Lauren Elizabeth Wilson

Identifying the MAGUK Protein Carma-1 as a Central Regulator of Humoral Immune Responses and Atopy by Genome-Wide Mouse Mutagenesis

In a genome-wide ENU mouse mutagenesis screen a recessive mouse mutation, unmodulated, was isolated with profound defects in humoral immune responses, selective deficits in B cell activation by antigen receptors and T cell costimulation by CD28, and gradual development of atopic dermatitis with hyper-IgE. Mutant B cells are specifically defective in forming connections between antigen receptors and two key signaling pathways for immunogenic responses, NF-kappaB and JNK, but signal normally to calcium, NFAT, and ERK. The mutation alters a conserved leucine in the coiled-coil domain of CARMA-1/CARD11, a member of the MAGUK protein family implicated in organizing multimolecular signaling complexes. These results define Carma-1 as a key regulator of the plasticity in antigen receptor signaling that underpins opposing mechanisms of immunity and tolerance.

Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride.

A 63 residue peptide, p7, encoded by hepatitis C virus was synthesised and tested for ion channel activity in lipid bilayer membranes. Ion channels formed by p7 had a variable conductance: some channels had conductances as low as 14 pS. The reversal potential of currents flowing through the channels formed by p7 showed that they were permeable to potassium and sodium ions and less permeable to calcium ions. Addition of Ca2+ to solutions made channels formed by p7 less potassium‐ or sodium‐selective. Hexamethylene amiloride, a drug previously shown to block ion channels formed by Vpu encoded by HIV‐1, blocked channels formed by p7. In view of the increasing number of peptides encoded by viruses that have been shown to form ion channels, it is suggested that ion channels may play an important role in the life cycle of many viruses and that drugs that block these channels may prove to be useful antiviral agents.

SARS coronavirus E protein forms cation-selective ion channels.

Abstract Severe Acute Respiratory Syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). Coronaviruses including SARS-CoV encode an envelope (E) protein, a small, hydrophobic membrane protein. We report that, in planar lipid bilayers, synthetic peptides corresponding to the SARS-CoV E protein forms ion channels that are more permeable to monovalent cations than to monovalent anions. Affinity-purified polyclonal antibodies recognizing the N-terminal 19 residues of SARS-CoV E protein were used to establish the specificity of channel formation by inhibiting the ion currents generated in the presence of the E protein peptides.

Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication

Abstract All coronaviruses encode a small hydrophobic envelope (E) protein, which mediates viral assembly and morphogenesis by an unknown mechanism. We have previously shown that the E protein from Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) forms cation-selective ion channels in planar lipid bilayers (Wilson, L., McKinlay, C., Gage, P., Ewart, G., 2004. SARS coronavirus E protein forms cation-selective ion channels. Virology 330(1), 322–331). We now report that three other E proteins also form cation-selective ion channels. These E proteins were from coronaviruses representative of taxonomic groups 1–3: human coronavirus 229E (HCoV-229E), mouse hepatitis virus (MHV), and infectious bronchitis virus (IBV), respectively. It appears, therefore, that coronavirus E proteins in general, belong to the virus ion channels family. Hexamethylene amiloride (HMA) – an inhibitor of the HIV-1 Vpu virus ion channel – inhibited the HCoV-229E and MHV E protein ion channel conductance in bilayers and also inhibited replication of the parent coronaviruses in cultured cells, as determined by plaque assay. Conversely, HMA had no antiviral effect on a recombinant MHV with the entire coding region of E protein deleted (MHVΔE). Taken together, the data provide evidence of a link between inhibition of E protein ion channel activity and the antiviral activity of HMA.

Autoimmunity, Self-Tolerance and Immune Homeostasis: From Whole Animal Phenotypes to Molecular Pathways

Current therapy for autoimmune disease is based on broad-spectrum immune suppression, rather than specific correction of defective tolerance mechanisms. On the preventive front, we are not yet able to identify individuals at risk of autoimmune disease or predict clinical course. To develop more specific therapeutic and diagnostic tools, we will need a map of the cellular and molecular pathways and genes that underpin immunological self-tolerance, illuminating the points where the process goes wrong and where it can be corrected.

Validation of coronavirus E proteins ion channels as targets for antiviral drugs.

Coronaviruses are divided into three groups, depending on the sequence homology and antigen cross-reactivity. Groups 1 and 2 contain the mammalian coronaviruses, and group 3 consists of the avian coronaviruses. All coronavirus groups encode E protein, a small, 9–12 kDa integral membrane protein. Although, there is little sequence homology between the coronavirus groups, all E proteins share structural homology, they all contain an N-terminus, which consists of a short 7–9 amino acid hydrophilic region, and a 21–29 amino acid hydrophobic transmembrane domain, followed by a hydrophilic C-terminal region. The exact functions and mechanisms of the coronavirus E proteins are yet to be established, although E proteins have been shown to be important for coronavirus replication, mediating viral assembly, and morphogenesis. Coronavirus E proteins share several characteristics with viral ion channels, which are small hydrophobic virus-encoded proteins. Virus ion channels have a highly hydrophobic domain that forms at least one amphipathic α-helix that oligomerizes to form an ion-conductive pore in membranes. Virus ion channels function to modify the cells permeability to ions and have been shown to mediate viral entry/exit or virus assembly and budding. The first identified viral ion channel, hence the best characterized, is the M2 protein encoded by influenza A. M2 forms proton selective ion channels that mediates viral uncoating and protects acid-sensitive hemagglutinin glycoprotein during transport to the cell surface. Although influenza B does not encode the M2 ion channel, it has been demonstrated to have two ion channel forming proteins, NB and BM2, whose role in viral replication are currently being investigated. Since the identification of the M2 ion channel, several other viruses have been demonstrated to encode viral ion channels. The HIV-1 accessory protein Vpu has also been shown to have ion channel activity, which mediates the release of viral particles from the plasma membrane. Most recently, the 6K proteins from the alphaviruses, Ross River Virus and Barmah Forest Virus, have been shown to form cation-selective ion channels in planar