4wb4
wt SA11 NSP4_CCD
Structural highlights
FunctionO92323_9VIRU Plays an essential role in the virus replication cycle by acting as a viroporin. Creates a pore in the host reticulum endoplasmic and as a consequence releases Ca(2+) in the cytoplasm of infected cell. In turn, high levels of cytoplasmic calcium trigger membrane trafficking and transport of viral ER-associated proteins to viroplasms, sites of viral genome replication and immature particle assembly.[HAMAP-Rule:MF_04091] The secreted form acts as an enterotoxin that causes phospholipase C-dependent elevation of the intracellular calcium concentration in host intestinal mucosa cells. Increased concentration of intracellular calcium disrupts the cytoskeleton and the tight junctions, raising the paracellular permeability. Potentiates chloride ion secretion through a calcium ion-dependent signaling pathway, inducing age-dependent diarrhea. To perform this enterotoxigenic role in vivo, NSP4 is released from infected enterocytes in a soluble form capable of diffusing within the intestinal lumen and interacting with host plasma membrane receptors on neighboring epithelial cells such as integrins ITGA1/ITGB1 and ITGA2/ITGB1.[HAMAP-Rule:MF_04091] Publication Abstract from PubMedRotavirus (RV) nonstructural protein 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 SA11 NSP4 forms a tetramer that binds Ca2+ at its core, the CCD of a human strain ST3 forms a pentamer lacking the bound Ca2+ despite the residues (E120 and Q123) that coordinate Ca2+-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 Ca2+-binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca2+ or pH. Biochemical, biophysical and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high affinity binding to Ca2+ at neutral pH and forms a tetramer, at low pH, it does not bind Ca2+ and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca2+ 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 Ca2+ may form a basis for its pleiotropic functions during RV replication. IMPORTANCE: The non-structural 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 Ca2+-bound tetrameric state and a Ca2+-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 Ca2+-bound tetramer to a Ca2+-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. Structural Plasticity of the Coiled-coil Domain of Rotavirus NSP4.,Sastri NP, Viskovska M, Hyser JM, Tanner MR, Horton LB, Sankaran B, Prasad BV, Estes MK J Virol. 2014 Sep 17. pii: JVI.02227-14. PMID:25231315[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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