Inhibition of the Human Respiratory Syncytial Virus Small Hydrophobic Protein and Structural Variations in a Bicelle Environment

Abstract

The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single ␣-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the ␣-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. IMPORTANCEThe human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.

Pentameric viral ion channels: from structure to function

“…Also, His residues are not required for channel activity, as the channel was active even when both His residues were changed to Phe. 84 These results are consistent with a dramatic reduction of channel activity at lower pH observed previously for synthetic SH-TM (residues 18-43) in planar lipid bilayers, 83 where only His22 was present. Overall, BLM results showed only minor variations in conductance and selectivity with pH.…”

“…84 Although modest, this K d compares favorably with the 16 µM for amantadine inhibition of influenza M2 protein, 86 the ∼10 µM reported for HMA inhibition of SARS-CoV E, 52 50-100 µM for rimantadine inhibition of HCV p7, 50 or the .100 µM of HMA for p7 inhibition. 85 SH protein conductance changed from 0.3±0.07 nS in control conditions to 0.12±0.06 nS Figure 5 Common features in SARS-Cov e and RSv SH channels.…”

“…Overall, BLM results showed only minor variations in conductance and selectivity with pH. 84 Similarly, ion selectivity was low, and appeared modulated by His51, with His22 contributing a more structural role. This is consistent with the observed electrophoretic pattern of His mutants; 75 mutant H22A showed aberrant mobility and/or aggregation, whereas H51A showed mobility similar to the WT form.…”

“…Later, the structure of these extramembrane domains has been confirmed using a DHPC/DLPC (1,2-Dihexanoylsn-Glycero-3-Phosphocholine/1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) bicellar system. 84 However, in that "bicelle" model, the TM α-helix was extended one more turn, until the other His residue, His51, whereas Pro-58 is located at the tip of a C-terminal loop ( Figure 3C). …”

Viroporins: structure, function and potential as antiviral targets

“…Conductance of such molecules may, at first glance, argue against selective channel properties, and is likely indicative of channel-pore dualism and the plasticity inherent to viroporin channel structures. However, indirect substrates Li et al (2014) often possess relatively small Stokes radii (e.g. 0.4-0.6 nm for carboxyfluorescein), consistent with their being able to pass through the lumenal apertures of many viroporins, based upon structural data and/or computer models.…”