Structure and ion channel activity of the human respiratory syncytial virus (hRSV) small hydrophobic protein transmembrane domain

Abstract

The small hydrophobic (SH) protein from the human respiratory syncytial virus (hRSV) is a glycoprotein of ;64 amino acids with one putative a-helical transmembrane domain. Although SH protein is important for viral infectivity, its exact role during viral infection is not clear. Herein, we have studied the secondary structure, orientation, and oligomerization of the transmembrane domain of SH (SH-TM) in the presence of lipid bilayers. Only one oligomer, a pentamer, was observed in PFO-PAGE. Using polarized attenuated total reflection-Fourier transform infrared (PATR-FTIR) spectroscopy, we show that the SH-TM is a-helical. The rotational orientation of SH-TM was determined by site-specific infrared dichroism (SSID) at two consecutive isotopically labeled residues. This orientation is consistent with that of an evolutionary conserved pentameric model obtained from a global search protocol using 13 homologous sequences of RSV. Conductance studies of SH-TM indicate ion channel activity, which is cation selective, and inactive below the predicted pK a of histidine. Thus, our results provide experimental evidence that the transmembrane domain of SH protein forms pentameric a-helical bundles that form cation-selective ion channels in planar lipid bilayers. We provide a model for this pore, which should be useful in mutagenesis studies to elucidate its role during the virus cycle.

Viroporins: structure, function and potential as antiviral targets

“…SH is commonly thought to form pentameric oligomers (Collins & Mottet, 1993;Gan et al, 2008Gan et al, , 2012, although hexamers have also been reported (Carter et al, 2010). Solution NMR structures have been reported for the pentameric bundles, yet have not been added to the PDB (Gan et al, 2008(Gan et al, , 2012.…”

“…SH is commonly thought to form pentameric oligomers (Collins & Mottet, 1993;Gan et al, 2008Gan et al, , 2012, although hexamers have also been reported (Carter et al, 2010). Solution NMR structures have been reported for the pentameric bundles, yet have not been added to the PDB (Gan et al, 2008(Gan et al, , 2012. Both SH TMD peptides and full-length protein form cation-selective channels in vitro (Gan et al, 2008), as well as promoting bacterial membrane permeability (Perez et al, 1997) and mediating dye release from liposomes (Carter et al, 2010).…”

“…Solution NMR structures have been reported for the pentameric bundles, yet have not been added to the PDB (Gan et al, 2008(Gan et al, , 2012. Both SH TMD peptides and full-length protein form cation-selective channels in vitro (Gan et al, 2008), as well as promoting bacterial membrane permeability (Perez et al, 1997) and mediating dye release from liposomes (Carter et al, 2010). The effect of low pH upon channel opening appears to be context dependent, with conserved His22, His51 and Trp15 residues implicated in channel gating/opening.…”

“…The effect of low pH upon channel opening appears to be context dependent, with conserved His22, His51 and Trp15 residues implicated in channel gating/opening. However, deletion of both His residues is required to generate non-functional channels and it remains unclear as to the precise effect of pH upon channel opening (Gan et al, 2008(Gan et al, , 2012. Recently, pyronin B was identified as an inhibitor of SH activity in liposome dye-release assays, suspended bilayers and RSV spread in culture .…”

Pentameric viral ion channels: from structure to function

“…68,82 However, both synthetic TM domain (residues 18-43) and full-length SH protein have been shown to form homopentamers in a variety of detergents. 75,83 These oligomers may have been responsible for early reports showing increased entry in bacteria of low molecular weight compounds after SH protein expression. Abbreviations: SARS-Cov, severe acute respiratory syndrome coronavirus; SH, small hydrophobic; HMA, hexamethylene amiloride; TROSY-HSQC, transverse relaxationoptimized spectroscopy-heteronuclear single quantum coherence.…”