N- and C-Terminal Cooperation in Rotavirus Enterotoxin: Novel Mechanism of Modulation of the Properties of a Multifunctional Protein by a Structurally and Functionally Overlapping Conformational Domain

Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic α-helix located between aa 73 and 85 (AAH(73-85)) at the N terminus of ΔN72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH(73-85), or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis

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

The rotavirus non-structural protein NSP4 functions as the viral enterotoxin and intracellular receptor for the double-layered particles (DLP). The full-length protein cannot be expressed and/or purified to homogeneity from bacterial or insect cells. However, a bacterially expressed and purified mutant lacking the N-terminal 72 aa(\triangle N72) was recently obtained from strains Hg18 and SA11 exhibiting approximately 17–20-, 150–200- and 13166–15800-fold lower $DD_{50}$ (50% diarrhoea-inducing dose) values in suckling mice compared with that reported for the partially pure, full-length protein, a C-terminal M175I mutant and a synthetic peptide comprising aa 114– 135, respectively, suggesting the requirement for a unique conformation for optimal functions of the purified protein. The stretch of approximately 40 aa from the C terminus of the cytoplasmic tail of the endoplasmic reticulum-anchored NSP4 is highly flexible and exhibits high sequence variation compared with the other regions, the significance of which in diarrhoea induction remain unresolved. Here, it was shown that every amino acid substitution or deletion in the flexible C terminus resulted in altered conformation, multimerization, trypsin resistance and thioflavin T (ThT)binding, and affected DLP binding and the diarrhoea-inducing ability of the highly diarrhoeagenic SA11 and Hg18 $\triangle$N72 in suckling mice. These studies further revealed that high ThT fluorescence correlated with efficient diarrhoea induction, suggesting the importance of an optimal ThT-recognizable conformation in diarrhoea induction by purified NSP4. These results based on biological properties provide a possible conformational basis for understanding the influence of primary sequence variations on diarrhoea induction in newborn mice by purified NSP4s that cannot be explained by extensive sequence analyses

DenHunt - A Comprehensive Database of the Intricate Network of Dengue-Human Interactions

<div><p>Dengue virus (DENV) is a human pathogen and its etiology has been widely established. There are many interactions between DENV and human proteins that have been reported in literature. However, no publicly accessible resource for efficiently retrieving the information is yet available. In this study, we mined all publicly available dengue–human interactions that have been reported in the literature into a database called DenHunt. We retrieved 682 direct interactions of human proteins with dengue viral components, 382 indirect interactions and 4120 differentially expressed human genes in dengue infected cell lines and patients. We have illustrated the importance of DenHunt by mapping the dengue–human interactions on to the host interactome and observed that the virus targets multiple host functional complexes of important cellular processes such as metabolism, immune system and signaling pathways suggesting a potential role of these interactions in viral pathogenesis. We also observed that 7 percent of the dengue virus interacting human proteins are also associated with other infectious and non-infectious diseases. Finally, the understanding that comes from such analyses could be used to design better strategies to counteract the diseases caused by dengue virus. The whole dataset has been catalogued in a searchable database, called DenHunt (<a href="http://proline.biochem.iisc.ernet.in/DenHunt/" target="_blank">http://proline.biochem.iisc.ernet.in/DenHunt/</a>).</p></div

Novel Pentameric Structure of the Diarrhea-Inducing Region of the Rotavirus Enterotoxigenic Protein NSP4

A novel pentameric structure which differs from the previously reported tetrameric form of the diarrhea-inducing region of the rotavirus enterotoxin NSP4 is reported here. A significant feature of this pentameric form is the absence of the calcium ion located in the core region of the tetrameric structures. The lysis of cells, the crystallization of the region spanning residues 95 to 146 of NSP4 (NSP4(95-146)) of strain ST3 (ST3: NSP4(95-146)) at acidic pH, and comparative studies of the recombinant purified peptide under different conditions by size-exclusion chromatography (SEC) and of the crystal structures suggested pH-, Ca(2+)-, and protein concentration-dependent oligomeric transitions in the peptide. Since the NSP4(95-146) mutant lacks the N-terminal amphipathic domain (AD) and most of the C-terminal flexible region (FR), to demonstrate that the pentameric transition is not a consequence of the lack of the N- and C-terminal regions, glutaraldehyde cross-linking of the Delta N72 and Delta N94 mutant proteins, which contain or lack the AD, respectively, but possess the complete C-terminal FR, was carried out. The results indicate the presence of pentamers in preparations of these longer mutants. Detailed SEC analyses of Delta N94 prepared under different conditions, however, revealed protein concentration-dependent but metal ion-and pH-independent pentamer accumulation at high concentrations which dissociated into tetramers and lower oligomers at low protein concentrations. While calcium appeared to stabilize the tetramer, magnesium in particular stabilized the dimer. Delta N72 existed primarily in the multimeric form under all conditions. These findings of a calcium-free NSP4 pentamer and its concentration-dependent and largely calcium-independent oligomeric transitions open up a new dimension in an understanding of the structural basis of its multitude of functions

Association of dengue viral interacting human proteins with infectious diseases and non-infectious diseases.

<p>The dengue virus interacting human proteins and their associated a) infectious and b) non-infectious diseases are visualized as a network using Cytoscape. The diseases are represented as triangles and are color coded based on the disease group they belong to. The genes that are associated with only in one disease are represented as violet colored circles and those that are associated with more than one disease are represented as green colored circles. c) Pathway analysis of the dengue virus interacting proteins that are associated with both infectious and non-infectious diseases. KEGG pathways that had 20 or more dengue virus proteins associated with both infectious and non-infectious diseases using KEGG Mapper—Search Pathway is shown as a bar graph.</p

Consolidated list of different types of dengue—human interactions available at the DenHunt database.

<p>The type of interactions, the total number of unique interactions for each type of interaction, the number of references that describe each type of interaction and the total number of human proteins involved in all the interactions are given in the table.</p

Representation of dengue viral interacting proteins in NF-κB and RIG-I-like receptor signaling pathway.

<p>The dengue virus interacting proteins are mapped to the KEGG pathways using the “Search&Colour Pathway” tool from the KEGG database. The proteins that are involved in direct interactions are colored orange, indirect interactions are colored in pink, up-regulated proteins are colored red and down-regulated proteins are colored green. The viral components are depicted as blue hexagons. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004965#pntd.0004965.g003" target="_blank">Fig 3a</a> represents NF-κB and 3b RIG-I-like receptor signaling pathway.</p

Visualization of the network of direct dengue-human interactions.

<p>The network of direct physical interactions of dengue–human components was visualized using Cytoscape. Pink hexagons represent dengue viral components, blue circles represent human proteins that interact with one dengue component and green circles represent human proteins that interact with more than one dengue protein.</p