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Lin, Shuo; Tian, Peng

doi:10.1023/a:1024451314534

Cited by 33 publications

(4 citation statements)

References 28 publications

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“…In our structure as well as in the structure determined by Chacko et al (14), Ca 2ϩ is coordinated entirely by the side chain oxygen atoms of E120 and Q123 despite slightly altered side chain conformations. The studies by Bowman et al (13) The E120 and Q123 residues of the NSP4 CCD that coordinate Ca 2ϩ binding are completely conserved among the NSP4 sequences of group A rotaviruses (35). In addition, the CCD itself, with the essential signatures of a heptad repeat, is the most conserved region of NSP4.…”

Section: Discussionmentioning

confidence: 99%

Structural Plasticity of the Coiled-Coil Domain of Rotavirus NSP4

Sastri

Viskovska

Hyser

et al. 2014

J Virol

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Rotavirus (RV) nonstructural protein 4 (NSP4) is a virulence factor that disrupts cellular Ca2؉ homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian strain SA11 NSP4 forms a tetramer that binds Ca 2؉ at its core, the CCD of human strain ST3 forms a pentamer lacking the bound Ca 2؉ despite the residues (E120 and Q123) that coordinate Ca 2؉ binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH, suggesting that preference for a particular oligomeric state is pH dependent and that pH could influence Ca 2؉ binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca 2؉ or pH. Biochemical, biophysical, and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high-affinity binding to Ca 2؉ at neutral pH and forms a tetramer, it does not bind Ca 2؉ at low pH and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca 2؉ binding is necessary for the tetramer formation, as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca 2؉ may form a basis for its pleiotropic functions during RV replication. IMPORTANCEThe nonstructural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis, and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states, a Ca 2؉ -bound tetrameric state and a Ca 2؉ -free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical, and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca 2؉ -bound tetramer to a Ca 2؉ -free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4.

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Section: Discussionmentioning

confidence: 99%

Structural Plasticity of the Coiled-Coil Domain of Rotavirus NSP4

Sastri

Viskovska

Hyser

et al. 2014

J Virol

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“…Recent studies from our laboratory [3, 6] also suggested that sequence variations observed in the flexible C-terminus compared to the other regions of NSP4s in different strains [33] (Fig. 1B ) would affect conformation, ThT binding and biological properties of the recombinant protein.…”

Section: Discussionmentioning

confidence: 99%

“…Rotaviruses can be either symptomatic or asymptomatic, and in some cases NSP4 and a few other viral proteins could cooperatively determine virus virulence [25-32]. Analysis of NSP4 sequences from more than 175 strains failed to identify any residue or motif that could be associated with the virulence phenotype [33]. Mutations at positions 131, 135 and 138 which were reported to result in loss of a porcine rotavirus virulence and diarrhea-inducing ability of the protein [34], did not correlate with the attenuated phenotype of a vaccine strain [32] or the asymptomatic human or feline strains [35-37].…”

Section: Introductionmentioning

confidence: 99%

Conformational Differences Unfold a Wide Range of Enterotoxigenic Abilities Exhibited by rNSP4 Peptides from Different Rotavirus Strains

Sastri¹,

Pamidimukkala²,

Marathahalli³

et al. 2011

TOVJ

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NSP4 has been recognized as the rotavirus-encoded enterotoxin. However, a few studies failed to support its diarrheagenic activity. As recombinant NSP4 (rNSP4) peptides of different lengths were used in the limited number of studies, a comparison of relative diarrheagenic potential of NSP4 from different strains could not be possible. To better understand the diarrheagenic potential of NSP4 from different strains, in this report we have evaluated the enterotoxigenic activity of the deletion mutant ΔN72 that lacks the N-terminal 72 residues and the biologically relevant ΔN112 peptide which when derived from SA11 rotavirus strain were previously shown to be highly diarrheagenic in newborn mice. Detailed comparative analysis of biochemical and biophysical properties and diarrheagenic activity of the recombinant ΔN72 peptides from seventeen different strains under identical conditions revealed wide differences among themselves in their resistance to trypsin cleavage, thioflavin T (ThT) binding, multimerization and conformation without any correlation with their diarrhea inducing abilities. These results support our previously proposed concept for the requirement of a unique conformation for optimal biological functions conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail.

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“…Rotavirus infections can be symptomatic or asymptomatic depending on the virus and the host. However, an analysis of NSP4 sequences from a large number of symptomatic and asymptomatic strains did not reveal any amino acid or motif that is consistently associated with the virulence of the virus in the animal host or the diarrhoea-inducing ability of the purified protein in the newborn mouse model system (Lin & Tian, 2003;Jagannath et al, 2000). Significantly, the region between amino acids 135 and 141 exhibits a high degree of sequence variation and mutations in this interspecies-variable domain (ISVD) have been observed to affect the virulence of the virus as well as the diarrhoea-inducing and double-layered particlebinding activities of the protein (Zhang et al, 1998;Jagannath et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

A new pentameric structure of rotavirus NSP4 revealed by molecular replacement

Chacko

Jeyakanthan

Ueno

et al. 2011

Acta Crystallogr D Biol Crystallogr

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The region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 has been purified and crystallized and diffraction data have been collected to a resolution of 2.6 Å. Several attempts to solve the structure by the molecular-replacement method using the available tetrameric structures of this domain were unsuccessful despite a sequence identity of 73% to the already known structures. A more systematic approach with a dimer as the search model led to an unexpected pentameric structure using the program Phaser. The various steps involved in arriving at this molecular-replacement solution, which unravelled a case of subtle variation between different oligomeric states unknown at the time of solving the structure, are presented in this paper.

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Cited by 33 publications

References 28 publications

Structural Plasticity of the Coiled-Coil Domain of Rotavirus NSP4

Structural Plasticity of the Coiled-Coil Domain of Rotavirus NSP4

Conformational Differences Unfold a Wide Range of Enterotoxigenic Abilities Exhibited by rNSP4 Peptides from Different Rotavirus Strains

A new pentameric structure of rotavirus NSP4 revealed by molecular replacement

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