Genetic Characterization of the Rotavirus Nonstructural Protein, NSP4

Kirkwood, Carl D.; Palombo, Enzo A.

doi:10.1006/viro.1997.8727

Cited by 71 publications

(51 citation statements)

References 25 publications

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“…The protease(s) responsible for the cleavage between aa 111 and aa 112 is not yet clear. Over 100 sequences for NSP4 have been determined (7,10,20,24,25), and a comparison of NSP4 sequences from available rotavirus strains shows that sequences at aa 111 (mainly E; a few D, A, or T; and rarely R and K residues), 112 (M), 113 (I), and 114 (D, E) are highly conserved. However, no known protease recognizes these specific amino acids, and so the responsible protease may cleave in a sequence-independent manner.…”

Section: Discussionmentioning

confidence: 99%

A Functional NSP4 Enterotoxin Peptide Secreted from Rotavirus-Infected Cells

Zhang

Zeng

Morris³

et al. 2000

J Virol

153

120

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Previous studies have shown that the nonstructural glycoprotein NSP4 plays a role in rotavirus pathogenesis by functioning as an enterotoxin. One prediction of the mechanism of action of this enterotoxin was that it is secreted from virus-infected cells. In this study, the media of cultured (i) insect cells infected with a recombinant baculovirus expressing NSP4, (ii) monkey kidney (MA104) cells infected with the simian (SA11) or porcine attenuated (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisWestern blotting, immunoprecipitation and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparent molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 aa112-175). The secretion of NSP4 aa112-175 was not affected by treatment of cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network. A partial gene fragment coding for NSP4 aa112-175 was cloned and expressed using the baculovirus-insect cell system. Purified NSP4 aa112-175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expressed endogenously, similarly to full-length NSP4. NSP4 aa112-175 caused diarrhea in neonatal mice, as did full-length NSP4. These results indicate that NSP4 aa112-175 is a functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.Rotaviruses are major pathogens causing life-threatening dehydrating gastroenteritis in young children and animals. Despite extensive studies of different animal models, rotavirus pathogenesis remains incompletely understood. A nonstructural protein, NSP4, encoded by rotavirus genome segment 10, is a transmembrane, endoplasmic reticulum (ER)-specific glycoprotein with pleotropic functions in viral replication and pathogenesis (15). NSP4 serves as an intracellular receptor for newly made double-layered particles and interacts with viral capsid proteins during viral morphogenesis (1). NSP4 has been shown previously to be an enterotoxin that causes diarrhea in mouse pups, suggesting a role for NSP4 in rotavirus pathogenesis (3, 21). Mutations in NSP4 have also been associated with altered virus virulence by comparing the sequences and biological activities of NSP4 from two pairs of virulent and avirulent porcine rotaviruses, thus supporting a role for NSP4 in rotavirus pathogenesis (46). Increasing evidence indicates that this enterotoxin functions to activate a signal transduction pathway that increases intracellular calcium levels in cells by mobilizing calcium from the ER and ultimately resulting in chloride secretion (3,11,33,38,39). Recent studies have shown that NSP4 induces diarrhea by activating an ...

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Section: Discussionmentioning

confidence: 99%

A Functional NSP4 Enterotoxin Peptide Secreted from Rotavirus-Infected Cells

Zhang

Zeng

Morris³

et al. 2000

J Virol

153

120

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“…Sequence alignments of NSP4 genes from human and animal rotavirus strains (14) indicate that the C-terminal 20 amino acids are reasonably well conserved, and circular dichroism spectroscopy has demonstrated the presence of a high proportion of random conformation in this region of the protein (26). A number of potential trypsin and protease V8 cleavage sites are contained within the upstream ␣-helical region (Fig.…”

Section: Figmentioning

confidence: 99%

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

O’Brien

Taylor

Bellamy

2000

J Virol

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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 ...

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“…The non-structural glycoprotein, NSP4, plays an important role in rotavirus morphogenesis, pathogenesis, and enterotoxic activity. Sequence analyses of the NSP4 genes revealed the presence of at least six distinct NSP4 genetic groups among human and animal rotaviruses, termed genetic groups A-F [Horie et al, 1997;Kirkwood and Palombo, 1997;Ciarlet et al, 2000;Mori et al, 2002;Khamrin et al, 2008]. Up to now, the precise role(s) of the NSP5/6 proteins encoded by gene 11 has only been partially characterized.…”

Section: Introductionmentioning

confidence: 99%

Molecular characterization of VP4, VP6, VP7, NSP4, and NSP5/6 genes identifies an unusual G3P[10] human rotavirus strain

Khamrin

Maneekarn

Peerakome

et al. 2008

Journal of Medical Virology

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An unusual strain of human rotavirus G3P [10] (CMH079/05) was detected in a stool sample of a 2-year-old child admitted to the hospital with severe diarrhea in Chiang Mai, Thailand. Analysis of the VP7 gene sequence revealed highest identities with unusual human rotavirus G3 strain CMH222 at 98.7% on the nucleotide and 99.6% on the amino acid levels. Phylogenetic analysis of the VP7 sequence confirmed that the CMH079/05 strain formed a cluster with G3 rotavirus reference strains and showed the closest lineage with the CMH222 strain. Analysis of partial VP4 gene of CMH079/05 revealed highest degree of sequence identities with P[10] rotavirus prototype strain 69M at nucleotide and amino acid levels of 92.9% and 94.6%, respectively. Phylogenetic analysis of the VP4 sequence revealed that CMH079/05 and 69M clustered closely together in a monophyletic branch separated from other rotavirus genotypes. To our knowledge, this is a novel G-P combination of G3 and P[10] genotypes. In addition, analyses of VP6, NSP4, and NSP5/6 genes revealed these uncommon genetic characteristics: (i) the VP6 gene differed from the four other known subgroups; (ii) the NSP4 gene was identified as NSP4 genetic group C, an uncommon group in humans; and (iii) the NSP5/6 gene was most closely related with T152, a G12P[9] rotavirus previously isolated in Thailand. The finding of uncommon G3P [10] rotavirus in this pediatric patient provided additional evidence of the genetic diversity of human group A rotaviruses in Chiang Mai, Thailand.

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Genetic Characterization of the Rotavirus Nonstructural Protein, NSP4

Cited by 71 publications

References 25 publications

A Functional NSP4 Enterotoxin Peptide Secreted from Rotavirus-Infected Cells

A Functional NSP4 Enterotoxin Peptide Secreted from Rotavirus-Infected Cells

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

Molecular characterization of VP4, VP6, VP7, NSP4, and NSP5/6 genes identifies an unusual G3P[10] human rotavirus strain

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