Virus M2 Research Articles

Adamantan derivatives capable of inhibiting the reproduction of a Rimantadine resistant strain of influenza A(H1N1)pdm09 virus (Influenza A virus, Alphainfluenzavirus, Orthomyxoviridae).

Adamantanthane-type drugs such as rimantadine and amantadine have long been used to treat diseases caused by influenza A virus. However, as a result of the mutations, influenza viruses have become resistant to aminoadamantans. The target for these drugs was the protein channel M2. Influenza A virus M2 viroporin in the protein shell forms fairly specific ion channels with a diameter of about 11 Å, specializing in transporting protons inside the viral particle (virion). Restoration of the antiviral properties of adamantanthane-type drugs consists in the selection of advanced functional groups bound by the carbocycle to find new sites of binding to the protein target M2. The purpose of the study is to identify the antiviral properties of new adamantanum derivatives to the pandemic strain of influenza A virus in vitro. Compounds of aminoadamantans with amino acids and other organic molecules were obtained by classical peptide synthesis methods. The structure of the compound was tested by means of physical and chemical methods. Antiviral properties of synthetic compounds were studied in vitro on monolayer MDCK cells infected with pandemic strain of influenza A/California/07/2009 virus in two schemes of administration of investigated compounds and virus. The reference strain of the influenza virus A/California/07/2009(H1N1) was sensitive to the compounds under test in varying degrees. The antiviral activity of the compounds was expressed in a 50% inhibitory concentration (IС50) ranging from 0.5 to 2.5 мкM, which is generally a good indicator for the Rimantadine/Amantadine resistant strain. The values of the IС50 for compounds introduced two hours before contact with the virus were slightly higher than those for single-moment introduction of the substance and virus. The effect of increasing the inhibitory concentration in the prophylactic scheme of compounds was valid for all compounds of the experiment. The presented synthetic compounds are active against the variant of influenza A virus resistant to Rimantadine and Amantadine preparations. The obtained compounds can be used as model structures for creation of a new drug of direct action against advanced strains of influenza A virus.

PH Gradient Mitigation in the Leaf Cell Secretory Pathway Attenuates the Defense Response of Nicotiana benthamiana to Agroinfiltration.

Partial neutralization of the Golgi lumen pH by the ectopic expression of influenza virus M2 proton channel is useful to stabilize acid-labile recombinant proteins in plant cells, but the impact of pH gradient mitigation on host cellular functions has not been investigated. Here, we assessed the unintended effects of M2 expression on the leaf proteome of Nicotiana benthamiana infiltrated with the bacterial gene vector Agrobacterium tumefaciens. An isobaric tags for relative and absolute quantification quantitative proteomics procedure was followed to compare the leaf proteomes of plants agroinfiltrated with either an "empty" vector or an M2-encoding vector. Leaves infiltrated with the empty vector had a low soluble protein content compared to noninfiltrated control leaves, associated with increased levels of stress-related proteins but decreased levels of photosynthesis-associated proteins. M2 expression partly compromised these effects of agroinfiltration to restore soluble protein content in the leaf tissue, associated with restored levels of photosynthesis-associated proteins and reduced levels of stress-related proteins in the apoplast. These data illustrate the cell-wide influence of the Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana responding to microbial challenge. They also underline the relevance of assessing the eventual unintended effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.

Experimental validation and computational modeling of anti-influenza effects of quercetin-3-O-\u03b1-L-rhamnopyranoside from indigenous south African medicinal plant Rapanea melanophloeos

BackgroundInfluenza A virus (IAV) is still a major health threat. The clinical manifestations of this infection are related to immune dysregulation, which causes morbidity and mortality. The usage of traditional medication with immunomodulatory properties against influenza infection has been increased recently. Our previous study showed antiviral activity of quercetin-3-O-α-L-rhamnopyranoside (Q3R) isolated from Rapanea melanophloeos (RM) (L.) Mez (family Myrsinaceae) against H1N1 (A/PR/8/34) infection. This study aimed to confirm the wider range of immunomodulatory effect of Q3R on selective pro- and anti-inflammatory cytokines against IAV in vitro, to evaluate the effect of Q3R on apoptosis pathway in combination with H1N1, also to assess the physical interaction of Q3R with virus glycoproteins and RhoA protein using computational docking.MethodsMDCK cells were exposed to Q3R and 100CCID50/100 μl of H1N1 in combined treatments (co-, pre- and post-penetration treatments). The treatments were tested for the cytokines evaluation at RNA and protein levels by qPCR and ELISA, respectively. In another set of treatment, apoptosis was examined by detecting RhoA GTPase protein and caspase-3 activity. Molecular docking was used as a tool for evaluation of the potential anti-influenza activity of Q3R.ResultsThe expressions of cytokines in both genome and protein levels were significantly affected by Q3R treatment. It was shown that Q3R was much more effective against influenza when it was applied in co-penetration treatment. Q3R in combination with H1N1 increased caspase-3 activity while decreasing RhoA activation. The molecular docking results showed strong binding ability of Q3R with M2 transmembrane, Neuraminidase of 2009 pandemic H1N1, N1 and H1 of PR/8/1934 and Human RhoA proteins, with docking energy of − 10.81, − 10.47, − 9.52, − 9.24 and − 8.78 Kcal/mol, respectively.ConclusionsQuercetin-3-O-α-L-rhamnopyranoside from RM was significantly effective against influenza infection by immunomodulatory properties, affecting the apoptosis pathway and binding ability to viral receptors M2 transmembrane and Neuraminidase of 2009 pandemic H1N1 and human RhoA cellular protein. Further research will focus on detecting the detailed specific mechanism of Q3R in virus-host interactions.

Discovery of M2 channel blockers targeting the drug-resistant double mutants M2-S31N/L26I and M2-S31N/V27A from the influenza A viruses

Influenza virus infections are a persistent threat to human health due to seasonal outbreaks and sporadic pandemics. Amantadine and rimantadine are FDA-approved influenza antiviral drugs and work by inhibiting the viral M2 proton channel. However, the therapeutic potential for the antiviral amantadine/rimantadine was curtailed by the emergence of drug-resistant mutations in its target protein M2. In this study, we identified four amantadine-resistant M2 mutants among avian and human influenza A H5N1 strains circulating between 2002 and 2019: the single S31N and V27A mutants, and the S31N/L26I and S31N/V27A double mutants. Herein, utilizing two-electrode voltage clamp (TEVC) assays, we screened a panel of structurally diverse M2 inhibitors against these single and double mutant channels. Three compounds 6, 7, and 15 were found to significantly block all three M2 mutants: M2-S31N, M2-S31N/L26I, and M2-S31N/V27A. Using recombinant viruses generated from reverse genetics, we further showed that these compounds also inhibited the replication of recombinant viruses harboring either the single S31N or double S31N/L26I and S31N/V27A mutants. This work represents the first example in developing antivirals by targeting the drug-resistant double mutants of M2 proton channels.

Influenza A virus M2 protein triggers mitochondrial DNA-mediated antiviral immune responses

Cytosolic mitochondrial DNA (mtDNA) activates cGAS-mediated antiviral immune responses, but the mechanism by which RNA viruses stimulate mtDNA release remains unknown. Here we show that viroporin activity of influenza virus M2 or encephalomyocarditis virus (EMCV) 2B protein triggers translocation of mtDNA into the cytosol in a MAVS-dependent manner. Although influenza virus-induced cytosolic mtDNA stimulates cGAS- and DDX41-dependent innate immune responses, the nonstructural protein 1 (NS1) of influenza virus associates with mtDNA to evade the STING-dependent antiviral immunity. The STING-dependent antiviral signaling is amplified in neighboring cells through gap junctions. In addition, we find that STING-dependent recognition of influenza virus is essential for limiting virus replication in vivo. Our results show a mechanism by which influenza virus stimulates mtDNA release and highlight the importance of DNA sensing pathway in limiting influenza virus replication.

Subunit vaccines based on recombinant yeast protect against influenza A virus in a one-shot vaccination scheme

Here we report on new subunit vaccines based on recombinant yeast of the type Kluyveromyces lactis (K. lactis), which protect mice from a lethal influenza A virus infection. Applying a genetic system that enables the rapid generation of transgenic yeast, we have developed K. lactis strains that express the influenza A virus hemagglutinin, HA, either individually or in combination with the viral M1 matrix protein. Subcutaneous application of the inactivated, but otherwise non-processed yeast material shows a complete protection of BALB/c mice in prime/boost and even one-shot/single dose vaccination schemes against a subsequent, lethal challenge with the cognate influenza virus. The yeast vaccines induce titers of neutralizing antibodies that are readily comparable to those induced by an inactivated virus vaccine. These data suggest that HA and M1 are produced with a high antigenicity in the yeast cells. Based on these findings, multivalent, DIVA-capable, yeast-based subunit vaccines may be developed as promising alternatives to conventional virus-based anti-flu vaccines for veterinary applications.

Physicochemical Study of the Influenza a Virus M2 Protein and Aluminum Salt Adjuvant Interaction As a Vaccine Candidate Model

Aim: The present study evaluated the structural changes resulting from the interaction between a recombinant influenza A virus M2 protein and aluminum hydroxide adjuvant to investigate the antigen for further immunological studies. Materials & methods: Membrane protein II was produced from the H1N1 subtype of human influenza A virus. The interaction between M2 protein and alum inum hydroxide adjuvant was evaluated by physicochemical techniques including scanning electron microscope, UV-Vis spectra, Fourier-transform infrared spectroscopy and circular dichroism spectroscopy. Results: Physicochemical methods showed high-level protein adsorption and accessibility to the effective parts of the protein. Conclusion: It was concluded that M2 protein secondary structural perturbations, including the α-helix-to-β-sheet transition, enhanced its mechanical properties toward adsorption.

Envelope-deforming antiviral peptide derived from influenza virus M2 protein

Molecules interfering with lipid bilayer function exhibit strong antiviral activity against a broad range of enveloped viruses, with a lower risk of resistance development than that for viral protein-targeting drugs. Amphipathic peptides are rich sources of such membrane-interacting antivirals. Here, we report that influenza viruses were effectively inactivated by M2 AH, an amphipathic peptide derived from the M2 protein of the influenza virus. Although overall hydrophobicity (<H>) of M2 AH was not related to antiviral activity, modification of the hydrophobic moment (<μH>) of M2 AH dramatically altered the antiviral activity of this peptide. M2 MH, a derivative of M2 AH with a <μH> of 0.874, showed a half maximal inhibitory concentration (IC50) of 53.3 nM against the A/PR/8/34 strain (H1N1), which is 16-times lower than that of M2 AH. The selectivity index (IC50/CC50), where CC50 is the half maximal cytotoxic concentration, was 360 for M2 MH and 81 for M2 AH. Dynamic light scattering spectroscopy and electron microscopy revealed that M2 AH-derived peptides did not disrupt liposomes but altered the shape of viruses. This result suggests that the shape of virus envelope was closely related to its activity. Thus, we propose that deforming without rupturing the membranes may achieve a high selectivity index for peptide antivirals.

The effect of amantadine on an ion channel protein from Chikungunya virus.

Viroporins like influenza A virus M2, hepatitis C virus p7, HIV-1 Vpu and picornavirus 2B associate with host membranes, and create hydrophilic corridors, which are critical for viral entry, replication and egress. The 6K proteins from alphaviruses are conjectured to be viroporins, essential during egress of progeny viruses from host membranes, although the analogue in Chikungunya Virus (CHIKV) remains relatively uncharacterized. Using a combination of electrophysiology, confocal and electron microscopy, and molecular dynamics simulations we show for the first time that CHIKV 6K is an ion channel forming protein that primarily associates with endoplasmic reticulum (ER) membranes. The ion channel activity of 6K can be inhibited by amantadine, an antiviral developed against the M2 protein of Influenza A virus; and CHIKV infection of cultured cells can be effectively inhibited in presence of this drug. Our study provides crucial mechanistic insights into the functionality of 6K during CHIKV-host interaction and suggests that 6K is a potential therapeutic drug target, with amantadine and its derivatives being strong candidates for further development.

Deep Mutational Scan of the Highly Conserved Influenza A Virus M1 Matrix Protein Reveals Substantial Intrinsic Mutational Tolerance.

Influenza A virus matrix protein M1 is involved in multiple stages of the viral infectious cycle. Despite its functional importance, our present understanding of this essential viral protein is limited. The roles of a small subset of specific amino acids have been reported, but a more comprehensive understanding of the relationship between M1 sequence, structure, and virus fitness remains elusive. In this study, we used deep mutational scanning to measure the effect of every amino acid substitution in M1 on viral replication in cell culture. The map of amino acid mutational tolerance we have generated allows us to identify sites that are functionally constrained in cell culture as well as sites that are less constrained. Several sites that exhibit low tolerance to mutation have been found to be critical for M1 function and production of viable virions. Surprisingly, significant portions of the M1 sequence, especially in the C-terminal domain, whose structure is undetermined, were found to be highly tolerant of amino acid variation, despite having extremely low levels of sequence diversity among natural influenza virus strains. This unexpected discrepancy indicates that not all sites in M1 that exhibit high sequence conservation in nature are under strong constraint during selection for viral replication in cell culture.IMPORTANCE The M1 matrix protein is critical for many stages of the influenza virus infection cycle. Currently, we have an incomplete understanding of this highly conserved protein's function and structure. Key regions of M1, particularly in the C terminus of the protein, remain poorly characterized. In this study, we used deep mutational scanning to determine the extent of M1's tolerance to mutation. Surprisingly, nearly two-thirds of the M1 sequence exhibits a high tolerance for substitutions, contrary to the extremely low sequence diversity observed across naturally occurring M1 isolates. Sites with low mutational tolerance were also identified, suggesting that they likely play critical functional roles and are under selective pressure. These results reveal the intrinsic mutational tolerance throughout M1 and shape future inquiries probing the functions of this essential influenza A virus protein.

The L46P mutant confers a novel allosteric mechanism of resistance toward the influenza A virus M2 S31N proton channel blockers.

The Food and Drug Administration-approved influenza A antiviral amantadine inhibits the wild-type (WT) AM2 channel but not the S31N mutant predominantly found in circulating strains. In this study, serial viral passages were applied to select resistance against a newly developed isoxazole-conjugated adamantane inhibitor that targets the AM2 S31N channel. This led to the identification of the novel drug-resistant mutation L46P located outside the drug-binding site, which suggests an allosteric resistance mechanism. Intriguingly, when the L46P mutant was introduced to AM2 WT, the channel remained sensitive toward amantadine inhibition. To elucidate the molecular mechanism, molecular dynamics simulations and binding free energy molecular mechanics-generalized born surface area (MM-GBSA) calculations were performed on WT and mutant channels. It was found that the L46P mutation caused a conformational change in the N terminus of transmembrane residues 22-31 that ultimately broadened the drug-binding site of AM2 S31N inhibitor 4, which spans residues 26-34, but not of AM2 WT inhibitor amantadine, which spans residues 31-34. The MM-GBSA calculations showed stronger binding stability for 4 in complex with AM2 S31N compared with 4 in complex with AM2 S31N/L46P, and equal binding free energies of amantadine in complex with AM2 WT and AM2 L46P. Overall, these results demonstrate a unique allosteric resistance mechanism toward AM2 S31N channel blockers, and the L46P mutant represents the first experimentally confirmed drug-resistant AM2 mutant that is located outside of the pore where drug binds. SIGNIFICANCE STATEMENT: AM2 S31N is a high-profile antiviral drug target, as more than 95% of currently circulating influenza A viruses carry this mutation. Understanding the mechanism of drug resistance is critical in designing the next generation of AM2 S31N channel blockers. Using a previously developed AM2 S31N channel blocker as a chemical probe, this study was the first to identify a novel resistant mutant, L46P. The L46P mutant is located outside of the drug-binding site. Molecular dynamics simulations showed that L46P causes a dilation of drug-binding site between residues 22 and 31, which affects the binding of AM2 S31N channel blockers, but not the AM2 WT inhibitor amantadine.

Regulation of influenza A virus mRNA splicing by CLK1

Influenza A virus carries eight negative single-stranded RNAs and uses spliced mRNAs to increase the number of proteins produced from them. Several genome-wide screens for essential host factors for influenza A virus replication revealed a necessity for splicing and splicing-related factors, including Cdc-like kinase 1 (CLK1). This CLK family kinase plays a role in alternative splicing regulation through phosphorylation of serine-arginine rich (SR) proteins. To examine the influence that modulation of splicing regulation has on influenza infection, we analyzed the effect of CLK1 knockdown and inhibition. CLK1 knockdown in A549 cells reduced influenza A/WSN/33 virus replication and increased the level of splicing of segment 7, which encodes the viral M1 and M2 proteins. CLK1−/− mice infected with influenza A/England/195/2009 (H1N1pdm09) virus supported lower levels of virus replication than wild-type mice. Screening of newly developed CLK inhibitors revealed several compounds that have an effect on the level of splicing of influenza A gene segment M in different models and decrease influenza A/WSN/33 virus replication in A549 cells. The promising inhibitor KH-CB19, an indole-based enaminonitrile with unique binding mode for CLK1, and its even more selective analogue NIH39 showed high specificity towards CLK1 and had a similar effect on influenza mRNA splicing regulation. Taken together, our findings indicate that targeting host factors that regulate splicing of influenza mRNAs may represent a novel therapeutic approach.

Influenza virus matrix protein M1 interacts with SLD5 to block host cell cycle.

Influenza virus matrix 1 protein (M1) is highly conserved and plays essential roles at many stages of virus life cycle. Here, we used a yeast two-hybrid system to identify the host protein SLD5, a component of the GINS complex, which is essential for the initiation of DNA replication in eukaryotic cells, as a new M1 interacting protein. M1 from several different influenza virus strains all interacted with SLD5. Overexpression of SLD5 suppressed influenza virus replication. Transient, stable, or inducible expression of M1 induced host cell cycle blockade at G0/G1 phase. Moreover, SLD5 partially rescued M1 expression- or influenza virus infection-induced G0/G1 phase accumulation in cell lines and primary mouse embryonic fibroblasts. Importantly, SLD5 transgenic mice exhibited higher resistance and improved lung epithelial regeneration after virus infection compared with wild-type mice. Therefore, influenza virus M1 blocks host cell cycle process by interacting with SLD5. Our finding reveals the multifunctional nature of M1 and provides new insight for understanding influenza virus-host interaction.

By Binding CD80 and CD86, the Vaccinia Virus M2 Protein Blocks Their Interactions with both CD28 and CTLA4 and Potentiates CD80 Binding to PD-L1.

In this article we report that the M2 protein encoded by the vaccinia virus is secreted as a homo-oligomer by infected cells and binds two central costimulation molecules, CD80 (B7-1) and CD86 (B7-2). These interactions block the ligation of the two B7 proteins to both soluble CD28 and soluble cytotoxic T-lymphocyte associated protein 4 (CTLA4) but favor the binding of soluble PD-L1 to soluble CD80. M2L gene orthologues are found in several other poxviruses, and the B7-CD28/CTLA4 blocking activity has been identified for several culture supernatants of orthopoxvirus-infected cells and for a recombinant myxoma virus M2 protein homolog (i.e., Gp120-like protein, or Gp120LP). Overall, these data indicate that the M2 poxvirus family of proteins may be involved in immunosuppressive activities broader than the NF-κB inhibition already reported (R. Gedey, X. L. Jin, O. Hinthong, and J. L. Shisler, J Virol 80:8676-8685, 2006, https://doi.org/10.1128/JVI.00935-06). A Copenhagen vaccinia virus with a deletion of the nonessential M2L locus was generated and compared with its parental virus. This M2L-deleted vaccinia virus, unlike the parental virus, does not generate interference with the B7-CD28/CTLA4/PD-L1 interactions. Moreover, this deletion did not affect any key features of the virus (in vitro replication, oncolytic activities in vitro and in vivo, and intratumoral expression of a transgene in an immunocompetent murine model). Altogether, these first results suggest that the M2 protein has the potential to be used as a new immunosuppressive biotherapeutic and that the M2L-deleted vaccinia virus represents an attractive new oncolytic platform with an improved immunological profile.IMPORTANCE The vaccinia virus harbors in its genome several genes dedicated to the inhibition of the host immune response. Among them, M2L was reported to inhibit the intracellular NF-κB pathway. We report here several new putative immunosuppressive activities of M2 protein. M2 protein is secreted and binds cornerstone costimulatory molecules (CD80/CD86). M2 binding to CD80/CD86 blocks their interaction with soluble CD28/CTLA4 but also favors the soluble PD-L1-CD80 association. These findings open the way for new investigations deciphering the immune system effects of soluble M2 protein. Moreover, a vaccinia virus with a deletion of its M2L has been generated and characterized as a new oncolytic platform. The replication and oncolytic activities of the M2L-deleted vaccinia virus are indistinguishable from those of the parental virus. More investigations are needed to characterize in detail the immune response triggered against both the tumor and the virus by this M2-defective vaccinia virus.

Elucidating Relayed Proton Transfer through a His–Trp–His Triad of a Transmembrane Proton Channel by Solid-State NMR

Proton transfer through membrane-bound ion channels is mediated by both water and polar residues of proteins, but the detailed molecular mechanism is challenging to determine. The tetrameric influenza A and B virus M2 proteins form canonical proton channels that use an HxxxW motif for proton selectivity and gating. The BM2 channel also contains a second histidine (His), H27, equidistant from the gating tryptophan, which leads to a symmetric H19xxxW23xxxH27 motif. The proton-dissociation constants (pKa's) of H19 in BM2 were found to be much lower than the pKa's of H37 in AM2. To determine if the lower pKa's result from H27-facilitated proton dissociation of H19, we have now investigated a H27A mutant of BM2 using solid-state NMR. 15N NMR spectra indicate that removal of the second histidine converted the protonation and tautomeric equilibria of H19 to be similar to the H37 behavior in AM2, indicating that the peripheral H27 is indeed the origin of the low pKa's of H19 in wild-type BM2. Measured interhelical distances between W23 sidechains indicate that the pore constriction at W23 increases with the H19 tetrad charge but is independent of the H27A mutation. These results indicate that H27 both accelerates proton dissociation from H19 to increase the inward proton conductance and causes the small reverse conductance of BM2. The proton relay between H19 and H27 is likely mediated by the intervening gating tryptophan through cation–π interactions. This relayed proton transfer may exist in other ion channels and has implications for the design of imidazole-based synthetic proton channels.

Influenza virus M1 interacts with SLD5 to block host cell cycle and inhibit lung epithelial regeneration

Abstract Influenza virus matrix 1 protein (M1) is highly conserved and plays essential roles at many stages of virus life cycle. Here we used a yeast two-hybrid system to identify the host protein SLD5, a component of the GINS complex, which is essential for the initiation of DNA replication in eukaryotic cells, as a new M1 interacting protein. M1 from several different influenza virus strains all interacted with SLD5. Overexpression of SLD5 inhibited influenza virus replication. Transient, stable or inducible expression of M1 induced host cell cycle blockade at G0/G1 phase. Importantly, SLD5 partly rescued M1 expression- or influenza virus infection-induced G0/G1 phase accumulation in cell lines and primary mouse embryonic fibroblasts. Importantly, SLD5 transgenic mice exhibited higher resistance and improved lung epithelial regeneration after virus infection compared to wild type mice. Therefore, influenza virus M1 inhibits host cell cycle process by interacting with SLD5. Our finding reveals the multifunctional nature of M1 and provides new insight for understanding influenza virus-host interaction.

Identification of H1N1 influenza virus-derived T-cell epitopes and the contribution in the cross-reactivities to avian influenza virus

Influenza viruses, such as 2009 pandemic A(H1N1) influenza virus (2009 pH1N1) and avian influenza A (H7N9) virus (H7N9), which spread across the species, pose a great threat to human health and cause socioeconomic losses. Since 2009, circulation of 2009 pH1N1 has become a seasonal influenza virus. The sustaining epidemics have resulted in certain T-cell immune level among healthy populations. T-cell epitopes are mainly derived from conserved internal proteins of influenza virus compared with B-cell epitopes, influenza virus-specific cross-reactive CD4+ and CD8+ T-cells broadly exist among the population, providing protection from subsequent infections by heterotypic viruses. Cytotoxic T lymphocytes (CTLs) specific for influenza A viruses mostly target internal, nonglycosylated proteins including M1, which are enriched with immunodominant CTL epitopes and markedly conserved among diverse strains compared to HA and NA. In comparison to M1 protein of 2009 pH1N1, the H7N9 M1 has 4 segments with clustering substitutions. In this study, we defined the cross-reactivity immunity between 2009 pH1N1 and H7N9 and identified four T-cell epitopes: H1-M42 (LMEWLKTR), H1-M102 (KLKREITFHGAK), H1-M202s (AMEVANQTR) and H1-M244 (MGVQMQRFK) in the mutant segments in 2009 pH1N1. T-cell responses were investigated using freshly isolated PBMCs from individuals ( n = 16) through IFN-γ ELISPOT with these peptides derived from influenza virus M1 protein that are not conserved between 2009 pH1N1 and H7N9. We investigated the baseline of pre-existing immunity targeting M1 mutant segments of avian H7N9 influenza virus compared to 2009 pH1N1 among a healthy population. There was a certain level of T-cell immunity to H7N9 in the healthy population, but still weaker than that to 2009 pH1N1. Peptides derived from H7N9 which had mutations induced significant lower T-cell responses. The limited pre-existing T-cell immunity against H7N9 in healthy populations was partially contributed by H7N9 amino acid mutations in novel identified epitopes. Furthermore, we found that H1-M102 and H1-M244 could bind with HLA-A*1101 stably after renaturation in vitro , while the binding of H1-M42 and H1-M202s with HLA-A*3303 were relatively weak. Combined refolding and functional studies based on T-cell epitopes derived from influenza virus illustrated that minor mutation of an epitope can lead to a profound effect on the antigenicity of the peptide, which may also influence both HLA binding and TCR docking. Our study on T-cell immunity against influenza viruses provides an important reference for understanding the preexisting immune responses to avian influenza virus, and benefits the development of universal influenza vaccine. At the same time, considering that a few amino acid mutations will change the immunogenicity of the peptide completely, the study of antigenic variability of influenza virus major T cell immunogens such as M1, NP and PB1 are crucial in the development of universal vaccines.

Targeting M2e to DEC-205 induces an enhanced serum antibody-dependent heterosubtypic protection against influenza A virus infection

The ectodomain of the influenza A virus (IAV) M2 protein (M2e) is highly conserved, and it represents a promising candidate for the development of an “universal vaccine”. However, the low immunogenicity associated to M2e in a natural infection or in response to seasonal vaccines has led to explore new approaches to enhance it. In recent years, it has become clear that targeting antigens to dendritic cells (DC) is an efficient way to enhance immune responses against pathogens. In this work, the M2e peptide was chemically cross-linked to a monoclonal antibody (mAb) specific for DEC-205 (α-DEC-205:M2e), present on DC. BALB/c mice were inoculated subcutaneously (s.c.) three times with the conjugate equivalent to 1 µg of M2e, in the presence of polyinosinic-polycytidylic acid (poly I:C) as adjuvant. As controls, other groups of mice were inoculated under the same conditions with M2e cross-linked to an isotype control mAb (isotype:M2e), 5 µg of free M2e peptide, ovalbumin (OVA) cross-linked to the α-DEC-205 mAb (α-DEC-205:OVA) or poly I:C alone. Immunization with α-DEC-205-M2e induced high levels of serum antibodies (Abs) compared to isotype:M2e or to free M2e peptide, and in all cases IgG1 was predominant over IgG2a Abs. Furthermore, immunization with the α-DEC-205:M2e conjugate did not prevent morbidity, but it induced up to 76% protection against a heterosubtypic IAV lethal challenge. Contrasting with the 20 to 40% protection induced by isotype:M2e or by free M2e peptide. The protection induced by α-DEC-205:M2e conjugate was dependent on non-neutralizing serum Abs and independent of effector CD4+ T cells. These results show that targeting M2e to DEC-205 is a very effective alternative to induce strong heterosubtypic protection against an IAV infection.

Identification of NMS-873, an allosteric and specific p97 inhibitor, as a broad antiviral against both influenza A and B viruses

Influenza virus infection causes substantial morbidity and mortality worldwide. The limited efficacy of oseltamivir in delayed treatment, coupled with the increasing incidences of oseltamivir-resistant strains, calls for next-generation of antiviral drugs. In this study, we discovered NMS-873, an allosteric and specific p97 inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. NMS-873 shows potent antiviral activity with low-nanomolar EC50s against multiple human influenza A and B viruses, including adamantine-, oseltamivir-, or double resistant strains. Our data further showed that silencing of p97 via siRNA or inhibiting p97 by NMS-873 both inhibited virus replication and retained viral ribonucleoproteins (vRNPs) in the nucleus, confirming p97 is the drug target. Mechanistic studies have shown that the nuclear retention of vRNP with NMS-873 treatment is a combined result of two effects: the reduced viral M1 protein level (indirect effect), and the disruption of p97-NP interactions (direct effect). Taken together, our results suggest that p97 could be a novel antiviral target and its inhibitor, NMS-873, is a promising antiviral drug candidate.

Computational investigation of influenza A virus M2 protein inhibitionmechanism by ion channel blockers

The M2 protein of the influenza A virus, responsible for flu, is a homotetramer transmembrane protein, forming a transmembrane ion channel, where His 37s act as pH sensors and Trp 41s and Asp 44s act as channel gates. Opening of this channel leads to transfer of virus RNA into the human host. Thus, blocking this transfer is an important pharmaceutical strategy to stop infection. As a result of viral drug resistance, commercially available channel inhibitors, rimantadine (RIM) and amantadine (AMA), are not as effective as they used to be. Understanding binding sites and outcomes of binding will lead to new inhibitor design studies. Here, utilizing molecular docking with classical and constant pH molecular dynamics simulations, two novel binding sites for RIM and AMA molecules were revealed. Both structural and chemical effects of inhibitors on M2 protein on the closed, inactive M2 channel structure were investigated. Upon binding of the inhibitor molecules, decrease in ion channel cross-distances was observed. Meanwhile, RIM binding did not alter protonation of His 37s and Asp 44s, whereas AMA binding drastically increased the protonation population of two residues from different monomers, creating a more basic channel at physiological pH.