowing rolling of the virus to find the endocytosis site, for virus infection as a co-receptor or alternative receptor of Sia receptors, or for inhibition of virus infection as a noncompetitive inhibitor of Sia receptors. Not only nonsialyl phosphorylated glycans but also negatively charged nonsialyl phospholipids (PI and PS) display significant binding to influenza A viruses. Although acidic phospholipids were shown to bind to HA spikes of influenza A viruses, the binding site on the HA spike should be further identified. These phospholipids were shown to have both virus binding activity and inhibitory activities against hemagglutination and fusion, and the PI with 14-methyloctadecanoic acid and palmitic acid is the most effective phospholipid inhibitor of influenza virus infection. On the cell surface, PI and PS are enriched on the inner plasma membrane leaflet; hence, these phospholipids cannot be receptors for influenza virus. However, PI and PS can be found in the airway surfactant 31,32 and mucus33, which are natural barriers to microorganisms. Whereas Sia decoy receptors in mucus may be removed by influenza virus sialidase activity, these acidic phospholipids with fatty acids possibly lock HAs from detachment, and PI and PS may thus trap the virus to stop the infection and spread of the virus.
The recent discovery that MHC class II proteins from a wide range of host species including humans can be receptors for infection of bat H17-H18 viruses29,30 indicates the possibility of wide spread of these viruses to other animals. Since MHC class II proteins are typically found on antigen-presenting immune cells, these bat viruses may infect immune cells, possibly leading to evasion of immune surveillance and impairment of immune response34. Thus, we need to prepare for prevention and control of the emergence of new variants with the potential to infect other animals and humans.
The results of studies on binding of viruses/HAs to host molecules, including sugars, proteins and lipids, are important for finding a way to efficiently control virus infection and transmission.
