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.

HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We use NMR to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An N-terminal coiled-coil and a C-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design.

Structural characterization of transmembrane proteins in isotropic bicelles has become an increasingly popular application of solution NMR spectroscopy, as the fast-tumbling bicelles are membrane-like yet can often yield spectral quality comparable to those of detergent micelles. While larger bicelles are closer to the true lipid bilayer, it remains unclear how large the bicelles need to be to allow accurate assessment of protein transmembrane partition in lipid bilayer. Here, we address the above question from the perspective of protein residing in the bicelles, through systematic measurement of protein chemical shift and transmembrane partition at different lipid:detergent ratios (q), ranging from 0.3 to 0.7, using the transmembrane domain of human Fas receptor as model system. We found that the lipid environment of the bicelles, as reflected by the protein chemical shift, begins to be perturbed when the q is reduced to below 0.6. We also implemented a solvent paramagnetic relaxation enhancement (PRE) approach for bicelles to show that the protein transmembrane partition in bicelles with q = 0.5 and 0.7 are very similar, but at q = 0.3 the solvent PRE profile is significantly different. Our data indicate that q values between 0.5 and 0.6 are good compromise between high resolution NMR and closeness to the membrane environment, and allow accurate characterization of protein position in lipid bilayer.

HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. Recent studies showed that the transmembrane domain (TMD) of the Env forms a trimer in lipid bilayer and that disruption of the TMD could significantly alter the antigenic properties of the Env. The TMD structure has several peculiar features that remain difficult to explain. One is the presence of an arginine R696 (three in the trimer) in the middle of the TM helix. Additionally, the N- and C-terminal halves of the TM helix form trimeric cores of opposite nature (hydrophobic for the N half and hydrophilic for the C half). Here we determined the membrane partition and solvent accessibility of the TMD in bicelles that mimic a lipid bilayer. Solvent paramagnetic relaxation enhancement analysis showed that the R696 is indeed positioned close to the center of the bilayer, but, surprisingly, can exchange rapidly with water as indicated by hydrogen-deuterium exchange measurements. The solvent accessibility of R696 is likely mediated by the hydrophilic core, which also showed fast water exchange. In contrast, the N-terminal hydrophobic core showed extremely slow solvent exchange, suggesting the trimer formed by this region is extraordinarily stable. Our data explain how R696 is accommodated in the middle of the membrane while reporting the overall stability of the Env TMD trimer in lipid bilayer.

The p7 protein of the hepatitis C virus (HCV) can oligomerize in membrane to form cation channels. Previous studies showed that the channel assembly in detergent micelles adopts a unique flower-shaped oligomer, but the unusual architecture also presented problems for understanding how this viroporin resides in the membrane. Moreover, the oligomeric state of p7 remains controversial, as both hexamer and heptamer have been proposed. Here we address the above issues using p7 reconstituted in bicelles that mimic a lipid bilayer. We found, using a recently developed oligomer-labeling method, that p7 forms hexamers in the bicelles. Solvent paramagnetic relaxation enhancement analyses showed that the bilayer thickness around the HCV ion channel is substantially smaller than expected, and thus a significant portion of the previously assigned membrane-embedded region is solvent exposed. Our study provides an effective approach for characterizing the transmembrane partition of small ion channels in near lipid bilayer environment.

Tumor selective, replication competent viruses are being tested for cancer gene therapy. This approach introduces a new therapeutic paradigm due to potential replication of the therapeutic agent and induction of a tumor-specific immune response. However, the experimental outcomes are quite variable, even when studies utilize highly inbred strains of mice and the same cell line and virus. Recognizing that virotherapy is an exercise in population dynamics, we utilize mathematical modeling to understand the variable outcomes observed when B16ova malignant melanoma tumors are treated with vesicular stomatitis virus in syngeneic, fully immunocompetent mice. We show how variability in the initial tumor size and the actual amount of virus delivered to the tumor have critical roles on the outcome of therapy. Virotherapy works best when tumors are small, and a robust innate immune response can lead to superior tumor control. Strategies that reduce tumor burden without suppressing the immune response and methods that maximize the amount of virus delivered to the tumor should optimize tumor control in this model system.

The p7 membrane protein encoded by Hepatitis C virus (HCV) assembles into a homo-hexamer that selectively conducts cations. An earlier solution NMR structure of the hexameric complex revealed a funnel-like architecture and suggests that a ring of conserved asparagines near the narrow end of the funnel are important for cation interaction. NMR based drug-binding experiments also suggest that rimantadine can allosterically inhibit ion conduction via a molecular wedge mechanism. These results suggest the presence of dilation and contraction of the funnel tip that are important for channel activity and that the action of the drug is attenuating this motion. Here, we determined the conformational dynamics and solvent accessibility of the p7 channel. The proton exchange measurements show that the cavity-lining residues are largely water accessible, consistent with the overall funnel shape of the channel. Our relaxation dispersion data show that residues Val7 and Leu8 near the asparagine ring are subject to large chemical exchange, suggesting significant intrinsic channel breathing at the tip of the funnel. Moreover, the hinge regions connecting the narrow and wide regions of the funnel show strong relaxation dispersion and these regions are the binding sites for rimantadine. Presence of rimantadine deceases the conformational dynamics near the asparagine ring and the hinge area. Our data provide direct observation of µs – ms dynamics of the p7 channel and support the molecular wedge mechanism of rimantadine inhibition of the HCV p7 channel.

No abstract