Microbiology and Virology Research Group, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand1
Author for correspondence: John Taylor. Fax +64 9 373 7414. e-mail ja.taylor{at}auckland.ac.nz
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NSP4 has been identified as a viral enterotoxin based on its ability to promote diarrhoea in infant mice when added intra-peritoneally and intra-ileally (Ball et al., 1996 ; Horie et al., 1999
). The enterotoxic behaviour of NSP4 is mediated by its interaction with a putative plasma membrane receptor triggering a phospholipase C-mediated increase in intracellular Ca2+ leading to enhanced Cl- secretion in epithelial cells (Dong et al., 1997
; Morris et al., 1999
).
The cytopathic and enterotoxic activities of NSP4 would appear to arise through different mechanisms but neither are fully understood. Given the intracellular location of this glycoprotein [NSP4 is an integral membrane protein resident in the endoplasmic reticulum (ER)], it is also unclear how NSP4 might be released into the cell medium, a likely prerequisite for enterotoxic activity. A peptide derived from the cytoplasmic domain of NSP4 (amino acids 114135) has been shown to possess both enterotoxic activity and cause the permeabilization of liposomal membrane vesicles in vitro (Ball et al., 1996 ; Tian et al., 1996
). Membrane destabilization activity may account for the NSP4 cytopathic effect through disruption of the ER leading to an increase in cytoplasmic Ca2+ concentration (Tian et al., 1996
).
We have previously shown that a membrane-proximal domain of NSP4 that is rich in basic amino acid residues and distinct from the enterotoxic peptide sequence (Fig. 1) is important in mediating the cytopathic effects of NSP4 in MA104 cells (Newton et al., 1997
). Modelling this amino acid sequence (residues 5474) as an
-helix reveals the potential for a highly amphipathic structure to form in which five lysine residues are clustered on one face of the helix. Since cationic amphipathic helices are motifs common to many polypeptides with membrane-destabilizing properties (Epand et al., 1995
), we predicted that this region of NSP4 might mediate disruption of the ER membrane in vivo. A direct demonstration of membrane-destabilizing activity of NSP4 in mammalian cells is hampered by the intracellular location of the protein and thus relies on indirect methods such as measurement of Ca2+ release from the intracellular store. Addition of purified NSP4 to ER-derived membrane vesicles represents an alternative strategy but given the integral membrane status of the protein, its extraction and solubilization require the use of detergents that would effect the permeability of membrane vesicles subsequently incubated with the purified protein. In this study we have used inducible expression of NSP4 in E. coli to examine potential membrane destabilization and to map the region(s) within the protein that mediate this activity. Inducible expression in bacteria has proved a valid and useful system for the identification of polypeptides from animal viruses with membrane-destabilizing properties (Arroyo et al., 1995
; Carrasco, 1994
; Guinea & 1994
; Lama & Carrasco, 1992
; Sanz et al., 1994
). Furthermore, the lipid composition of the bacterial inner membrane is similar to the membrane of the ER, both of which lack appreciable quantities of cholesterol, a significant component of the plasma membrane of mammalian cells.
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Exponentially growing cultures of E. coli strain BL21(DE3) pLysS transformed with appropriate NSP4-expressing plasmids were induced by the addition of 1 mM IPTG. The host bacterial strain is a lysogen that inducibly expresses T7 RNA polymerase under the control of a lac promoter (Studier & Moffat, 1986 ). In addition, the host constitutively synthesizes T7 lysozyme, an inhibitor of T7 RNA polymerase activity, and thus the expression of potentially toxic gene products is tightly repressed in the uninduced state. Expression of NSP4 following induction was monitored by the labelling of polypeptides during induction with [35S]methionine, followed by SDSPAGE and autoradiographic analysis (Fig. 2
). Selective labelling of the plasmid-encoded gene products was achieved by addition of rifampicin to induced cultures 10 min prior to addition of the radiolabel. In each case, a new polypeptide species was synthesized whose apparent molecular mass was consistent with that anticipated for either the full-length NSP4 polypeptide (unglycosylated), or the relevant truncated variants (Fig. 2
). Expression levels varied considerably, with peptides representing soluble regions of the cytoplasmic domain (NSP486175 and NSP486139), accumulating to the highest levels, while peptides containing hydrophobic domains were poorly expressed.
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A second assay for membrane-destabilizing activity measured the release of [3H]uridine from the cells. Prior to induction, bacteria were grown in medium containing 5 µCi/ml [3H]uridine. Cells were pelleted, washed twice and then resuspended at the original density in medium lacking [3H]uridine. NSP4 expression was then induced by the addition of 1 mM IPTG and release of radioactivity into the medium used as an indicator of membrane integrity. This assay confirmed the potent membrane-destabilizing activity exhibited by NSP448139 and NSP448175. Synthesis of these variants released ~8090% of the label within 30 min. A much slower but sustained release of [3H]uridine was observed upon expression of both full-length NSP4 and NSP44891. The remaining proteins examined had little or no apparent effect on the permeability of the bacterial membrane.
The ability of the cytoplasmic tail to enhance the lytic potential of the minimal membrane-permeabilizing region located within resides 4891 is notable since neither NSP486175 nor NSP486139 affected the stability of the membrane, despite accumulating to much greater levels than any of the other variants. The cytoplasmic tail of NSP4 contains an -helical coiled-coil domain, located between residues 97137 (hatched box in Fig. 1
), which is proposed to mediate subunit oligomerization (Taylor et al., 1996
). Extension of the membrane-destabilizing peptide (NSP44891) at its C terminus to include this coiled-coil domain might facilitate the oligomerization of the resultant polypeptide and thus increase its membrane-destabilizing potential. Oligomerization of the amphipathic sequence might facilitate the formation of a pore or channel in the membrane, similar to other proteins known to alter the permeability of bacterial and eukaryotic membranes (Epand et al., 1995
). However the extremely low level of expression of NSP448139 and NSP448175 and the rapid lysis of cells expressing these polypeptides prevented their isolation and subsequent analysis of the oligomeric state.
A 22 amino acid synthetic peptide representing the enterotoxic peptide (residues 114135) has previously been reported to cause disruption of liposomes and microsomal membrane vesicles (Tian et al., 1996 ). However, in our experiments, membrane-destabilizing activity could not be attributed to the truncated NSP4 variants that contained the enterotoxic peptide sequence (unless the amphipathic region between residues 4891 was also included). This difference could be due to different conformations adopted by the 22 amino acid synthetic peptide and the recombinant fragments of NSP4 expressed in this study. However, it should be noted that results derived from the bacterial expression system cannot necessarily be equated with results obtained from liposome permeability studies, particularly when expression of the same 22 amino acid enterotoxic peptide was not directly tested in our experiments. Nevertheless, we have identified a distinct region within NSP4 with the potential to form an amphipathic structure and demonstrated here that recombinant peptides containing this sequence can mediate membrane permeabilization in E. coli.
Localized disruption of the ER membrane might affect the permeability of this organelle. Studies of Ca2+ homeostasis in Sf9 cells expressing NSP4 support this notion and demonstrate an increase in the rate of Ca2+ leakage from the ER in the presence of thapsigargin, which prevents refilling of the stores (Tian et al., 1995 ). A similar effect is observed following expression of the coxsackievirus protein 2B in HeLa cells (van Kuppeveld et al., 1997
). Analysis of mutant 2B proteins showed that both a region of sequence predicted to form a cationic
-helix and an adjacent hydrophobic domain were critical in mediating this effect. The similar effects that the rotavirus and coxsackievirus proteins have on the permeability of the ER membrane could reflect an underlying similarity in the organization of structural domains. For NSP4, the precise conformation adopted by the membrane-destabilization domain awaits further investigation.
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References |
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Received 10 February 2000;
accepted 18 April 2000.