Insights into the specific feature of the electrostatic recognition binding mechanism between BM2 and BM1: a molecular dynamics simulation study.

Matrix protein 2 (M2) and matrix protein 1 (M1) of the influenza B virus are two important proteins, and the interactions between BM2 and BM1 play an important role in the process of virus assembly and replication. However, the interaction details between BM2 and BM1 are still unclear at the atomic level. Here, we constructed the BM2-BM1 complex system using homology modelling and molecular docking methods. Molecular dynamics (MD) simulations were used to illustrate the binding mechanism between BM2 and BM1. The results identify that the eight polar residues (E88B, E89B, H119BM1, E94B, R101BM1, K102BM1, R105BM1, and E104B) play an important role in stabilizing the binding through the formation of hydrogen bond networks and salt-bridge interactions at the binding interface. Furthermore, based on the simulation results and the experimental facts, the mutation experiments were designed to verify the influence of the mutation of residues both within and outside the effector domain. The mutations directly or indirectly disrupt interactions between polar residues, thus affecting viral assembly and replication. The results could help us understand the details of the interactions between BM2 and BM1 and provide useful information for the anti-influenza drug design.

Palmitoylation of the hemagglutinin of influenza B virus by ER-localized DHHC enzymes 1, 2, 4, and 6 is required for efficient virus replication.

IMPORTANCE: Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.

The Transmembrane Conformation of the Influenza B Virus M2 Protein in Lipid Bilayers.

Influenza A and B viruses cause seasonal flu epidemics. The M2 protein of influenza B (BM2) is a membrane-embedded tetrameric proton channel that is essential for the viral lifecycle. BM2 is a functional analog of AM2 but shares only 24% sequence identity for the transmembrane (TM) domain. The structure and function of AM2, which is targeted by two antiviral drugs, have been well characterized. In comparison, much less is known about the structure of BM2 and no drug is so far available to inhibit this protein. Here we use solid-state NMR spectroscopy to investigate the conformation of BM2(1-51) in phospholipid bilayers at high pH, which corresponds to the closed state of the channel. Using 2D and 3D correlation NMR experiments, we resolved and assigned the 13C and 15N chemical shifts of 29 residues of the TM domain, which yielded backbone (φ, ψ) torsion angles. Residues 6-28 form a well-ordered α-helix, whereas residues 1-5 and 29-35 display chemical shifts that are indicative of random coil or ß-sheet conformations. The length of the BM2-TM helix resembles that of AM2-TM, despite their markedly different amino acid sequences. In comparison, large 15N chemical shift differences are observed between bilayer-bound BM2 and micelle-bound BM2, indicating that the TM helix conformation and the backbone hydrogen bonding in lipid bilayers differ from the micelle-bound conformation. Moreover, HN chemical shifts of micelle-bound BM2 lack the periodic trend expected for coiled coil helices, which disagree with the presence of a coiled coil structure in micelles. These results establish the basis for determining the full three-dimensional structure of the tetrameric BM2 to elucidate its proton-conduction mechanism.

Identification of immunodominant CD8 epitope in the stalk domain of influenza B viral hemagglutinin.

Human infections by type B influenza virus constitute about 25% of all influenza cases. The viral hemagglutinin is comprised of two subunits, HA1 and HA2. While HA1 is constantly evolving in an unpredictable fashion, the HA2 subunit is highly conserved, making it a potential candidate for a universal vaccine. However, immunodominant epitopes in the HA2 subunit remain largely unknown. To delineate MHC Class I epitopes, we first identified 9-mer H-2Kd-restricted CD8 T cell epitopes in the HA2 domain by in silico analyses, followed by evaluating the immunodominance of these peptides in mice challenged with the virus. Of three peptides selected through in silico analysis, the universally conserved peptide, YYSTAASSL (B/HA2-190), possessed the highest predicted binding affinity to MHC Class I and was most effective in inducing IL-2 and TNF-α in mouse splenocytes. Importantly, the peptide demonstrated best capability of stimulating peptide-specific ex-vivo cytotoxicity against target cells. Taken together, this finding would be of value for assessment of cell-mediated immune responses elicited by vaccines based on the highly conserved HA2 stalk domain.

The Functional Study of the N-Terminal Region of Influenza B Virus Nucleoprotein.

Influenza nucleoprotein (NP) is a major component of the ribonucleoprotein (vRNP) in influenza virus, which functions for the transcription and replication of viral genome. Compared to the nucleoprotein of influenza A (ANP), the N-terminal region of influenza B nucleoprotein (BNP) is much extended. By virus reconstitution, we found that the first 38 residues are essential for viral growth. We further illustrated the function of BNP by mini-genome reconstitution, fluorescence microscopy, electron microscopy, light scattering and gel shift. Results show that the N terminus is involved in the formation of both higher homo-oligomers of BNP and BNP-RNA complex.

N 2 gas plasma inactivates influenza virus by inducing changes in viral surface morphology, protein, and genomic RNA.

We have recently treated with N2 gas plasma and achieved inactivation of bacteria. However, the effect of N2 gas plasma on viruses remains unclear. With the aim of developing this technique, we analyzed the virucidal effect of N2 gas plasma on influenza virus and its influence on the viral components. We treated influenza virus particles with inert N2 gas plasma (1.5 kpps; kilo pulses per second) produced by a short high-voltage pulse generated from a static induction thyristor power supply. A bioassay using chicken embryonated eggs demonstrated that N2 gas plasma inactivated influenza virus in allantoic fluid within 5 min. Immunochromatography, enzyme-linked immunosorbent assay, and Coomassie brilliant blue staining showed that N2 gas plasma treatment of influenza A and B viruses in nasal aspirates and allantoic fluids as well as purified influenza A and B viruses induced degradation of viral proteins including nucleoprotein. Analysis using the polymerase chain reaction suggested that N2 gas plasma treatment induced changes in the viral RNA genome. Scanning electron microscopy analysis showed that aggregation and fusion of influenza viruses were induced by N2 gas plasma treatment. We believe these biochemical changes may contribute to the inactivation of influenza viruses by N2 gas plasma.

Flu channel drug resistance: a tale of two sites.

The M2 proteins of influenza A and B virus, AM2 and BM2, respectively, are transmembrane proteins that oligomerize in the viral membrane to form proton-selective channels. Proton conductance of the M2 proteins is required for viral replication; it is believed to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. In addition to the role of M2 in proton conductance, recent mutagenesis and structural studies suggest that the cytoplasmic domains of the M2 proteins also play a role in recruiting the matrix proteins to the cell surface during virus budding. As viral ion channels of minimalist architecture, the membrane-embedded channel domain of M2 has been a model system for investigating the mechanism of proton conduction. Moreover, as a proven drug target for the treatment of influenza A infection, M2 has been the subject of intense research for developing new anti-flu therapeutics. AM2 is the target of two anti-influenza A drugs, amantadine and rimantadine, both belonging to the adamantane class of compounds. However, resistance of influenza A to adamantane is now widespread due to mutations in the channel domain of AM2. This review summarizes the structure and function of both AM2 and BM2 channels, the mechanism of drug inhibition and drug resistance of AM2, as well as the development of new M2 inhibitors as potential anti-flu drugs.

Solution structure and functional analysis of the influenza B proton channel.

Influenza B virus contains an integral membrane protein, BM2, that oligomerizes in the viral membrane to form a pH-activated proton channel. Here we report the solution structures of both the membrane-embedded channel domain and the cytoplasmic domain of BM2. The channel domain assumes a left-handed coiled-coil tetramer formation with a helical packing angle of -37 degrees to form a polar pore in the membrane for conducting ions. Mutagenesis and proton flux experiments identified residues involved in proton relay and suggest a mechanism of proton conductance. The cytoplasmic domain of BM2 also forms a coiled-coil tetramer. It has a bipolar charge distribution, in which a negatively charged region interacts specifically with the M1 matrix protein that is involved in packaging the genome in the virion. This interaction suggests BM2 also recruits matrix proteins to the cell surface during virus budding, making BM2 an unusual membrane protein with the dual roles of conducting ions and recruiting proteins to the membrane.

Interactions between histidine and tryptophan residues in the BM2 proton channel from influenza B virus.

The BM2 protein of influenza B virus forms a transmembrane proton channel essential for the virus infection. We investigated the structure and mechanism of the BM2 proton channel by using a 31-mer peptide (BM2-TMP) representing the putative transmembrane domain of BM2, with special focus on His19, Trp23 and His27. Like the full-length protein, BM2-TMP formed a transmembrane proton channel activated at acidic pH with a midpoint of transition at pH 6.4 +/- 0.1. Mutation of His19 to Ala almost abolished the channel activity, whereas the His27-to-Ala mutant retained partial activity. The proton selectivity of the channel was lost upon substitution of Phe for Trp23. Comparison of CD, fluorescence and Raman spectra measured for wild-type and mutated BM2-TMP at varied pH showed the pK(a) of the imidazole ring to be approximately 6.5 for His19 and approximately 7.6 for His27. Analysis of the pH-dependent fluorescence and Raman intensities suggested the occurrence of cation-pi interaction between the protonated imidazole ring of His and the indole ring of Trp. The His19-Trp23 cation-pi interaction below pH 6.5 is likely to trigger the opening of the proton channel, whereas His27 is not essential but enhances the channel activity through interaction with Trp23, which constitutes the proton-selective gate.

S acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine.

S acylation of cysteines located in the transmembrane and/or cytoplasmic region of influenza virus hemagglutinins (HA) contributes to the membrane fusion and assembly of virions. Our results from using mass spectrometry (MS) show that influenza B virus HA possessing two cytoplasmic cysteines contains palmitate, whereas HA-esterase-fusion glycoprotein of influenza C virus having one transmembrane cysteine is stearoylated. HAs of influenza A virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate. MS analysis of recombinant viruses with deletions of individual cysteines, as well as tandem-MS sequencing, revealed the surprising result that stearate is exclusively attached to the cysteine positioned in the transmembrane region of HA.

Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses.

Influenza A viruses cause a highly contagious respiratory disease in humans and are responsible for periodic widespread epidemics with high mortality rates. The influenza A virus NS1 protein (NS1A) plays a key role in countering host antiviral defense and in virulence. The 73-residue N-terminal domain of NS1A (NS1A-(1-73)) forms a symmetric homodimer with a unique six-helical chain fold. It binds canonical A-form double-stranded RNA (dsRNA). Mutational inactivation of this dsRNA binding activity of NS1A highly attenuates virus replication. Here, we have characterized the unique structural features of the dsRNA binding surface of NS1A-(1-73) using NMR methods and describe the 2.1-A x-ray crystal structure of the corresponding dsRNA binding domain from human influenza B virus NS1B-(15-93). These results identify conserved dsRNA binding surfaces on both NS1A-(1-73) and NS1B-(15-93) that are very different from those indicated in earlier "working models" of the complex between dsRNA and NS1A-(1-73). The combined NMR and crystallographic data reveal highly conserved surface tracks of basic and hydrophilic residues that interact with dsRNA. These tracks are structurally complementary to the polyphosphate backbone conformation of A-form dsRNA and run at an approximately 45 degrees angle relative to the axes of helices alpha2/alpha2'. At the center of this dsRNA binding epitope, and common to NS1 proteins from influenza A and B viruses, is a deep pocket that includes both hydrophilic and hydrophobic amino acids. This pocket provides a target on the surface of the NS1 protein that is potentially suitable for the development of antiviral drugs targeting both influenza A and B viruses.

Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus.

The N-terminal domains of the NS1 protein of influenza B virus (NS1B protein) and the NS1 protein of influenza A virus (NS1A protein) share one function: binding double-stranded RNA (dsRNA). Here we show that the N-terminal domain of the NS1B protein possesses an additional function that is not shared by its NS1A counterpart: binding the ubiquitin-like ISG15 protein that is induced by influenza B virus infection. Homology modeling predicts that the dimeric six-helical N-terminal domain of the NS1B protein differs from its NS1A protein counterpart in containing large loops between helices 1 and 2 (loops 1 and 1') and between helices 2 and 3 (loops 2 and 2'). Mutagenesis establishes that residues located in loop 1/1' together with residues located in polypeptide segment 94-103 form the ISG15 protein-binding site of NS1B protein. Loop 1/1' is not required for dsRNA binding, which instead requires arginine residues R50, R53, R50', and R53' located in antiparallel helices 1 and 1'. Further, we demonstrate that the binding sites for RNA and protein are independent of each other. In particular, ISG15 and dsRNA can bind simultaneously; the binding of the ISG15 protein does not have a detectable effect on the binding of dsRNA, and vice versa.

Transmembrane peptide NB of influenza B: a simulation, structure, and conductance study.

The putative transmembrane segment of the ion channel forming peptide NB from influenza B was synthesized by standard solid-phase peptide synthesis. Insertion into the planar lipid bilayer revealed ion channel activity with conductance levels of 20, 61, 107, and 142 pS in a 0.5 M KCl buffer solution. In addition, levels at -100 mV show conductances of 251 and 413 pS. A linear current-voltage relation reveals a voltage-independent channel formation. In methanol and in vesicles the peptide appears to adopt an alpha-helical-like structure. Computational models of alpha-helix bundles using N = 4, 5, and 6 NB peptides per bundle revealed water-filled pores after 1 ns of MD simulation in a solvated lipid bilayer. Calculated conductance values [using HOLE (Smart et al. (1997) Biophys. J. 72, 1109-1126)] of ca. 20, 60, and 90 pS, respectively, suggested that the multiple conductance levels seen experimentally must correspond to different degrees of oligomerization of the peptide to form channels.

The N-terminal extension of the influenza B virus nucleoprotein is not required for nuclear accumulation or the expression and replication of a model RNA.

The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues. We have deleted the N-terminal 51 and 69 residues of the influenza B/Ann Arbor/1/66 virus NP and show that nuclear accumulation of the protein is unaffected. This indicates that the nuclear localization signal is not located at the extreme N terminus, as in influenza A virus NP. To determine if the N-terminal mutants could support the expression and replication of a model influenza B virus RNA, the genes encoding the subunits of the viral RNA-dependent RNA polymerase (PA, PB1, and PB2) were cloned. Coexpression of NP and the P proteins in 293 cells was found to permit the expression and replication of a transfected model RNA based on segment 4 of B/Maryland/59, in which the hemagglutinin-coding region was replaced by a chloramphenicol acetyltransferase gene. The expression and replication of the synthetic RNA were not affected by the replacement of NP with NP mutants lacking the N-terminal 51 or 69 residues, indicating that the N-terminal extension is not required for transcription or replication of the viral RNA. In addition, we report that the influenza B virus NP cannot be functionally replaced by type A virus NP in this system.

Probing the structure of influenza B hemagglutinin using site-directed mutagenesis.

The crystal structure of the hemagglutinin (HA) of influenza virus A/Aichi/68 (H3N2) from the X-31 reassortant virus was reported in 1981, but as yet there are no X-ray diffraction structures for hemagglutinins of other types or even subtypes of influenza virus. We have used site-directed mutagenesis to probe the structure of the hemagglutinin of influenza B/Hong Kong/8/73. We investigated a region in the globular head domain that is helical in the influenza A HA structure, targeting sidechains that in the H3 HA point toward solvent (Thr196) or into the receptor-binding pocket (Gln197). None of the mutations affected hemagglutination activity, but mutations T196P or Q1971 eliminated binding of a monoclonal antibody. The data suggest that this region of the influenza B HA forms a surface structure different from the alpha-helix of the influenza A HA structure and that it accounts for much of the antigenic activity of influenza B HA.

Disassembly of influenza C viruses, distinct from that of influenza A and B viruses requires neutral-alkaline pH.

Influenza C (Flu C) viruses comprise an internal ribonucleoprotein (RNP) and an outer lipoprotein envelope with surface spike glycoproteins and the M1 protein matrix. The lipoprotein envelope and spike glycoproteins are solubilized by nonionic detergent in a pH-independent manner. In contrast, disassembly of the M1 protein matrix appears to depend on pH. Treatment of Flu C viruses with nonionic detergent in neutral or alkaline medium (pH 9.0-7.4) results in disintegration of the virion M1 matrix and leads to a significant release of RNP free of the M1 protein. In acidic medium (pH 6.0-5.0), the M1 matrix is not removed and the viral core-like complex of RNP along with the M1 matrix cover is released. Since Flu A and B viruses were characterized by acid-dependent disassembly of the virion M1 matrix, Flu C viruses seem to resemble the paramyxoviruses, which also show a neutral-alkaline pH dependence on matrix disintegration. These observations suggest that uncoating mechanisms of influenza C viruses and paramyxoviruses in target cells may be similar.

[Inhibition of the reproduction of the influenza B virus by aprotinin]. / Ingibirovanie reproduktsii virusa grippa B aprotininom.

Injection of aprotinin, a natural inhibitor of proteinases, into the allantoic cavity of chick embryos infected with influenza B/Lee/40 or B/HK/73 virus resulted in inhibition of proteolytic cleavage of virus hemagglutinin HA into HA1 and HA2, thereby decreasing the level of proteolytic activation of the synthesized virus particles. As a result of this inhibition in aprotinin-treated embryos, multicycle virus reproduction was limited and virus yields decreased considerably. The experimental results indicate the potential of chemotherapeutic inhibition of infection caused by influenza B viruses using antiproteinase agents interfering with proteolytic activation of virions.