Membrane Permeability Changes Induced inEscherichia coliby the SH Protein of Human Respiratory Syncytial Virus

Abstract

The small hydrophobic (SH) protein of human respiratory syncytial virus (HRSV) has been efficiently expressed in Escherichia coli. In analogy to small hydrophobic proteins encoded by other RNA viruses, membrane permeability changes to low-molecular-weight compounds were detected in bacteria expressing HRSV SH protein. These changes implied, at least, the entry of both the protein synthesis inhibitor hygromycin B and the beta-galactoside substrate o-nitrophenyl-beta-d-galactopyranoside and the exit of preloaded [3H]uridine from bacterial cells. Site-directed mutagenesis indicated that the C-terminal end of SH is needed for induction of membrane permeability changes. In addition, amino acid substitution at residue 32 (Ile to Lys) abolished that activity. This was correlated with a drastic increase in SH electrophoretic mobility and a decrease of the predicted values of alpha-helix for all residues of the SH transmembrane domain. Other sequence changes have either partial effect or no effect on the membrane permeability changes induced by the SH protein. However, none of the mutations abrogated the association of SH protein with bacterial membranes, indicating that incorporation of SH protein to membranes is not sufficient to induce the observed changes. Membrane permeability changes then might provide a useful test for the identification of key amino acid residues in this unique HRSV gene product.

Viroporins: structure, function and potential as antiviral targets

“…Thus, whilst clearly an indirect measure of channel activity, such indirect assays conducted upon multiple channels [e.g. IAV M2, HCV p7, classical swine fever virus (CSFV) p7, HPV E5, respiratory syncytial virus (RSV) SH and picornavirus 2B] have provided important insights into their activity as well as their inhibition by small molecules, with results generally consistent with those observed in culture (Agirre et al, 2002;Aldabe et al, 1996;Atkins et al, 2014;Carter et al, 2010;Gladue et al, 2012;Guinea & Carrasco, 1994;Lama & Carrasco, 1992;Perez et al, 1997;Sanz et al, 1994;StGelais et al, 2007;Wetherill et al, 2012;Wozniak et al, 2010).…”

“…This effect is highly likely to dysregulate cellular trafficking and the resultant surface expression of various proteins. Expression of M2 and p7 in isolation has also been shown to induce apoptosis via distinct mechanisms, although the relevance of this in the context of full infectious virus culture is currently unclear (Aweya et al, 2013 Lastly, as discussed above, expression of a variety of viroporins has been shown to induce generalized cellular permeability to a variety of small molecules, most notably hygromycin B, to which cells are otherwise impermeant (González & Carrasco, 1998;Guinea & Carrasco, 1994;Lama & Carrasco, 1992;Perez et al, 1997;Sanz et al, 1994). Whether or not such permeability has functional relevance to the virus life cycle, again this allows indirect assessment of viroporin function through hygromycin B effects on translation.…”

“…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.…”

Mumps virus small hydrophobic protein targets ataxin-1 ubiquitin-like interacting protein (ubiquilin 4)

“…A main feature of paramyxovirus SH proteins is their capacity to inhibit the TNF-a-mediated apoptosis pathway. Beyond that property, the SH protein of HRSV has been reported to act as an ion channel-forming viroporin as well (Kochva et al, 2003;Perez et al, 1997). Some viroporins have the capacity to suppress the induction of apoptosis by manipulating intracellular Ca 2+ homeostasis, as has been described for the 2B protein of coxsackievirus (Campanella et al, 2004).…”

“…Furthermore, a recombinant PIV5, in which the original SH has been replaced by that of HRSV or MuV, was able to block TNF-a-induced apoptosis (Fuentes et al, 2007;Wilson et al, 2006). In addition, the SH protein of HRSV has been predicted to act as a viroporin (Perez et al, 1997;Kochva et al, 2003). Viroporins are a group of highly hydrophobic small viral proteins that are able to homo-oligomerize and induce membrane permeability (Gonzalez & Carrasco, 2003).…”