Viral Ion Channel Research Articles

Comparative Efficacy of Hemagglutinin, Nucleoprotein, and Matrix 2 Protein Gene-Based Vaccination against H5N1 Influenza in Mouse and Ferret

Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge.

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.

Electrophysiology of Viral Envelope Protein Ion Channels in Lipid Membranes Across Apertures in Polystyrene and Silicon

Viral ion channels, such as the coronavirus envelope proteins (E protein), belong to a family of channels that have attracted a considerable amount of interest during recent years. However, not many studies on the electrophysiology have been performed; mainly due to the fact that these channels are membrane proteins that do not easily express in the outer membrane of bacteria. Moreover, the channel currents are small (on the order of 10-300 pS) when compared with bacterial outer membrane channels.

Viral ion channels teach us lessons on protein sorting

Viral ion channels teach us lessons on protein sorting

Mimicking biopolymers on a molecular scale: nano(bio)technology based on engineered proteins

Proteins are ubiquitous biopolymers that adopt distinct three-dimensional structures and fulfil a multitude of elementary functions in organisms. Recent systematic studies in molecular biology and biotechnology have improved the understanding of basic functional and architectural principles of proteins, making them attractive candidates as concept generators for technological development in material science, particularly in biomedicine and nano(bio)technology. This paper highlights the potential of molecular biomimetics in mimicking high-performance proteins and provides concepts for applications in four case studies, i.e. spider silk, antifreeze proteins, blue mussel adhesive proteins and viral ion channels.

Influenza Viruses: Basic Biology and Potential Drug Targets

Influenza A and influenza B viruses are continuing causes of morbidity and mortality on an annual basis. Influenza A viruses have historically caused periodic pandemics in the human population, sometimes with devastating consequences, such as in 1918. Fears of a new pandemic have increased in recent years because of continuing outbreaks of highly pathogenic H5N1 avian influenza viruses in birds with occasional, but often lethal infection of humans. Despite their importance as human pathogens, the antiviral drugs approved to treat influenza virus infections are currently limited to two targets, the viral neuraminidase and the viral ion channel, M2. The use of the M2 inhibitors amantadine and rimantadine is further limited by the propensity of these drugs to select for drug resistant variants. However, the replication cycle of influenza viruses has been intensively studied and is receiving increased attention. New opportunities exist to develop novel antiviral strategies targeting these viruses.

Strategies of Development of Antiviral Agents Directed Against Influenza Virus Replication

In this review, we will discuss drug design based on proven and potential anti-influenza drug targets including viral hemagglutinin (HA), neuraminidase (NA), M2 ion channel, 3P polymerase complex, and host factors such as kinases. We have summarized influenza inhibitors based on their mode of actions. For instance, included are descriptions of (1) inhibitors of HA cleavage, such as nafamostat, camostat, gabexate, epsilon-aminocapronic acid and aprotinin, (2) inhibitors of fusion and entry, such as benzoquinones and hydroquinones, CL 385319, BMY-27709, stachyflin, and their analogues, (3) inhibitors of viral RNPs/polymerase/endonuclease, such as T-705, L-735,822, flutimide and their analogues, (4) inhibitors of MEK, such as PD 0325901, CI-1040 and ARRY-142886, and (5) inhibitors of NA such as DANA, FANA, zanamivir, and oseltamivir, etc. Although amantadine and rimantadine are not recommended for treating influenza virus infections because of drug resistance problem, these viral M2 ion channel blockers established a proof-of-concept that the endocytosis of virion into host cells can be a valid drug target because M2 protein is involved in the endocytosis process. The influenza polymerase complex not only catalyzes RNA polymerization but also encodes the "cap snatching" activity. After being exported from the nucleus to the cytoplasm, the newly synthesized vRNPs are assembled into virions at the plasma membrane. The progeny virions will then leave the host cells through the action of NA. The strategies for discovery of small molecule inhibitors of influenza virus replication based on each particular mechanism will be discussed. Finally, the lessons learned from the design of NA inhibitors (NAI) are also included. Many exciting opportunities await the cadre of virologists, medicinal chemists, and pharmacologists to design novel influenza drugs with favorable pharmacological and pharmacokinetic properties to combat this threatening infectious disease.

Viral Ion channels – still white spots in virus research.

Viral Ion channels – still white spots in virus research.

Towards a Mechanism of Function of the Viral Ion Channel Vpu from HIV-1

Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo- oligomeric Vpu bundles. Reconstitution of both full length Vpu1–81 and a short, the transmembrane (TM) domain comprising peptide Vpu1-32 into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied. The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.

Controlling influenza virus replication by inhibiting its proton channel

Influenza A and viruses encode minimalistic proton-selective ion channels known as A/M2 and BM2, respectively. The A/M2 channel is the target of the antiviral drug, amantadine. The structural and mechanistic aspects of proton conductance of the viral ion channels are described and the review makes a case for the development of more effective antivirals.

Evidence for the Formation of a Heptameric Ion Channel Complex by the Hepatitis C Virus P7 Protein in Vitro

The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex.

Adamantane Resistance in Influenza A

THE GLOBAL BURDEN OF INFLUENZA INFECTION IS STAGgering. In a typical year, approximately 20% of the world’s population is infected and more than a half million individuals die of influenza-associated complications. During influenza pandemics, morbidity and mortality are catastrophic—20 million to 100 million individuals died in the 1918 influenza pandemic, 2 million in the 1955 pandemic, and 1 million in the 1968 pandemic. Vaccination is the primary means for preventing influenza infections. In addition, 2 classes of drugs are currently available for treatment and prophylaxis: the adamantanes (amantadine and rimantadine) and the neuraminidase inhibitors (zanamivir and oseltamivir). Adamantanes are effective against susceptible strains of influenza A virus, whereas neuraminidase inhibitors are effective against susceptible strains of both A and B virus. All 4 agents can reduce the duration of infection by 1 to 2 days if treatment is initiated within 48 hours of symptom onset. Amantadine, rimantadine, and oseltamivir also have been approved by the US Food and Drug Administration (FDA) for prophylaxis and appear to be 70% to 90% effective at preventing symptomatic influenza infection. In the absence of protective humoral immunity from prior exposure to antigenically compatible virus or vaccine, these drugs are the only treatments available to mitigate or prevent illness. Adamantanes inhibit influenza propagation by blocking the viral M2 protein ion channel, which prevents fusion of the virus and host-cell membranes and release of viral RNA into the cytoplasm of infected cells. Approximately 30% of individuals treated with adamantanes for influenza virus infection excrete viruses resistant to these drugs. Resistance to adamantanes develops rapidly as a result of single amino acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the M2 protein. Adamantane-resistant mutants are not impaired in virulence, in vitro replication, or human-to-human transmission and maintain drug resistance for multiple generations in culture. Cross-resistance between amantadine and rimantadine is complete and adamantane-resistant virus can be shed for prolonged periods in immunocompromised patients. Nonetheless, through the mid-1990s the rate of resistance among untreated patients remained less than 1%. In this issue of JAMA, Bright and colleagues at the Centers for Disease Control and Prevention (CDC) report that the overwhelming majority of currently circulating influenza A(H3N2) viruses in the United States exhibit highlevel resistance to adamantanes due to an S31N (serine to asparagine at position 31) substitution in the viral M2 protein. Using the combination of 2 sequencing methods and a bioassay to confirm phenotypic resistance, Bright et al demonstrated resistance in 92% of 209 isolates submitted to the CDC during the first few months of the 2005-2006 influenza season. Based on an earlier analysis of 120 of these influenza A(H3N2) isolates, the CDC issued a Health Alert on January 14, 2006, and recommended that neither amantadine nor rimantadine should be used for the treatment or prophylaxis of influenza A infections in the United States for the remainder of the 2005-2006 influenza season. The resistance pattern they described extends beyond the United States: according to Bright et al, 43 of 47 isolates (91%) recently tested in Canada are resistant to adamantanes, and isolates from the 2004-2005 influenza season from some regions of Asia have shown high rates of resistance to adamantanes. The startling prevalence of resistance raises obvious questions: what happened, what does this mean, and what are physicians, other health care professionals, and public health officials, individually and collectively, going to do? The answers are in the epidemiology. The rate of adamantane resistance began to increase in Asia in the 1997-1998 influenza season and increased markedly in China to 57.5% in 2002-2003 and 73.8% in 2003-2004. Misuse of adamantanes most likely contributed to this rapid increase in resistance. In China, Russia, and some other countries, amantadine and rimantadine are both available without a prescription and are included in over-the-counter “antiflu” and “cold” preparations at a range of doses. In North America, the increase in resistance began about 5 years after the initial increase in Asia. In the United States, resis-

Adamantane Resistance in Influenza A

THE GLOBAL BURDEN OF INFLUENZA INFECTION IS STAGgering. In a typical year, approximately 20% of the world’s population is infected and more than a half million individuals die of influenza-associated complications. During influenza pandemics, morbidity and mortality are catastrophic—20 million to 100 million individuals died in the 1918 influenza pandemic, 2 million in the 1955 pandemic, and 1 million in the 1968 pandemic. Vaccination is the primary means for preventing influenza infections. In addition, 2 classes of drugs are currently available for treatment and prophylaxis: the adamantanes (amantadine and rimantadine) and the neuraminidase inhibitors (zanamivir and oseltamivir). Adamantanes are effective against susceptible strains of influenza A virus, whereas neuraminidase inhibitors are effective against susceptible strains of both A and B virus. All 4 agents can reduce the duration of infection by 1 to 2 days if treatment is initiated within 48 hours of symptom onset. Amantadine, rimantadine, and oseltamivir also have been approved by the US Food and Drug Administration (FDA) for prophylaxis and appear to be 70% to 90% effective at preventing symptomatic influenza infection. In the absence of protective humoral immunity from prior exposure to antigenically compatible virus or vaccine, these drugs are the only treatments available to mitigate or prevent illness. Adamantanes inhibit influenza propagation by blocking the viral M2 protein ion channel, which prevents fusion of the virus and host-cell membranes and release of viral RNA into the cytoplasm of infected cells. Approximately 30% of individuals treated with adamantanes for influenza virus infection excrete viruses resistant to these drugs. Resistance to adamantanes develops rapidly as a result of single amino acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the M2 protein. Adamantane-resistant mutants are not impaired in virulence, in vitro replication, or human-to-human transmission and maintain drug resistance for multiple generations in culture. Cross-resistance between amantadine and rimantadine is complete and adamantane-resistant virus can be shed for prolonged periods in immunocompromised patients. Nonetheless, through the mid-1990s the rate of resistance among untreated patients remained less than 1%. In this issue of JAMA, Bright and colleagues at the Centers for Disease Control and Prevention (CDC) report that the overwhelming majority of currently circulating influenza A(H3N2) viruses in the United States exhibit highlevel resistance to adamantanes due to an S31N (serine to asparagine at position 31) substitution in the viral M2 protein. Using the combination of 2 sequencing methods and a bioassay to confirm phenotypic resistance, Bright et al demonstrated resistance in 92% of 209 isolates submitted to the CDC during the first few months of the 2005-2006 influenza season. Based on an earlier analysis of 120 of these influenza A(H3N2) isolates, the CDC issued a Health Alert on January 14, 2006, and recommended that neither amantadine nor rimantadine should be used for the treatment or prophylaxis of influenza A infections in the United States for the remainder of the 2005-2006 influenza season. The resistance pattern they described extends beyond the United States: according to Bright et al, 43 of 47 isolates (91%) recently tested in Canada are resistant to adamantanes, and isolates from the 2004-2005 influenza season from some regions of Asia have shown high rates of resistance to adamantanes. The startling prevalence of resistance raises obvious questions: what happened, what does this mean, and what are physicians, other health care professionals, and public health officials, individually and collectively, going to do? The answers are in the epidemiology. The rate of adamantane resistance began to increase in Asia in the 1997-1998 influenza season and increased markedly in China to 57.5% in 2002-2003 and 73.8% in 2003-2004. Misuse of adamantanes most likely contributed to this rapid increase in resistance. In China, Russia, and some other countries, amantadine and rimantadine are both available without a prescription and are included in over-the-counter “antiflu” and “cold” preparations at a range of doses. In North America, the increase in resistance began about 5 years after the initial increase in Asia. In the United States, resis-

Chemical Synthesis Approaches to the Engineering of Ion Channels

Chemoselective ligation strategies have previously provided synthetic access to water-soluble proteins with novel properties, and more recently these strategies have been used to prepare ion channels. Examples of ion channels prepared by total chemical synthesis include bacterial mechanosensitive channels, and viral ion channels. Chemical protein synthesis allows for the generation of ion channel proteins with both native, and engineered structural or conductance properties.

Viral ion channels: from self – assembly to drug – development

Viral ion channels: from self – assembly to drug – development

Hypothalamic Growth Hormone-Releasing Hormone (GHRH) Deficiency: Targeted Ablation of GHRH Neurons in Mice Using a Viral Ion Channel Transgene

Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.

Viral ion channels as models for ion transport and targets for antiviral drug action

Viral ion channels as models for ion transport and targets for antiviral drug action

Conditional ablation of T-cell development by a novel viral ion channel transgene.

A novel conditional-lethal transgene system is defined in which a mutated influenza A virus ion-channel protein, which is permeable to monovalent cations, is lethal to cells on heterotypic expression and whose activity can be blocked by an antiviral drug (amantadine), is used to reversibly disrupt T-cell development. In vivo expression of the M2 ion channel, as a transgene under control of the T-cell specific p56(Lck) proximal promoter, resulted in total ablation of T-cell development with the accumulation of three distinct populations of early progenitor cells (CD44(+) CD25(-); CD44(+) CD25(+); CD44(+) CD25(hi)) in the thymic rudiment. In vitro development of transgenic fetal thymic progenitors to single-positive T cells could be rescued by antiviral drug treatment. Moreover, there was a radical reduction in B-cell lymphopoiesis, evident at the pre-B-cell stage, with a twofold increase of lymphoid cells 'in cycle' in transgenic bone marrow, indicative of major changes in haematopoietic homeostasis. This system may provide a generic protocol for conditional, lineage-specific cell ablation with available tissue-specific promoters for any eukaryotic developmental system, and provide a window on early T-cell development.

Definitive assignment of proton selectivity and attoampere unitary current to the M2 ion channel protein of influenza A virus.

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer--1.2 aA (1.2 x 10(-18) A) at neutral pH and 2.7 to 4.1 aA at pH 5.7--which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na+ or K+ probes. Even low-pH-activated M2 did not conduct Na+ and K+. The proton selectivity of M2 was estimated to be at least 3 x 10(6) (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.

Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells.

We show that a cellular nuclear protein, the SR splicing factor SF2/ASF, controls the level of production of an essential influenza virus protein, the M2 ion channel protein. The M2 mRNA that encodes the ion channel protein is produced by alternative splicing of another viral mRNA, M1 mRNA. The production of M2 mRNA is controlled in two ways. First, a distal (stronger) 5' splice site in M1 mRNA is blocked by the complex of viral polymerase proteins synthesized during infection, allowing the cellular splicing machinery to switch to the proximal (weaker) M2 5' splice site. Second, utilization of the weak M2 5' splice site requires its activation by the cellular SF2/ASF protein. This activation is mediated by the binding of the SF2/ASF protein to a purine-rich splicing enhancer sequence that is located in the 3' exon of M1 mRNA. We demonstrate that activation of the M2 5' splice site is controlled by the SF2/ASF protein in vivo during influenza virus infection. Utilizing four cell lines that differ in their levels of production of the SF2/ASF protein, we show that during virus infection of these cell lines both M2 mRNA and the M2 ion channel protein are produced in amounts that are proportional to the different expression levels of the SF2/ASF protein.