Function Of NSP4 Research Articles

Role of Rotavirus Enterotoxin NSP4 in the Inflammatory Response in Murine Macrophage RAW 264.7 Cells

The rotavirus nonstructural glycoprotein, NSP4, has been identified as the first viral enterotoxin capable of inducing diarrhea. To investigate the biological function of NSP4 in the inflammatory process, a cDNA from human rotavirus (Wa strain) RNA segment 10 was amplified by RT-PCR, cloned into TA vector, and subsequently subcloned into pET23b expression plasmid. The expression of NSP4 protein was determined by SDS-PAGE and Western blotting, then, the protein was purified by affinity chromatography on Ni-NTA-agarose column. The inflammatory effects of NSP4, namely, production of nitric oxide (NO), proinflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α), and prostaglandin E2 (PGE2), was evaluated using NSP4-stimulated RAW 264.7 murine macrophages and compared with those observed after stimulation with lipopolysaccharide (LPS). The levels of IL-1β, IL-6, and TNF-α were significantly increased, and those of NO and PGE2 also increased in NSP4-stimulated RAW 264.7 cells. These findings indicate that NSP4 plays an important role in the inflammatory response observed during rotavirus infection.

Structures of Neutrophil Serine Protease 4 Reveal an Unusual Mechanism of Substrate Recognition by a Trypsin-Fold Protease

Trypsin-fold proteases, the largest mammalian protease family, are classified by their primary substrate specificity into one of three categories, trypsin-like, chymotrypsin-like, and elastase-like, based on key structural features of their active site. However, the recently discovered neutrophil serine protease 4 (NSP4, also known as PRSS57) presents a paradox: NSP4 exhibits a trypsin-like specificity for cleaving substrates after arginine residues, but it bears elastase-like specificity determining residues in the active site. Here we show that NSP4 has a fully occluded S1 pocket and that the substrate P1-arginine adopts a noncanonical "up" conformation stabilized by a solvent-exposed H-bond network. This uncommon arrangement, conserved in all NSP4 orthologs, enables NSP4 to process substrates after both arginine as well as post-translationally modified arginine residues, such as methylarginine and citrulline. These findings establish a distinct paradigm for substrate recognition by a trypsin-fold protease and provide insights into the function of NSP4.

Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for Activation of Apoptosis during Infection

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca(2+) homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61-83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.

Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells

BackgroundRotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types.MethodsTransport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab)2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls.ResultsOnly full-length (FL), endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells.ConclusionsThe intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study.

Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function

Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VIII. Pathological consequences of rotavirus infection and its enterotoxin.

Rotaviral infection in neonatal animals and young children leads to acute self-limiting diarrhea, but infected adults are mainly asymptomatic. Recently, significant in-roads have been made into our understanding of this disease: both viral infection and virally manufactured nonstructural protein (NSP)4 evoke intracellular Ca(2+) ([Ca(2+)]i) mobilization in native and transformed gastrointestinal epithelial cells. In neonatal mouse pup mucosa models, [Ca(2+)]i elevation leads to age-dependent halide ion movement across the plasma membrane, transepithelial Cl(-) secretion, and, unlike many microbial enterotoxins, initial cyclic nucleotide independence to secretory diarrhea. Similarities between rotavirus infection and NSP4 function suggest that NSP4 is responsible for these enterotoxigenic effects. NSP4-mediated [Ca(2+)]i mobilization may further facilitate diarrhea by signaling through other Ca(2+)-sensitive cellular processes (cation channels, ion and solute transporters) to potentiate fluid secretion while curtailing fluid absorption. Apart from these direct actions in the mucosa at the onset of diarrhea, innate host-mediated defense mechanisms, triggered by either or both viral replication and NSP4-induced [Ca (2+)]i mobilization, sustain the diarrheal response. This secondary component appears to involve the enteric nervous system and may be cyclic nucleotide dependent. Both phases of diarrhea occur in the absence of significant inflammation. Thus age-dependent rotaviral disease represents an excellent experimental paradigm for understanding a noninflammatory diarrhea.

The authentic sequence of rotavirus SA11 nonstructural protein NSP4

Recent studies demonstrate that the rotavirus nonstructural protein NSP4 functions as an enterotoxin and plays an important role in viral pathogenesis. Previous in vitro studies of NSP4 have used a cDNA clone of gene 10 derived from the prototypic rotavirus strain, SA11. We recently compared the sequence of the commonly used NSP4 cDNA with the sequence obtained from several SA11 isolates by direct sequencing of reverse transcription polymerase chain reaction products. One codon difference was identified between the cDNA clone and the SA11 virus isolates, and this resulted in a predicted amino acid substitution at position 47. The cDNA sequence specifies an asparagine at position 47, and the SA11 virus gene 10 encodes a hisitidine. To determine if this amino acid substitution altered the function of NSP4, we analyzed the ability of both NSP4-Asn47 and NSP4-His47 to regulate intracellular calcium levels and exhibit cell cytotoxicity. Our results indicate that the expression of NSP4-His47 from a recombinant baculovirus displays enhanced cytotoxicity and calcium flux.

Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis

Nonstructural proteins of Sindbis virus, nsP1, nsP2, nsP3, and nsP4, as well as intermediate polyproteins, are produced from two precursor polyproteins, P123 and P1234, by a proteolytic enzyme encoded in the C-terminal half of nsP2. We studied the requirements for and the functions of the intermediate and mature processing products for Sindbis virus RNA synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various combinations. A mutant defective in cleaving both the 1/2 and 2/3 sites, which makes only uncleavable P123 and mature nsP4 as final products, produced 10(-3) as much virus as did the wild-type virus after 10 h at 30 degrees C and was nonviable at 40 degrees C. A mutant defective in processing the 2/3 site, which makes nsP1, nsP4, and P23 as well as precursor P123, grew 10(-1) as efficiently as wild-type virus at 30 degrees C and 10(-3) as efficiently at 40 degrees C. Early minus-strand RNA synthesis by these mutants was as efficient as that by wild-type virus, whereas plus-strand RNA synthesis was substantially decreased compared with that by wild-type virus. A mutant defective in processing the 3/4 site was nonviable at either 30 or 40 degrees C. The 3/4 site mutant could be complemented by the mutant unable to cleave either the 1/2 or 2/3 site, which can provide mature nsP4. We interpret these results to signify that (i) mature nsP4 is required for RNA replication, (ii) nsP4 and uncleaved P123 function in minus-strand RNA synthesis, and (iii) cleavage of P123 is required for efficient plus-strand RNA synthesis. We propose that Sindbis virus RNA replication is regulated by differential proteolysis of P123. Early in infection, nsP4 and uncleaved P123 form transient minus-strand RNA replication complexes which vanish upon cleavage of P123. Later in infection, an elevated level of viral proteinase activity eliminates de novo synthesis of P123, and no further synthesis of minus-strand RNA is possible. In contrast, nsP4 and cleavage products from P123 form plus-strand RNA replication complexes which are stable and remain active throughout the infection cycle.

Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host-dependent manner

Two mutants of Sindbis virus have been isolated which grow inefficiently at 34.5 degrees C in mosquito cells yet replicate normally in chicken embryo fibroblast cells at the same temperature. In addition, these mutants exhibit temperature-sensitive growth in both cell types and are RNA- at the nonpermissive temperatures (K.J. Kowal and V. Stollar, Virology 114:140-148, 1981). To clarify the basis of this host restriction, we have mapped the causal mutations for these temperature-dependent, host-restricted mutants. Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase. These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187. Virus containing any of these mutations was restricted in its ability to replicate in mosquito but not chicken embryo fibroblast cells at 34.5 degrees C. In addition to its temperature-dependent, host-restricted phenotype, virus derived from one cDNA clone also exhibited decreased levels of nsP34 and nsP4 yet contained only a silent change in its genome. This C-to-U mutation occurred at nucleotide 5751, the first nucleotide after the opal termination codon separating nsP3 and nsP4. Our results suggest that this substitution decreases readthrough of the opal codon and diminishes production of nsP34 and nsP4. Such a decrease in synthesis rates might lead to levels of these products which are insufficient for viral RNA replication in mosquito cells at the higher temperature. This work provides the first evidence that nsP4 function can be strongly influenced by the host environment.

Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies

Plasmids were constructed which contained a large portion of each of the four nonstructural genes of Sindbis virus fused to the N-terminal two-thirds of the trpE gene of Escherichia coli. The large quantity of fusion protein induced from cells containing these plasmids was subsequently used as an antigen to generate polyclonal antisera in rabbits. Each antiserum was specific for the corresponding nonstructural protein and allowed ready identification of each nonstructural protein and of precursors containing the sequences of two or more nonstructural proteins. These antisera were used to determine the stability of the mature nonstructural proteins and to examine the kinetics of processing of the nonstructural proteins from their respective precursors in vivo. Pulse-chase experiments showed that the precursor P123 is cleaved with a half-life of approximately 19 min to produce P12 and nsP3; P12 is then cleaved with a half-life of approximately 9 min to produce nsP1 and nsP2. Thus, although the rate of cleavage between nsP1 and nsP2 is faster than that between nsP2 and nsP3, the latter cleavage must occur first and is therefore the rate-limiting step. The rate at which P34 is chased suggests that the cleavage between nsP3 and nsP4 is the last to occur; however the regulation of nsP4 function in Sindbis virus-infected cells may be even more complex than was previously thought. The products nsP1 and nsP2 (and nsP4) are relatively stable; nsP3, however, is unstable, with a half-life of about 1 h, and appears to be modified to produce heterodisperse, higher-molecular-mass forms. In general, the processing schemes used by Sindbis virus and Semliki Forest virus appear very similar, the major difference being that most nsP3 in Sindbis virus results from termination at an opal condon, whereas in Semliki Forest virus cleavage of the P34 precursor is required.