Person:

Chou, James

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

The Hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations1-4. We are curious as to what structural solution has the viroporin adopted to afford selective cation conduction because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The p7 activity can be inhibited by amantadine and rimantadine2,5, which also happen to be potent blockers of the influenza M2 channel6 and licensed drugs against influenza infections7. The adamantane derivatives were subjects of HCV clinical trials8, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here, we determined the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbors, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also alludes to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter while an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording showed that these residues are indeed critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Mitochondria from multiple, eukaryotic clades uptake and buffer large amounts of calcium (Ca2+) via an inner membrane transporter called the uniporter. Early studies demonstrated that this transport requires a mitochondrial membrane potential and that the uniporter is itself Ca2+ activated, and blocked by ruthenium red or Ru3601. Later, electrophysiological studies demonstrated that the uniporter is an ion channel with remarkably high conductance and selectivity2. Ca2+ entry into mitochondria is also known to activate the TCA cycle and appears to be critical for matching ATP production in mitochondria with its cytosolic demand3. MCU (mitochondrial calcium uniporter) is the pore forming and Ca2+ conducting subunit of the uniporter, but its primary sequence does not resemble any calcium channel known to date. Here, we report the structure of the core region of MCU, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer with the second transmembrane helix forming a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture and is stabilized by a coiled coil motif protruding in the mitochondrial matrix. The critical DxxE motif forms the pore entrance featuring two carboxylate rings, which appear to be the selectivity filter based on the ring dimensions and functional mutagenesis. To our knowledge, this is one of the largest structures characterized by NMR, which provides a structural blueprint for understanding the function of this channel.

The membrane protein M2 from influenza-A forms a single-pass transmembrane helix that assembles in lipid membrane as homotetramers whose primary function is to act as a proton transporter for viral acidification. A single residue, histidine 37 (His 37), is known to be responsible for selectivity and plays an integral role in the protein’s function. We report pH-dependent 15N MAS NMR spectra of His 37 within the influenza-A proton conduction domain of M2, M218–60, which has been previously shown to be a fully functional construct and was recently determined to adopt a dimer-of-dimers structure in lipids. By extracting the ratio of [His]/[HisH+] as a function of pH, we obtained two doubly degenerate proton disassociation constants, 7.63 ± 0.15 and 4.52 ± 0.15, despite a possible maximum of four. We also report the 1HNε chemical shifts at pH 6.5 recorded at 60 kHz MAS in a CP-based 1H–15N spectrum. We were unable to detect resonances indicative of direct proton sharing among His 37 side chains when the tetramer is in the +2 state. In the neutral state, His 37 is exclusively in the τ tautomer, indicating that the δ nitrogen is protonated solely as a function of pH. We also found that the plot of [HisH+]/[His] as a function of pH is qualitatively similar to previously reported proton conduction rates, indicating that proton conduction rate is proportional to the level of histidine protonation within the channel. Two-dimensional 13C–13C and 13C–15N correlations suggest that at low pH multiple conformations are populated as the spectra broaden and eventually disappear as the acidity is increased. A second highly resolved state at low pH was not observed.

Nuclear magnetic resonance (NMR) has been an important source of structural restraints for solving structures of oligomeric transmembrane domains (TMDs) of cell surface receptors and viral membrane proteins. In NMR studies, oligomers are assembled using inter-protomer distance restraints. But, for oligomers that are higher than dimer, these distance restraints all have two-fold directional ambiguity, and resolving such ambiguity often requires time-consuming trial-and-error calculations using restrained molecular dynamics (MD) with simulated annealing (SA). We report an Exhaustive Search algorithm for Symmetric Oligomer (ExSSO), which can perform near-complete search of the symmetric conformational space in a very short time. In this approach, the predetermined protomer model is subject to full angular and spatial search within the symmetry space. This approach, which can be applied to any rotationally symmetric oligomers, was validated using the structures of the Fas death receptor, the HIV-1 gp41 fusion protein, the influenza proton channel, and the MCU pore. The algorithm is able to generate approximate oligomer solutions quickly as initial inputs for further refinement using the MD/SA method.