Pleiotropic Properties of Rotavirus Nonstructural Protein 4 (NSP4) and Their Effects on Viral Replication and Pathogenesis

Chapter 2.4

Pleiotropic Properties of Rotavirus Nonstructural Protein 4 (NSP4) and Their Effects on Viral Replication and Pathogenesis

N.P. Sastri*

S.E. Crawford*

M.K. Estes*,**
*    Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, United States
**    Department of Medicine, Baylor College of Medicine, Houston, TX, United States


Rotavirus (RV), the causative agent of infantile diarrhea in a wide range of hosts, encodes a multifunctional nonstructural protein, NSP4. Interactions of NSP4 with both host factors and other viral proteins are critical for viral replication, morphogenesis, and pathogenesis. This review focuses on the strategies used by intracellular NSP4 (iNSP4) and extracellular NSP4 (eNSP4) to induce diarrhea, to modulate intracellular calcium homeostasis, apoptosis, and tight junction integrity, to regulate viral transcription, replication, and morphogenesis; as well as exploit cellular processes, such as protein trafficking and autophagy, required for RV biology. The molecular mechanisms underpinning the numerous roles for NSP4 are discussed with particular emphasis towards delineating both the functional domains and the structural forms of NSP4 required for its many activities. Defining the multiple functions of NSP4 is important to fully understand rotavirus replication and pathogenesis and will possibly lead to new approaches to prevent or treat disease.


multifunctional protein





structural plasticity

coiled-coil oligomer


1. Introduction

Rotaviruses (RV) remain major causes of life-threatening diarrheal disease in children under 5 years of age, resulting in nearly half a million deaths annually. Although RV vaccines are available and successful in many developed countries, vaccine efficacy and delivery are suboptimal in developing countries where they are needed most. RV-induced disease is life-threatening compared to disease caused by most other enteric microbes, emphasizing the need to more fully understand the pathophysiology of RV gastroenteritis. RV infection, replication and pathogenesis are carried out by the viral encoded six structural and six nonstructural proteins and the viral genome. One intriguing protein encoded in the viral genome is nonstructural protein 4 (NSP4). RV NSP4 has been the focus of much attention in our laboratory since it was first described as a nonstructural glycoprotein with an uncleaved signal sequence and a glycosylation pattern that indicated it does not traffic to the Golgi apparatus (Arias et al., 1982; Both et al., 1983; Ericson et al., 1983). At that time, few viral glycoproteins with uncleaved signal sequences were known. More importantly, the existence of a nonstructural glycoprotein, which contained only high mannose glycans that are trimmed and produced by a nonenveloped virus was certainly intriguing.

Broadly speaking, NSP4 is involved in at least three different functions: pathogenesis, replication, and morphogenesis. This chapter summarizes our current understanding of how NSP4 regulates these processes and new information about how it affects host cell physiologic pathways to enhance viral replication and cause disease. NSP4’s functions in multiple critical pathways are consistent with it being a master regulator of RV replication. NSP4’s central role in virus replication is further highlighted by the consequences of knockdown of NSP4, which affects viroplasm formation that is central to viral replication, modulates viral transcription, inhibits genome packaging into particles and reduces infectious particle assembly (Silvestri et al., 2005). The role of NSP4 in these events critical to replication may explain the inability to obtain mutants of the NSP4 gene using methods that have successfully selected mutants of the other 10 RV genes (Vende et al., 2013; Criglar et al., 2014). This chapter focuses on NSP4s from Group A rotaviruses (RVA) and updates information reviewed in recent years (Ruiz et al., 2000; Estes et al., 2001; Morris and Estes, 2001; Ball et al., 2005; Greenberg and Estes, 2009; Hagbom et al., 2012; Hu et al., 2012).

2. Classification and phylogeny of NSP4

The epidemiology and evolutionary patterns of RVs are being documented by studies of viruses from humans and a variety of animal species. RVs are broadly classified based on the serologic reactivity and genetic variability of the middle capsid structural protein VP6 into eight groups/species (RVA-RVH). RVA viruses are the most extensively studied and they are further classified based on antigenic and sequence differences of the two outer capsid proteins VP7 and VP4 into G (Glycosylated) and P (Protease-sensitive) types, respectively. Due to the unavailability of serological reagents for these neutralization antigens, and the increasing ease and availability of sequencing, including whole genome sequencing, each genomic segment of RVA is now classified based on sequencing data. A Rotavirus Classification Working Group (RCWG) established a system to identify and differentiate each genome segment according to particular cut-off points of nucleotide sequence identities. Each RV genome is designated by listing the segments in the order of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 with their genotypes being represented by Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx (Matthijnssens et al., 2008a,b2011) as discussed more thoroughly in Chapter 2.10.

Here, we review the current NSP4 genotype (called E for enterotoxin) classification and phylogenetic analysis. The initial classification of NSP4 into genotypes was based on amino acid clusters, and six groups (A–F) were identified (Horie et al., 1997; Ciarlet et al., 2000; Mori et al., 2002). The current method of classification based on nucleotide sequence has characterized 19 E genotypes, which supersede those earlier groupings of NSP4 protein sequences (Table 2.4.1).

Table 2.4.1

Summary of Properties of Different Enterotoxin Genotypes

Genotype/reference strain #AA Early group Diarrhea in mice (strain) References
E1/Human-tc/USA/Wa/1974/G1P1A[8] 175 B


(Wa, ST3, 116E)

Sastri et al. (2011)
E2/Human-tc/USA/DS-1/1976/G2P1B[8] 175 A



Hg18, I321, NCDV, S2, 1040, N136, N138, 2KD-851, 99-D/214)

Ball et al. (1996), Zhang et al. (1998), Seo et al. (2008), Chen et al. (2011), Sastri et al. (2011)
E3/Human-tc/JPN/AU-1/1982/G3P3[9] 175 C



Sastri et al. (2011)
E4/Pigeon-tc/JPN/PO-13/1983/G18P[17] 169 E


(PO-13, TY-1)

Mori et al. (2002)
E5/Rabbit-wt/USA/Alabama/2000/G3P[14] 175 A
E6/Human-wt/BGD/N26/2000/G12P[6] 175

E7/Mouse-wt/JPN/EW/1999/G16P[16] 175 D

Yes (EW, EHP)



Horie et al. (1997); Tsugawa et al. (2014), Sastri et al. (2011)
E8/Cow-lab/GBR/PP-1/1976/G3P[7] 175 B
E9/Pig-wt/THA/CMPP034/2000/G2P[27] 175

E10/Chicken-wt/JPN/Ch-1/2001/G19P[17] 168 F



Mori et al. (2002)
E11/Turkey-tc/IRL/Ty-3/1979/G7P[17] 169 E



Mori et al. (2002)
E12/Guanaco-wt/ARG/Chubut/1999/G8P[14] 175

E13/Human-tc/KEN/B10/1987/G3P[2] 175

E14/Horse-tc/GBR/L338/1991/G13P[18] 175

E15/Camel/KUW/s21/2010/G10P[15] 181

E16/Vicuna-wt/ARG/C75/2010/G8P[14] 175

E17/Sugar glider-tc/JPN/SG385/2012/G27P[36] 175

E18/Rat-wt/GER/KS-11-573/2011/G3P[3] 175

E19/Fox/ITA/288356/2010/G18P[17] 169

The results of analyses at both the nucleotide and amino acid levels are in complete concordance with each other but use of nucleotide sequences makes it clearer to assign different genotypes without overlaps as accepted by the RCWG. The cut-off value for designating a new E genotype is defined as 85% nucleotide identity. Analysis and understanding the genotype differences and distribution of all the RV genes provide a framework to analyze interspecies evolutionary relationships, gene reassortment events, functional gene linkage with other genome segments in reassortant progeny and the emergence of new RVs by interspecies transmission (Matthijnssens et al., 2008a) (see Chapter 2.10).

A phylogenetic tree (Fig.  2.4.1A) using nucleotide sequences from each representative genotype constructed with MEGA software (version 6.0) (Tamura et al., 2013) shows there are three distinct NSP4 clusters from genes from human viruses (E1, E2, E3) that are in agreement with the previously described gene constellations by RNA-RNA hybridization (called Wa- DS-1- and KUN-/AU-1-like genogroups). These three E genotypes are commonly detected among viruses from humans that contain common G and P types, whereas E6 contains a human NSP4 gene from unusual G12 strains (Rahman et al., 2007). E types from avian species (E4, E10, and E11) branch out and form a distinct constellation. Full genome classification of multiple genes has revealed genetic relatedness of human RV genes with those from animals supporting the occurrence of interspecies transmission and penetration of animal virus genes into human RVs. A common origin between human Wa-like and porcine RV strains is now recognized (Matthijnssens et al., 2008a). A close genetic relatedness between E1 sequences and human strains and an evolutionarily positively selected site in NSP4 have also been described (Malik et al., 2014). Interestingly, the most recently identified E genotype (E19) was isolated from a fox in Italy (Fox/Italy/288356/2010) and is closely related to avian strains (Boniotti, Unpublished), which further supports the transmission and circulation of new RV species in wild animals. To our knowledge, this is the first report that suggests the circulation of avian RVs in other animal species. This raises the question of whether a fox ate a bird. Full genotypic characterization of the fox isolate from Italy will allow conclusions about its evolution.


Figure 2.4.1 Analysis of currently identified NSP4 E genotypes.
(A) Phylogenetic analysis of NSP4 nucleotide sequences representing each genotype. A phylogenetic tree was constructed by the neighbor-joining method using MEGA software (Version 6.0) and boot strap values (2000 replicates) are shown at the nodes. Representative strains from each genotype are labeled to the right. GenBank accession numbers for the NSP4 nucleotide sequences used in this analysis are as follows: E1 (K02032), E2 (AF174305), E3 (D89873), E4 (AB009627), E5 (AB005472), E6 (DQ146691), E7 (AB003805), E8 (AF427521), E9 (KM820684), E10 (AB065287), E11 (AB065286), E12 (FJ347109), E13 (HM627562), E14 (JF712564), E15 (JX968472), E16 (JX070055), E17 (AB971769), E18 (KJ879457), E19 (unpublished and kindly provided by Beatrice Boniotti).(B) Amino acid sequence alignment of representative NSP4 genotypes. NSP4 amino acid sequences corresponding to the representative E genotypes shown in Fig. 2.4.1A were aligned by ClustalW. Mammalian strains (E1-E3, E5, E6, E8, E9, E12-E15, E17, E18) are shown on the top followed by mouse (E7), avian [pigeon (E4), chicken (E10), turkey (E11)], vicuna (E16), fox (E19), and camel (E15). Domains are indicated by lines over the alignment: hydrophobic domains H1, H2, and H3: blue line; viroporin domain: orange line; coiled-coil domain: solid green line; enterotoxin peptide: black dashed line showing the well-characterized aa 114–135 followed by a black dotted line that represents an extended domain with enterotoxin activity whose function might depend on regions other than the core domain. Conserved glycosylation sites at aa 8 and 18 are represented by “Y”; avian and the new fox genotypes have a conserved predicted glycosylation site at aa 17 (not shown). The interspecies variable domain (ISVD) is boxed with a blue dashed line. The conserved calcium-binding residues E120 and Q123 are indicated by asterisks. Residues that are identical to the top sequence (E1 genotype) are indicated by a dot. GenBank accession numbers used for this amino acid alignment are as follows: E1 (AAA47309), E2 (AAG09190), E3 (BAA24413), E4 (BAA24144), E5 (BAA36286), E6 (ABA34245), E7 (BAA33948), E8 (AAL31532), E9 (AIY56440), E10 (BAB83747), E11 (BAB83746), E12 (ACN86097), E13 (ADP68540), E14 (AEH96577), E15 (AGC24703), E16 (AFO84069), E17 (BAP91310), E18 (AIL24115), E19 (unpublished and kindly provided by Beatrice Boniotti).(C) Schematic diagram of NSP4 domain organization and interacting partner proteins. A linear diagram of SA11 NSP4 with amino acid numbers marking the N- and C-terminal amino acids of the following domains: hydrophobic domains H1, H2, and H3 are indicated by blue boxes; the viroporin domain, shown in orange, overlaps the H3 domain; the coiled-coil domain is shown in red; and the flexible region is shown in green. The H2 domain traverses the ER membrane (shown by blue lines) followed by an extended C-terminus present in the cytoplasm. Conserved glycosylation sites (CHO), indicated with a “Y,” reside in the ER lumen. The conserved calcium binding residues (E120 and Q123) are shown within the coiled-coil domain. NSP4 functional domains and cellular and viral protein interaction domains are shown above and below the linear schematic. ISVD,: interspecies variable domain; DLP, double-layered particle; CRAC, cholesterol recognition amino acid consensus; ECM, extracellular matrix.

Other phylogenetic analyses of whole RV genome sequences have sought to determine if there are genetic linkages between specific RV gene segments, which might support dependent functions. The first reported genetic linkage was between the NSP4 gene and the VP6 gene that seems to correlate with the host species of origin (Iturriza-Gomara et al., 2003). This linkage may have biological relevance because it is known that NSP4 functions as an intracellular receptor that binds VP6 as part of virus morphogenesis (Au et al., 1989; Bergmann et al., 1989; Meyer et al., 1989). Early studies of receptor interactions suggested there might be specific interactions between NSP4s and VP6s of different subgroups (Au et al., 1989). Other studies have confirmed this linkage in human RVs, and in some cases, also reported linkages between VP4, VP7, VP6, NSP4, and NSP5 (Araujo et al., 2007; Tavares Tde et al., 2008; Benati et al., 2010; Khamrin et al., 2010; Chaimongkol et al., 2012). By contrast, VP6 and NSP4 reportedly segregate independently for porcine RV strains (Ghosh et al., 2007).

3. NSP4 domain organization

A multiple amino acid sequence alignment of the current 19 E genotypes (Fig. 2.4.1B) provides information about similarities that may be related to protein function. Most mammalian NSP4 proteins contain 175 amino acids while NSP4s from avian, camel and fox species have smaller and larger proteins, respectively (Table 2.4.1 and Fig. 2.4.1B). This figure aligns NSP4 proteins from mammalian viruses on the top followed by those from mouse (E7), avian [pigeon (E4), chicken (E10), turkey (E11)], vicuna (E16), fox (E19) and camel (E15) at the bottom. Highly conserved residues in the mammalian viruses are evident that diverge in the NSP4 proteins from rodent, avian and camel viruses. Fig. 2.4.1C shows a schematic of the known functional domains in NSP4 in more detail. These figures illustrate that NSP4, which is synthesized as an ER transmembrane glycoprotein, consists of three hydrophobic domains (H1–H3) with two N-linked high mannose glycosylation sites oriented to the luminal side of the ER in the H1 domain (Chan et al., 1988; Bergmann et al., 1989). The H2 transmembrane domain traverses the ER bilayer and serves as an uncleaved signal sequence and aa 85–123 are reported to be involved in ER retention although this is likely to be an indirect involvement because these residues are located in the cytoplasmic domain of NSP4 (Mirazimi et al., 2003). The H3 domain consists of an amphipathic α-helix, which is part of the viroporin domain (Hyser et al., 2010). The remainder of NSP4 is an extended cytoplasmic domain containing a highly conserved coiled-coil region and a flexible C-terminus (Estes and Kapikian, 2007). The coiled-coil domain contains a calcium ion binding site coordinated by amino acids E120 and Q123, which are 100% conserved among all RVA viruses. This is consistent with the evidence showing that this region plays a critical role in conserved functions of NSP4 across different species. The number of multiple overlapping binding domains with distinct partners is striking (Fig. 2.4.1C). An interspecies variable domain (ISVD, aa 135–141) is clearly evident in the amino acid alignment. This alignment highlights that there are multiple other highly conserved domains without current functions, which suggest areas for future research.

The fact that NSP4 has been found in dimeric, tetrameric, pentameric, and higher ordered multimeric structures may explain how NSP4 is able to interact with multiple partners within the same region of the protein (Fig. 2.4.1C) (Maass and Atkinson, 1990; Bowman et al., 2000; Chacko et al., 2011; Sastri et al., 2011 2014). The coiled-coil domain interacts with multiple proteins including integrin I domains, caveolin, extracellular matrix proteins, a cholesterol recognition/interaction amino acid consensus sequence (CRAC) and VP4 (Boshuizen et al., 2004; Parr et al., 2006; Hyser et al., 2008; Seo et al., 2008; Schroeder et al., 2012). In addition, the flexible C-terminal domain interacts with both tubulin and newly formed, immature double-layered particles (DLPs) (Au et al., 1989; O’Brien et al., 2000; Xu et al., 2000). Whether specific structural forms are associated with these different interactions remains to be determined.

4. NSP4 structure

The coiled-coil domain (aa 95–137) is the only domain whose structure has been determined by crystallographic studies. Challenges for obtaining crystals of the full-length protein are due to the presence of the N-terminal hydrophobic domains and a C-terminal flexible region, which leads to full-length NSP4 being highly insoluble in aqueous solution. Thus, crystallization studies have focused on the soluble domains. An initial study, which used a synthetic peptide composed of aa 95–137 of the simian strain SA11 (Bowman et al., 2000), and later studies using bacterially expressed protein (aa 95–146) from SA11 and a human I321 strain reported that the coiled-coil domain forms a tetramer. In spite of crystallizing longer proteins, only aa 95–137 have been resolved in the structures, and the amino acids after 137 are disordered (Bowman et al., 2000; Deepa et al., 2007). The first structure from the SA11 NSP4 peptide showed a tetramer with a calcium ion bound and coordinated by aa E120 and Q123. In contrast, the structure of the NSP4 coiled-coil domain from a human RV ST3 strain formed a pentameric coiled-coil that lacked a calcium ion at its core (Chacko et al., 2011). In these studies, the tetramer was crystallized at neutral pH while the pentamer was crystallized at low pH. It was unclear whether the two oligomeric forms of the coiled-coil domain from the two different strains represented a calcium-dependent, strain-dependent, or pH-dependent phenomenon. This was tested by determining the crystal structure of the NSP4 coiled-coil domain from a single strain SA11 (Sastri et al., 2014); these studies showed that at neutral pH this domain exists as a tetramer, which binds a calcium ion at its core (Fig. 2.4.2, structure on the left). However, mutation of the calcium- coordinating amino acids resulted in formation of a pentamer that lacks a calcium ion at its core (Fig. 2.4.2, structure on the right). Further solution studies showed that the NSP4 coiled-coil domain can directly bind calcium at physiological pH and exists as a tetramer. In contrast, at low pH, the coiled-coil domain does not bind calcium and forms a pentamer. This structural plasticity of NSP4 regulated by pH and calcium may be the basis for its pleiotropic functions during RV replication and pathogenesis.


Figure 2.4.2 Wheel diagram showing NSP4 forms and functions.
The functions of both intracellular NSP4 (iNSP4, shown in blue) and extracellular NSP4 (eNSP4, shown in green) are indicated with arrows. Inside the wheel at the top: a model of the topology of the NSP4 viroporin, in which the pentalysine domain is predicted to mediate viroporin domain insertion into the ER membrane, NSP4 oligomerization and the amphipathic α-helix formation of a pore that allows the release of calcium into the cytoplasm. In the center: NSP4 domain organization (refer to Fig. 2.4.1C for details). At the bottom: two structures of the NSP4 coiled-coil domain. The calcium-bound tetramer that forms at pH 7.4 (left) and a pentamer that forms as a result of mutation of the calcium-coordinating amino acids (E120 and Q123) or at low pH 5.6, which abolishes calcium binding.

5. The pathophysiology of rotavirus-induced disease and characterization of the role of NSP4 in diarrhea induction

The spectrum of responses to RV infection varies and can be asymptomatic, mild or severe, which results in a life-threatening dehydrating illness. The incubation time is short (<48 h), and illness often begins with a sudden onset of nausea and vomiting, followed by diarrhea that leads to a high frequency of dehydration and a mean duration of illness lasting 5–6 days. Several studies have shown infection leads to extra-intestinal spread of virus with antigenemia and viremia, and limited systemic replication in a variety of sites occurs frequently (Blutt et al., 2007). It remains unclear whether this systemic spread and replication cause any specific pathology in the normal host, but fever has been associated with antigenemia (Sugata et al., 2008), and antigenemia is reported to be associated with more severe clinical manifestations of acute gastroenteritis (Hemming et al., 2014).

The mechanisms causing these symptoms remain incompletely understood although our understanding of the pathophysiological processes of RV-induced disease has improved in recent years. The outcome of infection is more complex than initially appreciated and is affected by a complex interplay of host and viral factors. Chapter 2.6 covers this topic in detail; so later we focus on information related to NSP4 in pathogenesis.

RVs infect intestinal enterocytes and the early events in infection are mediated by virus-epithelial cell interactions (see Chapter 2.2). The cause of RV-induced diarrhea is multifactorial, and mechanisms include (1) increased intestinal secretion stimulated by infection, (2) villus ischemia and activation of the enteric nervous system that may be evoked by release of a vasoactive agent from infected epithelial cells in the absence of significant pathologic lesions or enterocyte damage, (3) malabsorption that occurs secondary to the down regulation of host proteins or destruction of differentiated absorptive enterocytes, and (4) alterations in transepithelial fluid balance caused by loss of polarized epithelial cell tight junctions and cell integrity. Each of these mechanisms is associated with functions of NSP4 either expressed intracellularly (iNSP4) or secreted extracellularly (eNSP4). Fig. 2.4.2 shows the functions attributed to iNSP4 (blue) or eNSP4 (green).

A possible role for NSP4 in pathogenesis (diarrhea induction) was initially suggested in studies of a virulent porcine RV (SB-1A strain) and an avirulent human RV (DS-1) in gnotobiotic pigs (Hoshino et al., 1995). Pigs administered a single-gene reassortant virus that derived its 10th gene (that encodes NSP4) from the avirulent human DS-1 virus failed to develop diarrhea, and these pigs were also protected from subsequent challenge with a different virulent porcine RV. These results were unexpected and a mechanistic explanation for why the gene encoding NSP4 was a virulence gene associated with diarrhea was unclear.

Discovery that administration of NSP4 alone or synthetic peptides from NSP4 cause age-dependent diarrhea in mice without causing histologic changes in the intestine led to the recognition that NSP4 functions as an enterotoxin (Ball et al., 1996). Subsequent work showed that NSP4 is a novel secretory agonist eliciting both paracrine and autocrine signaling. Both trigger (by different mechanisms) calcium-dependent signaling pathways that alter epithelial cell permeability and augment endogenous secretory pathways (Estes and Morris, 1999; Morris et al., 1999; Estes et al., 2001). This led to a new explanation for RV-induced diarrhea and why diarrhea can be seen before or in the absence of histologic changes in several species (Mebus et al., 1975; Collins et al., 1989; Burns et al., 1995; Ward et al., 1996; Ciarlet and Estes, 1999). Such observations had been made previously in several settings, and the production of endogenous, neuroactive, hormonal substances of pathophysiological importance had been proposed (Osborne et al., 1988).

The ability of NSP4 from a number of mammalian and avian virus strains to induce diarrhea in neonatal mice has been demonstrated by multiple groups (Table 2.4.1). The enterotoxin domain was initially characterized using both purified protein and a synthetic peptide that contained aa 114–135 (Ball et al., 1996). Further studies revealed that this region has two distinct domains: amino acids 114–130 are essential for binding to α1 and α2 integrin I domains while amino acids 131–140, which are not associated with the initial binding to the I domain, elicit signaling (Fig. 2.4.3). NSP4 mutants that fail to bind or signal through integrin α2 are attenuated in diarrhea induction in neonatal mice (Seo et al., 2008). It is unclear whether interaction of NSP4 with other proteins is involved in diarrhea induction. In addition, the diarrheagenic peptide contains only a part of the active domain of the protein based on comparisons of the effective dose of the peptide and the full-length protein to cause diarrhea, but the exact size of the active toxin domain remains unknown.


Figure 2.4.3 NSP4 is a master regulator that modulates cellular signaling pathways during rotavirus infection.
(A) Following RV infection of an intestinal enterocyte, iNSP4 is cotranslationally inserted into the ER membrane. Functioning as a viroporin, iNSP4 mediates the release of calcium from ER into the cytoplasm. iNSP4 viroporin-mediated loss of ER calcium activates and translocates STIM1, which interacts and activates Orai1 for SOCE. Elevated cytoplasmic calcium initiates the autophagy process, which is necessary for RV replication and production of infectious particles. iNSP4 also induces apoptosis by activating intrinsic apoptotic pathway and releases cytochrome c, which activates procaspase-9 that triggers caspase-3 cleavage. Secreted eNSP4 binds integrins and triggers a PLC-mediated pathway that elevates cytoplasmic calcium. The increased cytoplasmic calcium activates calcium-activated chloride channels such as TMEM16A, which results in cellular Cl secretion. (B) Macrophage depicting paracrine signaling pathway elicted by eNSP4, which triggers the release of the proinflammatory cytokines TNF-α and IL-6 and activates p38 and JNK MAP kinases and NF-κB. NSP4 also induces NO release.

Based on the hypothesis that diarrhea induction should be predicated on a secreted form of NSP4, studies were performed to detect such forms, and several secreted forms of NSP4 have been described. In a first study, a 7 kDa molecular weight secreted form of NSP4, spanning amino acids 112–175, was isolated from the media of rotavirus-infected MA104 and intestinal HT-29 cells. This product was secreted in a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network (Zhang et al., 2000). Diarrhea was induced in neonatal mice using an expressed and purified form of this protein. Another secreted, fully glycosylated form of NSP4 was isolated from Caco-2 cells as discrete detergent-sensitive oligomers in a complex with phospholipids. Secretion of this form of NSP4 was dramatically inhibited by brefeldin A and monensin, suggesting that a Golgi-dependent pathway is involved in release of the protein, which was supported by its partial resistance to deglycosylation by endoglycosidase H. This form of NSP4 was found to bind cell surface glycosaminoglycans (Didsbury et al., 2011). A third full-length, glycosylated, endoglycosidase H-sensitive form of NSP4 was detected in the media of two cell types (MDCK and HT-29 cells) (Gibbons et al., 2011). Others detected impairment of lactase activity in the brush border of Caco-2 cells caused by secreted NSP4 in the media from virus-infected cells although the exact form of the protein was not characterized (Beau et al., 2007). It remains unclear whether the different forms of NSP4 detected in these studies relate to the use of different virus strains, cells or methods of isolation, and this issue remains to be resolved, perhaps by designing new experiments that take into account the new knowledge about the plasticity of the structure of NSP4 that can be influenced by pH and ionic conditions of buffers used in the isolation and purification of NSP4.

One continuing impediment preventing complete characterization of the activity of NSP4 is the lack of any mutants in gene 10 that encodes NSP4 and the lack of a reverse genetics system (see Chapter 2.3) to study site-directed mutants in the context of a viral infection. These limitations have forced studies of the functions of NSP4 to focus on examining the effects of purified protein or expressed proteins with engineered mutations by using a variety of cell biology and biochemical studies including RNA interference (RNAi) knockdowns and pharmacologic treatments. In another approach, sequence changes associated with modified function can be identified in the NSP4 gene of isogenic virulent/avirulent pairs of viruses. NSP4s from the avirulent viruses showed reduced enterotoxin activity; in one example, amino acids 136 and 138 were implicated in reduced enterotoxin activity (Zhang et al., 1998). These results also demonstrate that the enterotoxin domain extends beyond aa 114–135. More recent studies indicate that the diarrheagenic domain may extend to the C-terminus of NSP4 (Sastri et al., 2011). It is interesting to note that the NSP4 of the porcine/human reassortant viruses studied by Hoshino had sequence changes at aa 135 and 138 that might explain their phenotypes (Hoshino et al., 1995). Some studies have tried to examine sequences of RVs that are associated with symptomatic or asymptomatic infections and failed to find distinct sequences associated with illness (Lee et al., 2000; Rajasekaran et al., 2008; Sastri et al., 2011). This is not surprising because both host and virus properties affect infection outcome, and clear results are unlikely to be obtained unless isogenic virus strains are studied and compared.

Another novel approach to define virulence-associated genome mutations of murine RVs evaluated a cell culture-adapted murine EB RV strain that was serially passaged in mouse pups or in cell cultures, alternately and repeatedly, and then the genomes of viruses with different virulence phenotypes were fully sequenced. Mouse-passaged virus that regained virulence had aa substitutions in VP4 and at aa 37 in NSP4 (Tsugawa et al., 2014). This is an unexpected but interesting result because this aa is present in a highly conserved domain (aa 35–41) of the NSP4s of mammalian RVs that currently does not have a known function (Fig. 2.4.1B). A human vaccine strain of lamb RV also has a change at aa 36 in this highly conserved region of NSP4 (Mohan et al., 2003). These results raise the question of whether this domain may be correlated with virulence and suggest that it should be examined in additional functional studies.

A correlation of RV-induced diarrhea in neonatal mice and NSP4 has been confirmed using RNAi technology, specifically using a short hairpin RNA (shRNA) reactive with the NSP4 gene engineered into a lentivirus. No suckling mice developed diarrhea induced by bovine RV when the NSP4 was silenced by the shRNA while 100% of suckling mice without shRNA treatment or with treatment with a control shRNA developed diarrhea (Chen et al., 2011). These results provide an independent approach to confirm that NSP4 is associated with diarrhea induction in neonatal mice. The ability to prevent or reduce RV-induced diarrhea by treating mice with NSP4-specific antibody is additional independent evidence of the role of NSP4 in diarrhea induction (Ball et al., 1996; Estes et al., 2001; Hou et al., 2008).

6. Mechanism of NSP4 in diarrhea induction

The mechanism of diarrhea induction is best characterized through studies of the paracrine action of eNSP4 on uninfected intestinal epithelial cells. NSP4 binding to intestinal epithelial cell lines has been shown to initiate a signaling pathway that involves activation of phospholipase C (PLC), elevation in inositol 1,4,5-trisphophate (InsP3) and release of ER Ca2+ stores that increases cytoplasmic Ca2+ resulting in Ca2+-dependent Cl secretion (Tian et al., 1995; Ball et al., 1996; Dong et al., 1997). Crypt cells isolated from mice also respond in a similar manner (Morris et al., 1999). Studies in mice lacking the cystic fibrosis transmembrane regulator (CFTR) channel showed that age-dependent diarrheal disease results from an event downstream from increased cytoplasmic Ca2+ (Morris et al., 1999). Since the CFTR channel is a c-AMP-regulated Cl channel, the finding that diarrhea is observed in CFTR knockout mice following RV infection or NSP4 treatment indicated that a Cl channel different than CFTR mediates this effect (Angel et al., 1998; Morris et al., 1999). Cl secretion is age-dependent in CFTR mice, indicating that age-dependent disease may result from an age-dependent induction, activation or regulation of the Cl channel, which was proposed to be the calcium-activated chloride channel (CaCC). This pathway would be one that is activated when eNSP4 is released from virus-infected cells and the affected cells are neighboring, noninfected secretory crypt or epithelial cells (Fig. 2.4.3). These studies also illustrate that NSP4 is a novel secretory agonist because CFTR knockout mice do not respond to classical secretory agonists (Morris et al., 1999). Confirmatory data for this proposed model came from showing NSP4 induces diarrhea by activation of the proposed CaCC TMEM16A and inhibition of Na+ absorption (Ousingsawat et al., 2011) and that CaCC inhibitors prevent RV secretory diarrhea in neonatal mice (Ko et al., 2014). NSP4 also does not have a direct, specific effect on chloride transport in brush border membranes, confirming the hypothesis that its actions are through triggering signal transduction pathways (Lorrot and Vasseur, 2006).

Further studies using Ussing chamber experiments showed that the enteric nervous system (ENS) also plays a role in the fluid secretion evoked by RV in neonatal mice (Lundgren et al., 2000). Drugs that inhibit ENS functions attenuate the intestinal secretory response to RV in vitro and in vivo, suggesting that the ENS participates in RV-induced electrolyte and fluid secretion as shown previously for bacterial enterotoxins (Lundgren and Jodal, 1997; Farthing et al., 2004; Kordasti et al., 2004). Activation of secretory reflexes in the ENS system represents another proposed mechanism for RV fluid secretion. This hypothesis implies that nerve afferent fibers, located just underneath the intestinal epithelium, are activated by amines/peptides secreted from the entero-endocrine cells and/or by chemokines, prostaglandins, or nitric oxide (NO) released from enterocytes exposed to microorganisms (Fig. 2.4.3) (Lundgren and Svensson, 2001). In line with this, NSP4 and RV can stimulate the release of NO from HT-29 cells, and increased levels of NO2/NO3 are detected in the urine of RV-infected mice and young children (Rodriguez-Diaz et al., 2006). Finally, NSP4 can induce nitric oxide synthase (iNOS) and stimulate NO release from macrophages (Borghan et al., 2007). It is in this interesting context that Rollo et al. reported that RV evokes an age-dependent release of chemokines from epithelial cells, occurring only in mice less than 2 weeks old (Rollo et al., 1999). Thus, age-dependent diarrhea may be multifactorial, and mechanisms may include an infection-mediated intestinal chemokine response as well as through a NSP4-mediated activation of an age- and Ca2+-dependent plasma membrane Cl channel distinct from CFTR.

Subsequent studies indicated that the neurotransmitters serotonin and vasoactive intestinal peptide (VIP) are involved in RV diarrhea (Kordasti et al., 2004). RV can infect cultured human entero-chromaffin (EC) cells and stimulate serotonin secretion, which activates the vagal afferent nerves connected to the brain stem structures associated with nausea and vomiting; NSP4 alone also was shown to evoke release of serotonin from EC cells in a calcium-dependent manner, suggesting new mechanisms to explain the nausea and vomiting associated with RV infections (Fig. 2.4.3) (Hagbom et al., 2011 2012) (see Chapter 2.6). It is evident that NSP4 activates and triggers multiple signaling pathways that affect pathogenesis.

Another mechanism of RV-induced pathogenesis is loss of cell integrity and polarized epithelial cell tight junctions. A calcium-dependent depolymerization of microvillar actin occurs in RV-infected polarized human intestinal Caco-2 cells that results in alterations of protein trafficking (Jourdan et al., 1998; Brunet et al., 2000) and disruption of tight junctions with loss of cell monolayer integrity (Obert et al., 2000). Silencing NSP4 provided confirmation that disruption of actin in RV-infected MA104 cells is directly related to changes in the intracellular calcium concentration mediated by the expression of NSP4 (Zambrano et al., 2012). One mechanism of iNSP4-mediated, calcium-dependent actin reorganization appears to be through decreased phosphorylation of the actin remodeling protein cofilin (Berkova et al., 2007). In addition to iNSP4 disruption of actin, addition of eNSP4 to the apical media of polarized MDCK-1 cells causes filamentous actin (F-actin) redistribution accompanied by a reduction of transepithelial resistance (Tafazoli et al., 2001). These alterations of the actin cytoskeleton may contribute to RV pathogenesis.

7. iNSP4-mediated apoptosis

Many viruses induce apoptosis for release and dissemination of viral progeny. Apoptosis may also be a contributing factor to pathogenesis through destruction of differentiated absorptive enterocytes. In murine EDIM RV-infected mouse pups, apoptosis, shown by detection of caspase-3 in infected mature small intestinal enterocytes, was hypothesized to be responsible for the observed villous atrophy (Boshuizen et al., 2003). Further in vitro studies in human colon carcinoma HT-29 and fully differentiated Caco-2 cells indicated that RV infection induces apoptosis as demonstrated by peripheral condensation of chromatin and fragmentation of the nuclei (Superti et al., 1998; Chaibi et al., 2005). Apoptosis in RV-infected MA104 cells has been attributed to the activation of Bax, a proapoptotic member of the Bcl-2 family (Martin-Latil et al., 2007).

More recently, NSP4 and NSP1 have been proposed to regulate apoptosis during infection; NSP4 plays a proapoptotic role and NSP1 an antiapoptotic role (Bhowmick et al., 2012 2013). NSP4 has been implicated in activation of the intrinsic apoptosis pathway by depolarizing the mitochondria; this leads to the release of cytochrome c into the cytosol, causing the autocatalytic activation of procaspase-9 that triggers caspase-3 cleavage (Bhowmick et al., 2012). However, apoptosis is inhibited by NSP1 signaling through PI3K/AKT to upregulate XIAP, which inhibits caspase-3 and -9. This NSP1-activated signaling pathway is downregulated later in infection, resulting in apoptosis of the infected cell. In RV-infected and NSP4-expressing cells, nonglycosylated NSP4 (20 kDa) was detected in both the outer and inner mitochondria membranes. The mitochondrial targeting signal sequence was identified as the amphipathic α-helix within the viroporin domain, amino acids 61–83 (Bhowmick et al., 2012 2013).

To further confirm NSP4 localization in both the inner and outer mitochondrial membranes, NSP4 was shown to coimmunoprecipitiate with the outer mitochondrial membrane voltage-dependent anion channel (VDAC) and the inner mitochondrial membrane adenine nucleotide translocator (ANT) (Bhowmick et al., 2012). VDAC facilitates the exchange of ions and molecules between the mitochondria and cytosol and ANT exports ATP from the mitochondrial matrix. NSP4-mediated mitochondria depolarization is proposed to occur through NSP4 interaction with VDAC that may impair the ion supply to the mitochondria and/or interaction with ANT to inhibit ATP exportation. Future studies are needed to address these proposed mechanisms of nonglycosylated NSP4-mediated activation of the intrinsic apoptotic pathway.

8. iNSP4 viroporin-mediated elevation of cytoplasmic calcium

Perturbing the host Ca2+ signaling pathway is not only important in pathogenesis but also for viral replication and morphogenesis. RV infection induces dramatic changes in cellular calcium homeostasis causing a two- to fourfold elevation in cytoplasmic calcium ([Ca2+]cyto). Cellular calcium homeostasis is tightly regulated as calcium ions are ubiquitous intracellular signaling molecules responsible for controlling a plethora of cellular processes. To determine the viral protein(s) responsible for the elevation in [Ca2+]cyto, individual RV proteins were expressed in Sf9 insect cells or a variety of mammalian cell lines and [Ca2+]cyto was measured. NSP4 was identified as the sole RV protein responsible for the increase in [Ca2+]cyto by a PLC-independent mechanism (Tian et al., 1994 1995; Berkova et al., 2007; Diaz et al., 2008 2012). NSP4 expression in cells recapitulates all of the changes in calcium homeostasis observed in RV-infected cells, while silencing of NSP4 expression in infected cells inhibits these changes (Tian et al., 1994; Berkova et al., 2007; Diaz et al., 2008 2012Zambrano et al., 2008). A breakthrough in identifying the mechanism of iNSP4-mediated global disruption in cellular calcium homeostasis was attained when NSP4 was characterized as a viroporin in the ER (Fig. 2.4.2) (Hyser et al., 2010). Viroporins are small, hydrophobic proteins that contain a cluster of basic residues (Lys or Arg) and an amphipathic-α-helix that oligomerizes to create a transmembrane aqueous pore. The NSP4 viroporin domain is comprised of amino acids 47–90, which are critical for elevation of [Ca2+]cyto, since mutation of either the cluster of basic residues or amphipathic α-helix abolishes the observed elevation in [Ca2+]cyto (Hyser et al., 2010). Whether NSP4 viroporin functions as a channel or a pore to elevate cytoplasmic calcium requires further study. Although NSP4 mediates the release of calcium from the ER store into the cytoplasm, this alone is not sufficient to explain the dramatic increase in [Ca2+]cyto or the progressive increase of calcium permeability of the plasma membrane.

The mechanism of NSP4-mediated [Ca2+]cyto was further illuminated when NSP4-mediated depletion of calcium from the ER store was shown to involve store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1) (Hyser et al., 2013). STIM1 is an ER single transmembrane glyco/phosphoprotein that senses ER calcium levels through a low-affinity EF-hand calcium binding site located within its N-terminal domain in the ER lumen. Loss of calcium from the EF-hand induces a conformational change in STIM1 and formation of STIM1 oligomers. The STIM1 oligomers are retained in the ER but rapidly move to ER–PM junctions to activate a variety of calcium-release-activated calcium (CRAC) channels, including Orai1 and TRPC channels, for SOCE. In RV-infected and NSP4-expressing cells, STIM1 colocalizes with and activates Orai1 at the PM for SOCE. In contrast, a NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. These studies provided a mechanism for and confirmed early work indicating iNSP4 release of intracellular Ca2+ stimulated Ca2+ influx from the extracellular medium through the capacitative calcium entry pathway (Tian et al., 1995).

NSP4 viroporin-mediated STIM1 activation of SOCE (Fig. 2.4.3) is supported by pharmacological studies showing that ion channel inhibitors block calcium entry into RV-infected or NSP4-expressing cells. Due to the constitutive activation of STIM1 in RV-infected or NSP4-expressing cells, it is likely that a variety of calcium entry channels are activated, and Orai1, voltage-gated calcium channels and the sodium/calcium exchanger NCX have been implicated. The Orai1 inhibitor 2-APB partially blocks NSP4-mediated calcium entry (Diaz et al., 2012). Similarly, methoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibits the entry of calcium in virus-infected MA104 and HT-29 cells (Perez et al., 1999). Under normal conditions, NCX pumps Ca2+ out of cells using the Na+ gradient as the driving force, but if intracellular Na+ is elevated, as seen in RV-infected cells, NCX will pump Na+ out and bring Ca2+ into the cytoplasm, thus functioning in reverse mode. The inhibitory effect of KB-R7943, a commonly used blocker of NCX functioning in reverse mode, raises the possibility of Ca2+ entry through NCX in its reverse mode in RV-infected or NSP4-expressing cells (Diaz et al., 2012). Thus, while RV may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER.

Another consequence of NSP4 viroporin-mediated increase in [Ca2+]cyto is the activation of the initial stages of autophagy. Autophagy is a cellular degradation process involving an intracellular membrane trafficking pathway that recycles cellular components or eliminates intracellular microbes in lysosomes. RV infection initiates autophagy, characterized by an increase in lipidated LC3 II, a marker of autophagy (Crawford et al., 2012; Crawford and Estes, 2013; Arnoldi et al., 2014). Autophagy initiation is required for the production of infectious virus; pharmacological inhibition of autophagy, RNAi knockdown of autophagy genes and infection of cells genetically deficient in autophagy proteins results in reduced virus yields (Crawford et al., 2012; Crawford and Estes, 2013; Arnoldi et al., 2014). The mechanism of RV-initiated autophagy is through NSP4 viroporin-mediated increases in [Ca2+]cyto that activates a calcium signaling pathway involving CAMKK2 and AMPK (Crawford et al., 2012; Crawford and Estes, 2013). NSP4-initiated autophagy is inhibited by RNAi knockdown of NSP4, expression of a viroporin mutant that does not increase [Ca2+]cyto, and chelation of [Ca2+]cyto following NSP4 expression (Crawford et al., 2012; Crawford and Estes, 2013). Furthermore, a specific inhibitor of CAMKK2, STO-609, not only inhibits AMPK phosphorylation and autophagy induction, but also significantly reduces the yield of infectious virus (Crawford et al., 2012; Crawford and Estes, 2013). Induction of autophagy also leads to the colocalization of NSP4 and LC3 in puncta that traffic to viroplasms that is inhibited by STO-609 (Crawford et al., 2012; Crawford and Estes, 2013).

Although RV infection initiates autophagy, autophagy maturation is inhibited as demonstrated by a lack of autophagosomes and autophagolysosomes (Crawford et al., 2012; Crawford and Estes, 2013; Arnoldi et al., 2014). Instead of forming autophagosomes, LC3 colocalizes with NSP4 surrounding viroplasms (Berkova et al., 2006; Crawford et al., 2012; Crawford and Estes, 2013). Furthermore, colocalization analysis of NSP4 and endogenous LC3 following RV infection indicates that NSP4 colocalizes with LC3 in puncta and traffics to viroplasms. Induction of autophagy and trafficking of ER-localized NSP4 and VP7 to viroplasms is hindered by STO-609 (Crawford et al., 2012; Crawford and Estes, 2013). These results suggest that RV exploits the membrane trafficking function of autophagy to transport the ER-localized viral glycoproteins NSP4 and VP7 to viroplasms to facilitate infectious particle assembly, thus disrupting autophagosome and autophagolysosome formation. These results were confirmed by Arnoldi et al., (Arnoldi et al., 2014) who found that RV replication: (1) induces accumulation of LC3 II; (2) does not lead to accumulation of autophagosomes in spite of significant accumulation of lipidated LC3; and (3) takes advantage of LC3 II for improved production of infectious progeny virus. However, they did not detect LC3 colocalization with NSP4 surrounding viroplasms. We predict that the use of different LC3 antibodies or staining conditions are responsible for the lack of detecting LC3 colocalization with NSP4 surrounding viroplasms by Arnoldi et al. (2014). Nonetheless, RV infection induces autophagy, which is required for infectious particle assembly. Questions remain as to whether immature particles bud through ER membranes or ER-derived membranes.

9. Role of iNSP4 in viral morphogenesis

The ability of NSP4 to interact with multiple viral and cellular proteins may rely on the differential localization of this protein in infected cells during the RV replication process. In the ER, NSP4 interacts with the ER transmembrane chaperone calnexin, which accelerates NSP4 carbohydrate trimming by glucosidase I and II as well as affects the stability of infectious particles. TLPs produced in calnexin-silenced cells are less infectious, less dense in CsCl gradients (1.35 g/cm3) compared to control (1.36 g/cm3) and do not assemble properly suggesting that NSP4–calnexin interactions and ER carbohydrate quality control affect TLP assembly (Mirazimi et al., 1998; Maruri-Avidal et al., 2008).

NSP4 is essential to RV morphogenesis by serving as an intracellular receptor to DLPs (Au et al., 1989; O’Brien et al., 2000). RV genome replication and nascent particle assembly occurs in electron dense viroplasms located in the cytoplasm of the infected cell. NSP4-containing membranes are detected adjacent to viroplasms. The C-terminal cytoplasmic domain of NSP4, amino acids 161–175, binds the inner coat protein (VP6) of DLPs in viroplasms. This interaction triggers the budding of the DLP into the NSP4-containing membranes where the particles become transiently enveloped. The transient envelope is removed by an unknown mechanism and the outer capsid proteins, VP7 and VP4, are assembled onto the particle.

NSP4 has been detected in the ER-Golgi intermediate compartment (ERGIC) and in the plasma membrane (Berkova et al., 2006; Storey et al., 2007; Crawford et al., 2012; Ball et al., 2013; Crawford and Estes, 2013). The form and functional significance of detecting NSP4 in the ERGIC is unknown. However at the plasma membrane, a glycosylated, endoglycosidase H-sensitive form of NSP4 is detected at the apical surface or apical and basolateral surfaces of infected polarized MDCK or HT-29.F8 cells, respectively (Gibbons et al., 2011). It has been proposed that NSP4 is transported to the plasma membrane by interacting with both caveolin and cholesterol; caveolin forms a complex with chaperone proteins that deliver cholesterol from the ER to the plasma membrane, bypassing the Golgi apparatus (Uittenbogaard and Smart, 2000; Storey et al., 2007; Ball et al., 2013). NSP4 colocalizes with caveolin in the ER and the plasma membrane and a domain of NSP4, the hydrophobic face of the NSP4 amphipathic helix, interacts with both the N- and C-terminal domains of caveolin-1. NSP4 contains a cholesterol recognition/interaction amino acid consensus sequence (CRAC) and binds cholesterol at a 1:1 ratio (Fig. 2.4.1C). Transport of NSP4 to the plasma membrane is altered by treatment of cells with the cholesterol disrupting drugs filipin and nystatin. Since reactivity with different NSP4 antibodies revealed that the cytoplasmic tail of NSP4 is exposed on the exofacial surface of the plasma membrane, it was hypothesized that caveolin/cholesterol transport may be the mechanism by which glycosylated, endoglycosidase H-sensitive NSP4 is secreted into the media of infected cells (Gibbons et al., 2011; Schroeder et al., 2012). Further studies are needed to verify this hypothesis.

NSP4 also was found to colocalize with the enterocyte basement membrane during RV infection in suckling mice suggesting this may be a secreted form of NSP4 (Boshuizen et al., 2004). Although the form of NSP4 detected remains uncharacterized, protein interaction and mutation analysis studies mapped domains on NSP4 that interact with the extracellular matrix proteins laminin-3 and fibronectin (Boshuizen et al., 2004). The functional significance of such interactions remains to be fully understood. A nonglycosylated 20K form of NSP4 can also be detected in RV-infected cells (Ericson et al., 1982) and this may be the form recently reported to be associated with mitochondria (see earlier).

10. NSP4-activated immune responses

Roles for NSP4 in innate immune responses have been described. NSP4 has adjuvant properties when coadministered with model antigens in mice (Kavanagh et al., 2010). This response may be through NSP4 functioning through pathogen-associated molecular pattern (PAMP) activity (Ge et al., 2013). Treatment of macrophages with NSP4 purified from the media of RV-infected Caco-2 cells induces secretion of the pro-inflammatory cytokines TNF-α and IL-6 as well as activation of p38 and JNK mitogen-activated protein kinases (MAPKs) and nuclear factor NF-κB (Ge et al., 2013) (Fig. 2.4.3). Secretion of cytokines was mediated through Toll-like receptor 2 (TLR-2) and may provide a mechanism for the production of proinflammatory cytokines associated with the clinical symptoms of infection in humans and animals.

In addition, a role for NSP4 in injury to the extrahepatic biliary epithelium that may be associated with biliary atresia in neonatal mice has been found (Feng et al., 2011; Zheng et al., 2014). Biliary atresia is an obliterative cholangiopathy with progressive hepatobiliary disease. Although the pathogenic mechanisms of biliary atresia remain largely unknown, interferon (IFN)-γ- driven and CD8+ T-cell-dependent inflammatory injury to extrahepatic biliary epithelium is likely to be involved in the development of biliary atresia in a neonatal mouse model of this disease. Injection of NSP4 peptides, amino acids 144–152 or 157–170, into neonatal mice increased IFN-γ release by CD8+ T cells, elevated the population of hepatic memory CD8+ T cells, and augmented cytotoxicity of CD8+ T cells to rhesus RV-infected or naïve EHBE cells (Zheng et al., 2014). Furthermore, immunization of mouse dams with GST-NSP4, or the NSP4 peptides 144–152 or 157–170 decreased the incidence of rhesus RV-induced biliary atresia in their offspring. These results support a role for autoimmune responses to NSP4 in the pathogenesis of experimental biliary atresia.

11. Concluding remarks and summary

Due to their limited coding capacities, most viruses encode multifunctional proteins that accomplish a variety of tasks during infection. NSP4 is a pleiotropic protein that performs many functions as described in this review. Our knowledge of the molecular mechanisms by which iNSP4 and eNSP4 mediate RV-induced pathogenesis, replication, and morphogenesis has increased over the last years. However, additional studies are needed to elucidate the different structural forms of NSP4 in order to augment biochemical and functional studies addressing the many functions that NSP4 performs during RV infection. Many questions remain for the structure of NSP4 and how it facilitates multiple functions of NSP4 including the functions of eNSP4 and iNSP4 in infection and pathogenesis (Table 2.4.2).

Table 2.4.2

Questions for Future Research on NSP4

What is structure of full-length NSP4?

What are the functions of the conserved domains identified in the amino acid alignment of the E genotypes?

How do the different NSP4 interacting domains bind to so many different partners? Structure-dependent? Spatiotemporal-dependent? pH-dependent? Cooperative-binding?
How does eNSP4 traffic to the plasma membrane for secretion?

Is NSP4-integrin interaction necessary and sufficient for diarrhea induction?

Are there unknown functions for eNSP4?
How does iNSP4 traffic to different subcellular compartments?

Is the NSP4 viroporin an ion channel?

Does binding of other viral or cellular proteins to domains of iNSP4 modulate any of the iNSP4 functions?

Are other signaling pathways activated by the NSP4-mediated increase in cytoplasm calcium?

How does NSP4 interaction with DLPs trigger the budding process?

Does NSP4 play a role in removal of the transient envelope?

Does NSP4 mediate the assembly of the outer capsid proteins onto DLPs?

Are there unknown functions for iNSP4?

Knowledge of RV-induced disease pathogenesis is based primarily on studies using animal rotaviruses, which grow well in cultured cells and in animal models, or in vitro studies in immortalized or cancer cell lines. New models to understand human rotavirus infection are needed. New models that show promise are human intestinal organoids or enteroids produced from approved stem cell lines or from human intestinal biopsies or tissue, respectively; these models are the first physiologically active ex vivo model of the human intestine for studying RV-induced pathophysiology (Finkbeiner et al., 2012; Kovbasnjuk et al., 2013; Foulke-Abel et al., 2014; Saxena et al., 2015; Zachos et al., 2015). Use of these nontransformed human miniguts that include all epithelial cell types normally present in the human small intestine may identify the form of eNSP4 produced in the infected human intestinal cells and clarify whether the form of eNSP4 produced and its functions are strain-dependent. These systems will help us better understand the roles of eNSP4 and iNSP4 following infection with human RV strains and may identify new functions for these proteins.


We gratefully acknowledge partial support of our rotavirus research from NIH grants R01 AI080656 and P30 DK56338 (M.K.E.) and R37 AI36040 (B.V.V. Prasad). We also thank Dr Beatrice Boniotti, Istituto Zooprofilattico Sperimentale della Lombardia e dell’ Emilia Romagna “Bruno Ubertini,” Brescia, Italy for sharing the sequence of the NSP4 from a fox prior to its publication.


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Apr 25, 2018 | Posted by in MICROBIOLOGY | Comments Off on Pleiotropic Properties of Rotavirus Nonstructural Protein 4 (NSP4) and Their Effects on Viral Replication and Pathogenesis
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