Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle

Abstract

The rotavirus nonstructural glycoprotein NSP4 functions as the receptor for the inner capsid particle (ICP) which buds into the lumen of the endoplasmic reticulum during virus maturation. The structure of the cytoplasmic domain of NSP4 from rotavirus strain SA11 has been investigated by using limited proteolysis and mass spectrometry. Digestion with trypsin and V8 protease reveals a C-terminal protease-sensitive region that is 28 amino acids long. The minimal sequence requirements for receptor function have been defined by constructing fusions with glutathione S-transferase and assessing their ability to bind ICPs. These experiments demonstrate that 17 to 20 amino acids from the extreme C terminus are necessary and sufficient for ICP binding and that this binding is cooperative. These observations are consistent with a model for the structure of the NSP4 cytoplasmic region in which four flexible regions of 28 amino acids are presented by a proteaseresistant coiled-coil tetramerization domain, with only the last ϳ20 amino acids of each peptide interacting with the surface binding sites on the ICP.Rotavirus is the single most important cause of severe, dehydrating diarrhea in young children worldwide and accounts for as many as 125 pediatric deaths per year in the United States and 873,000 childhood deaths each year in developing countries (10). The mechanism by which rotavirus induces intestinal secretion of fluid and electrolytes is unknown, but proposals include destruction of enterocytes in the small intestine (11), alterations in transepithelial fluid balance (5), a toxin-like effect by a viral nonstructural protein, NSP4 (3), or activation of the enteric nervous system (15). The virus particle consists of a three-layer protein capsid that contains a segmented, double-stranded RNA genome. The assembly of mature rotavirus involves a unique budding process in which cytoplasmically assembled immature virus particles bud into the lumen of the rough endoplasmic reticulum (ER). The transfer is mediated by an interaction between the double-layered rotavirus inner capsid particle (ICP; approximate molecular mass, 5 ϫ 10 7 Da) and the cytoplasmic tail of NSP4 (1,2,19,24). This region of NSP4 contains a putative coiled-coil stem structure and a C-terminal receptor domain that is sensitive to proteolysis (26), but the precise size of the domain that interacts with the ICP and the number of interactions involved remain to be established.Here, we have used a combination of limited proteolysis and mass spectrometry to probe the structure of the C-terminal region of NSP4. The minimal receptor domain and characteristics of the NSP4-ICP interaction have been defined by using site-directed mutagenesis and quantitative analysis of binding kinetics. The results demonstrate that 28 residues form an exposed C-terminal domain, of which the last 17 to 20 (amino acids 156 to 175) are required for binding of the ICP to the receptor. In solid-phase binding assays that reflect some aspects of the in vivo interaction, multiple receptors ...

The flexible C terminus of the rotavirus non-structural protein NSP4 is an important determinant of its biological properties

“…The highly flexible and protease-sensitive nature of the C terminus (Deepa et al, 2007;Jagannath et al, 2006;O'Brien et al, 2000) and the multimerizationpromoting activity of the N-terminal helical regions from aa 52 to 85 (Jagannath et al, 2006;Newton et al, 1997) appear to impede crystallization of the complete CT (Deepa et al, 2007). The longest region that could be crystallized and for which the structure has been determined corresponds to aa 95-146 from the SA11 and I321 strains, which conclusively showed that the N-terminal region from aa 95 to 137 exists as tetrameric CCD and that the region C-terminal to aa 137 is highly flexible, and no structure could be assigned (Deepa et al, 2007).…”

“…It has been presumed that the C-terminal boundary lies around aa 135 or 138, as the synthetic peptide of aa 114-135 induced diarrhoea and mutations at positions 135 and 138 in the ISVD resulted in loss of virus virulence and diarrhoea-inducing ability of the protein (Zhang et al, 1998). Furthermore, the region C-terminal to the CCD exhibits a high degree of sequence variation compared with the other regions of the protein without correlation to virus virulence, and is highly flexible and susceptible to proteases due to its extended conformation (Jagannath et al, 2000(Jagannath et al, , 2006O'Brien et al, 2000). Hence, no role in diarrhoea induction has been considered.…”

“…The region of 20 aa from the C terminus (Fig. 1a) in the context of the full-length protein or in fusion with glutathione S-transferase (GST) appears to be sufficient for DLP binding (Au et al, 1993;O'Brien et al, 2000;Taylor et al, 1993).…”

Viroporins: structure, function and potential as antiviral targets

“…NSP4 (175 aa) is subdivided into an N-terminal helical domain, a coiled-coil region (aa 95-146) for which both tetrameric and pentameric crystal structures have been solved (Bowman et al, 2000;Chacko et al, 2011Chacko et al, , 2012aDeepa et al, 2007;Sastri et al, 2014), and a Cterminal double-layered particle receptor domain, which is essential for the assembly and egress of rotavirus capsids (O'Brien et al, 2000). Viroporin activity has recently been shown to exist for the N-terminal portion of the protein, which contains multiple predicted helical domains (Hyser et al, 2010(Hyser et al, , 2012.…”

Elisas for Rotavirus Diagnosis, Typing, and Analysis of Antibody Response

“…Upon fusion to the carboxy terminus of glutathione-S-transferase (GST), the C-terminal 20 amino acids of NSP4 were identified as sufficient for DLP-binding, however a C-terminal peptide failed to inhibit the receptor activity of the full-length protein, indicating that DLP binding may somehow depend also on other parts of the protein (Au et al, 1993;O'Brien et al, 2000;Taylor et al, 1993). The N-terminus of NSP4 is relevant for DLP binding, since a deletion mutant lacking the N-terminal 85 amino acids is severely affected in this function.…”