Presence Of Amantadine Research Articles

Biophysical investigation of liposome systems decorated with bioconjugated copolymers in the presence of amantadine.

Liposome-based technologies derived from lipids and polymers (e.g., PEGylated liposomes) have been recognized because of their applications in nanomedicine. However, since such systems represent myriad challenges and may promote immune responses, investigation of new biomaterials is mandatory. Here, we report on a biophysical investigation of liposomes decorated with bioconjugated copolymers in the presence (or absence) of amantadine (an antiviral medication). First, copolymers of poly(N,N-dimethylacrylamide-co-fluoresceinacrylate-co-acrylic acid-N-succinimide ester)-block-poly(N-isopropylacrylamide) (PDMA-b-PNIPAM) containing a fluorescence label were biofunctionalized with short peptides that resemble the sequence of the loops 220 and 130 of the binding receptor of the hemagglutinin (HA) protein of the influenza A virus. Then, the bioconjugated copolymers were self-assembled along with liposomes composed of 1,2 dimyristoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol (MSC). These biohybrid systems, with and without amantadine, were systematically characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryoTEM). Finally, the systems were tested in an in vitro study to evaluate cytotoxicity and direct immunofluorescence in Madin Darbin Canine Kidney (MDCK) cells. The biohybrid systems displayed long-term stability, thermo-responsiveness, hydrophilic-hydrophobic features, and fluorescence properties and were presumable endowed with cell targeting properties intrinsically integrated into the amino acid sequences of the utilized peptides, which indeed turn them into promising nanodevices for biomedical applications.

Can We Predict the Motor Performance of Patients With Parkinson's Disease Based on Their Symptomatology?

Introduction: Parkinson's disease hinders the ability of a person to perform daily activities. However, the varying impact of specific symptoms and their interactions on a person's motor repertoire is not understood. The current study investigates the possibility to predict global motor disabilities based on the patient symptomatology and medication.Methods: A cohort of 115 patients diagnosed with Parkinson's disease (mean age = 67.0 ± 8.7 years old) participated in the study. Participants performed different tasks, including the Timed-Up & Go, eating soup and the Purdue Pegboard test. Performance on these tasks was judged using timing, number of errors committed, and count achieved. K-means method was used to cluster the overall performance and create different motor performance groups. Symptomatology was objectively assessed for each participant from a combination of wearable inertial sensors (bradykinesia, tremor, dyskinesia) and clinical assessment (rigidity, postural instability). A multinomial regression model was derived to predict the performance cluster membership based on the patients' symptomatology, socio-demographics information and medication.Results: Clustering exposed four distinct performance groups: normal behavior, slightly affected in fine motor tasks, affected only in TUG, and affected in all areas. The statistical model revealed that low to moderate level of dyskinesia increased the likelihood of being in the normal group. A rise in postural instability and rest tremor increase the chance to be affected in TUG. Finally, LEDD did not help distinguishing between groups, but the presence of Amantadine as part of the medication regimen appears to decrease the likelihood of being part of the groups affected in TUG.Conclusion: The approach allowed to demonstrate the potential of using clinical symptoms to predict the impact of Parkinson's disease on a person's mobility performance.

Surface-confined amantadine–β-cyclodextrin inclusion complex: voltammetric study and application

A carbon paste electrode was modified with β-cyclodextrin (β-CD/CPE) and used in the competitive voltammetric determination of antiviral drug, amantadine. Ferrocene (FC) was used as the electrochemical probe which competed with amantadine for β-CD cavity. Study of the complexation of FC and amantadine with β-cyclodextrin was followed by cyclic voltammetry. Inclusion of FC in β-CD cavity at the surface of the modified electrode showed the adsorptive nature of current flow $$ \left( {\Delta E_{\text{p}} \sim 0\;{\text{V}}} \right) $$ . The formation constant of FC–β-CD complex was calculated from voltammetric data to be 2.03 × 103 M−1. In the presence of amantadine, the adsorptive current of FC decreased. The stability constant of amantadine–β-CD complex was calculated to be 9.40 × 103 M−1. A linear relationship between current decrease and amantadine concentration was observed in the range of 0.1–1.0 mM using differential pulse voltammetry. The limit of detection was 0.08 mM with a sensitivity of 1.34 μA/mM. The modified electrode was used successfully in the determination of amantadine in tablets (recovery % = 96 ± 1 %).

Molecular interactions between amantadine and model cell membranes.

Sum frequency generation (SFG) vibrational spectroscopy was applied to study molecular interactions between amantadine and substrate supported lipid bilayers serving as model cell membranes. Both isotopically asymmetric and symmetric lipid bilayers were used in the research. SFG results elucidated how the water-soluble drug, amantadine, influenced the packing state of each leaflet of a lipid bilayer and how the drugs affected the lipid flip-flop process. It is difficult to achieve such detailed molecular-level information using other analytical techniques. Especially, from the flip-flop rate change of isotopically asymmetric lipid bilayer induced by amantadine, important information on the drug-membrane interaction mechanism can be derived. The results show that amantadine can be associated with zwitterionic PC bilayers but has a negligible influence on the flip-flop behavior of PC molecules unless at high concentrations. Different effects of amantadine on the lipid bilayer were observed for the negatively charged DPPG bilayer; low concentration amantadine (e.g., 0.20 mM) in the subphase could immediately disturb the outer lipid leaflet and then the lipid associated amantadine molecules gradually reorganize to cause the outer leaflet to return to the original orderly packed state. Higher concentration amantadine (e.g., 5.0 mM) immediately disordered the packing state of the outer lipid leaflet. For both the high and low concentration cases, amantadine molecules only bind to the outer PG leaflet and cannot translocate to the inner layer. The presence of amantadine within the negatively charged lipid layers has certain implications for using liposomes as drug delivery carriers for amantadine. Besides, by using PC or PG bilayers with both leaflets deuterated, we were able to examine how amantadine is distributed and/or oriented within the lipid bilayer. The present work demonstrates that SFG results can provide an in-depth understanding of the molecular mechanisms of interactions between water-soluble drugs and model cell membranes.

EGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM)

The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500nm in two color channels with time resolution of below 100ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H+ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Endocytic Mechanism of Internalization of Dietary Peptide Lunasin into Macrophages in Inflammatory Condition Associated with Cardiovascular Disease

Cardiovascular disease (CVD) is the leading cause of death in the United States. Diet influences risk factors associated with CVD and atherosclerosis, a major vascular disease that arises from inflammation. Lunasin, a peptide derived from plant foods such as soybeans, contains a unique Arg-Gly-Asp cell-adhesion motif and inhibits the pathways involved in the inflammatory cascade. The objective was to determine the mechanism by which lunasin is internalized into human THP-1 macrophages, investigate the expression of endocytic membrane proteins in inflammatory conditions and to identify the pathways involved. While lipopolysaccharide (10 nM), vitronectin (130 nM) and a combination of these two molecules enhanced lunasin uptake and increased basal αVβ3 integrin expression, lunasin reduced αVβ3 expression by 25.5, 26.8 and 49.2%, respectively. The pretreatment of cells with brefeldin A (71 µM), an inhibitor of protein trafficking, inhibited lunasin internalization by up to 99.8%. Lunasin increased caveolin-1 expression by up to 204.8%, but did not modulate clathrin. The pretreatment of macrophages with nystatin (54 µM), an inhibitor of caveolae-dependent endocytosis, reduced lunasin internalization. The presence of amantadine (1 mM) and amiloride (1 mM), inhibitors of clathrin-mediated endocytosis and macropinocytosis, abolished lunasin cell entry. Lunasin elicited a transient reduction in intracellular levels of Ca2+ in LPS-induced macrophages. The results suggest that internalization of lunasin into macrophages is amplified in inflammatory conditions and is primarily mediated by endocytic mechanisms that involve integrin signaling, clathrin-coated structures and macropinosomes. Lunasin may be responsible for attenuation of CVD risk factors by interacting with pathways involved in endocytosis and inflammation.

The effect of amantadine on clomipramine induced sexual dysfunction in male rats.

Several studies have reported that Clomipramine has the ability to suppress male rat sexual behavior. Literature indicates that the activation of brain D(2) receptors causes facilitation of penile erection, and a number of reports have indicated dopamine's involvement in sexual function. Hence this study was undertaken to investigate the effect of Amantadine, a dopamine agonists on the Clomipramine induced sexual dysfunction. The study subjects involved a total of 48 males and 48 females, 4 months old Sprague-Dawley albino rats, all housed in a group of six males and females separately in plexi glass cages in an acclimatized colony room (25±0.5(0)C) maintained on a 12/12 hr light/dark cycle. The male rats were randomly divided into four groups of 12 male rats each. Group I served as controls. Group II, III, and IV were treated with Amantadine (9 mg/kg body weight, p.o) 30 min, prior to the treatment with 13.5 mg/kg, 27 mg/Kg and 54 mg/Kg bodyweight p.o of Clomipramine respectively for 60 days. The control group received vehicle 1 ml / kg p.o. The sexual behavior of the male rats was observed to determine the following parameters: mount latency, intromission latency, ejaculation latency, post ejaculatory pause, and intromission frequency. As well as the sexual behavior; serum testosterone and histopathology of the testes were also investigated in this study. The results indicate that Amantadine in all aspects failed to antagonize Clomipramine induced sexual dysfunction in male rats. Even the sexual competence of male rats treated with ½ therapeutic dose (TD) of Clomipramine failed to regain their sexual competence in the presence of Amantadine. Testicular damage and decline in testosterone levels continued in the presence of Amantadine. Overall, the results suggest that Amantadine could not be a safe antidote to antagonize Clomipramine induced sexual dysfunction.

Quantification of the Mg2+-induced potency shift of amantadine and memantine voltage-dependent block in human recombinant GluN1/GluN2A NMDARs

Clinically, amantadine and memantine are drugs whose therapeutic utility is linked to their ability to block N-methyl-D-aspartate receptors (NMDARs) in a voltage-dependent manner. Nevertheless many studies that have characterized the pharmacological actions of amantadine and memantine have done so in the absence of physiological levels of Mg2+ ions. This study quantifies the extent to which Mg2+ alters the potency of the block produced by both amantadine and memantine at human recombinant GluN1/GluN2A NMDARs. Human recombinant GluN1/GluN2A NMDARs were expressed in Xenopus laevis oocytes and two-electrode voltage-clamp recordings were made at −80, −60 and −40mV to quantify amantadine and memantine block in the absence and presence of Mg2+. Amantadine and memantine blocked human GluN1/GluN2A NMDARs in a voltage-dependent manner with IC50 values (at −80mV) of 49±6μM (n=7) and 1.0±0.3μM (n=7), respectively. In the presence of Mg2+ (1mM) the equivalent IC50 values were 165±10μM (n=6) and 6.6±0.3μM (n=5). Similarly in the presence of amantadine or memantine the potency of Mg2+ in blocking GluN1/GluN2A NMDARs was reduced. The decrease in the potencies of both amantadine and memantine in the presence of physiological concentrations of Mg2+ indicates that other targets (e.g. α7-nicotinic acetylcholine receptors and 5-HT3 receptors) in addition to NMDARs may well be sites of the therapeutic action of these channel blockers.

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the (1)H rotating-frame spin-lattice relaxation (T (1rho)) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by (2)H, (15)N and (13)C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor, amantadine, the protein exhibits significantly narrower linewidths at physiological temperature. We now investigate the origin of this line narrowing through temperature-dependent (1)H T (1rho) relaxation times in the absence and presence of amantadine. Analysis of the temperature dependence indicates that amantadine decreases the correlation time of motion from 2.8 +/- 0.9 mus for the apo peptide to 0.89 +/- 0.41 micros for the bound peptide at 313 K. Thus the line narrowing of the bound peptide is due to better avoidance of the NMR time scale and suppression of intermediate time scale broadening. The faster diffusion of the bound peptide is due to the higher attempt rate of motion, suggesting that amantadine creates better-packed and more cohesive helical bundles. Analysis of the temperature dependence of ln(T_1rho(-1)) indicates that the activation energy of motion increased from 14.0 +/- 4.0 kJ/mol for the apo peptide to 23.3 +/- 6.2 kJ/mol for the bound peptide. This higher activation energy indicates that excess amantadine outside the protein channel in the lipid bilayer increases the membrane viscosity. Thus, the protein-bound amantadine speeds up the diffusion of the helical bundles while the excess amantadine in the bilayer increases the membrane viscosity.

Effects of Double Combinations of Amantadine, Oseltamivir, and Ribavirin on Influenza A (H5N1) Virus Infections in Cell Culture and in Mice

An amantadine-resistant influenza A/Duck/MN/1525/81 (H5N1) virus was developed from the low-pathogenic North American wild-type (amantadine-sensitive) virus for studying treatment of infections in cell culture and in mice. Double combinations of amantadine, oseltamivir (or the cell culture-active form, oseltamivir carboxylate), and ribavirin were used. Amantadine-oseltamivir carboxylate and amantadine-ribavirin combinations showed synergistic interactions over a range of doses against wild-type virus in Madin-Darby canine kidney (MDCK) cell culture, but oseltamivir carboxylate-ribavirin combinations did not. Primarily additive interactions were seen with oseltamivir carboxylate-ribavirin combinations against amantadine-resistant virus. The presence of amantadine in drug combinations against the resistant virus did not improve activity. The wild-type and amantadine-resistant viruses were lethal to mice by intranasal instillation. The resistant virus infection could not be treated with amantadine up to 100 mg/kg body weight/day, whereas the wild-type virus infection was treatable with oral doses of 10 (weakly effective) to 100 mg/kg/day administered twice a day for 5 days starting 4 h prior to virus exposure. Drug combination studies showed that treatment of the amantadine-resistant virus infection with amantadine-oseltamivir or amantadine-ribavirin combinations was not significantly better than using oseltamivir or ribavirin alone. In contrast, the oseltamivir-ribavirin (25- and 75-mg/kg/day combination) treatments produced significant reductions in mortality. The wild-type virus infection was markedly reduced in severity by all three combinations (amantadine, 10 mg/kg/day combined with the other compounds at 20 or 40 mg/kg/day) compared to monotherapy with the three compounds. Results indicate a lack of benefit of amantadine in combinations against amantadine-resistant virus, but positive benefits in combinations against amantadine-sensitive virus.

In Vivo and In Vitro Alterations in Influenza A/H3N2 Virus M2 and Hemagglutinin Genes: Effect of Passage in MDCK-SIAT1 Cells and Conventional MDCK Cells

No mutations were detected in the hemagglutinin gene of influenza A/H3N2 virus isolates from patients undergoing short-term amantadine treatment. However, genetic changes occurred after serial passage in either MDCK or MDCK-SIAT1 cells. Our results showed that only a few mutations were observed in MDCK-SIAT1-passaged isolates in the presence of amantadine.

A Secondary Gate As a Mechanism for Inhibition of the M2 Proton Channel by Amantadine

The mechanism of inhibition of the influenza A virus M2 proton channel by the antiviral drug amantadine has been under intense investigation. The importance of a mechanistic understanding is heightened by the prevalence of amantadine-resistant mutations. To gain mechanistic insight at the molecular level, we carried out extensive molecular dynamics simulations of the tetrameric M2 proton channel in both apo and amantadine-bound forms in a lipid bilayer. The simulation of the apo form revealed that Val27 from the four M2 subunits can form a secondary gate near the channel entrance and break the water wire in the channel pore. This gate arises from physical occlusion and the elimination of hydrogen-bonding partners for water molecules. In the presence of amantadine, the secondary gate formed by Val27 and the drug molecule lying just below form an extended blockage, which breaks the water wire throughout the simulation. The location and orientation of amantadine inside of the channel pore as found in our simulation are supported by a host of experimental observations. Our study suggests a novel role for Val27 in the inhibition of the M2 proton channel by amantadine.

Proton Transport Behavior through the Influenza A M2 Channel: Insights from Molecular Simulation

The structural properties of the influenza A virus M2 transmembrane channel in dimyristoylphosphatidylcholine bilayer for each of the four protonation states of the proton-gating His-37 tetrad and their effects on proton transport for this low-pH activated, highly proton-selective channel are studied by classical molecular dynamics with the multistate empirical valence-bond (MS-EVB) methodology. The excess proton permeation free energy profile and maximum ion conductance calculated from the MS-EVB simulation data combined with the Poisson-Nernst-Planck theory indicates that the triply protonated His-37 state is the most likely open state via a significant side-chain conformational change of the His-37 tetrad. This proposed open state of M2 has a calculated proton permeation free energy barrier of 7 kcal/mol and a maximum conductance of 53 pS compared to the experimental value of 6 pS. By contrast, the maximum conductance for Na + is calculated to be four orders of magnitude lower, in reasonable agreement with the experimentally observed proton selectivity. The pH value to activate the channel opening is estimated to be 5.5 from dielectric continuum theory, which is also consistent with experimental results. This study further reveals that the Ala-29 residue region is the primary binding site for the antiflu drug amantadine (AMT), probably because that domain is relatively spacious and hydrophobic. The presence of AMT is calculated to reduce the proton conductance by 99.8% due to a significant dehydration penalty of the excess proton in the vicinity of the channel-bound AMT.

The Chemical and Dynamical Influence of the Anti-Viral Drug Amantadine on the M 2 Proton Channel Transmembrane Domain

The M 2 proton channel plays a vital role in the life cycle of the influenza A virus. His 37, the key residue in the M 2 transmembrane domain (M 2-TMD), plays a central role in the proton conductance mechanism. The anti-influenza drug, amantadine, inhibits the channel activity through binding to the pore of the M 2 channel. The nuclear spin relaxation data and polarization inversion spin exchange at the magic angle spectra show that both the polypeptide backbone and His 37 side chain are more constrained in the presence of amantadine. Using 15N cross polarization magic-angle spinning NMR spectroscopy, the protonation of His 37 of M 2-TMD in lipid bilayers was monitored in the absence and presence of amantadine as a function of pH. Binding amantadine lowers the His 37 pK a values by approximately three orders of magnitude compared with the first pK a of histidine in amantadine-free M 2-TMD. Amantadine’s influence on the His 37 chemical properties suggests a novel mechanism by which amantadine may inhibit proton conductance.

Sequence Determinants of a Transmembrane Proton Channel: An Inverse Relationship between Stability and Function

The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.

Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture.

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del(29-31) virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del(29-31) virus. We found that the M2-del(29-31) virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M2-del(29-31) virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.

Compatibility of various avian HA subtypes with the genome of human influenza A viruses

So far, with human influenza A viruses, only hemagglutinin (HA) subtypes 1, 2, or 3 were found. For pandemic planning, the question, whether other HA subtypes are compatible to create new human pandemic strains by reassortment, arises. Therefore, we crossed an amantadine-resistant variant of the human A/Singapore/57 (Sing) strain (H2N2) with various avian strains of different HA subtypes in the presence of amantadine and of anti-H2-antibodies. With A/Duck/Ukraine/63 (H3N8) and some other avian strains isolated recently in Hong Kong, large plaque-forming reassortants with the Sing-M-gene and the avian HA could be obtained in high yields, while with A/Chicken/Germany N/49 (Virus N) (H10N7) and some other avian strains, the yields were extremely low, and the remaining plaques were very small, and partly turbid and fuzzy. These observations may indicate that certain avian HA subtypes are not compatible to replace the HA-gene of the present human influenza A viruses to create a viable pandemic strain.

Amantadine blocks channel activity of the transmembrane segment of the NB protein from influenza B.

NB is short auxiliary protein with ca. 100 amino acids, encoded in the viral genome of influenza B. It is believed to be similar to M2 from influenza A and Vpu from HIV-1 in that it demonstrates ion channel activity. Channels formed by the protein can be blocked by amantadine. We have synthesized the putative transmembrane segment of NB (IRG S20 IIITICVSL I30 VILIVFGCI A40 KIFI (NB, Lee)). Reconstituted in a lipid bilayer, the peptide shows channel activity. The addition of amantadine leads to dose-dependent loss of channel activity. Channel blocking is reversible. Channel behaviour of the peptide in the presence of amantadine is in accordance with findings for the intact channel. Thus, the synthetic transmembrane peptide captures the ion channel activity of the intact NB protein.

Protonation of histidine and histidine-tryptophan interaction in the activation of the M2 ion channel from influenza a virus.

The M2 protein of influenza A virus forms a homotetramer ion channel in the lipid membrane. The channel is specific for proton conductance and is activated by low pH with a transition midpoint at pH 5.7. We have studied the structure of the transmembrane domain of the M2 ion channel by using UV resonance Raman spectroscopy, with special attention to the side chains of histidine (His37) and tryptophan (Trp41) residues. The Raman spectra provide direct evidence that the imidazole ring of His37 is protonated upon channel activation at low pH. Concomitantly, the UV resonance Raman scattering from Trp41 shows an unusual intensity change, which is ascribed to a cation-pi interaction between the protonated (cationic) imidazole ring of His37 and the indole ring of Trp41. The protonation of His37 and the Raman intensity change of Trp41 do not occur in the presence of amantadine that blocks the M2 ion channel. These observations clearly show that the protonation of His37 and concomitant cation-pi interaction with Trp41 is a key step in the activation of the M2 ion channel. The His37-Trp41 interaction associated with the channel activation is explained by assuming a conformational transition of His37 induced by electrostatic repulsion among the protonated imidazole rings of four His37 residues in the tetramer channel. Trp41 may play a role in stabilizing the channel open state through cation-pi interaction with His37. A molecular model for the activation of M2 ion channel is proposed on the basis of the gating mechanism.

Modified M2 proteins produce heterotypic immunity against influenza A virus

Vaccination with the influenza A transmembrane protein M2 provides enhanced viral clearance and recovery from influenza A virus infection in mice. However, the high degree of hydrophobicity of the protein limits its purification for vaccine purposes. We have attempted to alter the structure of the M2 protein to allow high level recombinant expression in Escherichia coli, to reduce its hydrophobicity and improve protein solubility, thus improving its properties as a vaccine subunit candidate. Constructs investigated include deletion of the transmembrane domain of M2 (residues 26–43) and an extended deletion (residues 26–55). A full-length M2 protein was not pursued because of poor expression, even in the presence of amantadine. Expressed as glutathione S-transferase fusion proteins and used to vaccinate mice, either deletion construct was found to raise M2-specific serum antibodies and enhance viral clearance in mice challenged with homologous and heterologous influenza A viruses. Enzymatic cleavage from the GST fusion domain produces soluble protein giving similar results. The results demonstrate that large alterations of M2 protein structure can improve its isolation and purification characteristics without detracting from its immunogenic properties.