A lack of consistent amino acid substitutions in NSP4 between rotaviruses derived from diarrheal and asymptomatically-infected kittens

Nonstructural glycoprotein NSP4 of group A rotavirus has recently been shown to be a viral enterotoxin, inducing diarrhea in neonatal mice. Literature is conflicting as to whether there is any consistent amino acid substitution between virulent (or symptomatic) and attenuated (or asymptomatic) rotavirus strains. We have sequenced and compared the NSP4 sequences derived from a total of 10 geographically- and serologically-related feline rotavirus strains from both diarrheal and asymptomatically-infected kittens. These NSP4 sequences were closely related to each other and there were differences at 19 amino acid residues, but none was segregated according to whether the strain was isolated from a diarrheal kitten or not. Thus, this study failed to lend support to the contention that mutations in NSP4 play a significant role in the pathogenesis of rotavirus diarrhea. Involvement of other genes may explain the outcome of infection in cats from which these 10 feline rotaviruses were isolated.     

A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells

Previous studies have shown that the nonstructural glycoprotein NSP4 plays a role in rotavirus pathogenesis by functioning as an enterotoxin. One prediction of the mechanism of action of this enterotoxin was that it is secreted from virus-infected cells. In this study, the media of cultured (i) insect cells infected with a recombinant baculovirus expressing NSP4, (ii) monkey kidney (MA104) cells infected with the simian (SA11) or porcine attenuated (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western blotting, immunoprecipitation and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparent molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 aa112-175). The secretion of NSP4 aa112-175 was not affected by treatment of cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network. A partial gene fragment coding for NSP4 aa112-175 was cloned and expressed using the baculovirus-insect cell system. Purified NSP4 aa112-175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expressed endogenously, similarly to full-length NSP4. NSP4 aa112-175 caused diarrhea in neonatal mice, as did full-length NSP4. These results indicate that NSP4 aa112-175 is a functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.     

Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene.

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.     

Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen.

A single-gene substitution reassortant 11-1 was generated from two porcine rotaviruses, OSU (serotype 5) and Gottfried (serotype 4). This reassortant derived 10 genes, including gene 4 encoding VP3, from the OSU strain and only gene 9, encoding a major neutralization glycoprotein (VP7), from the Gottfried strain and was thus designated VP3:5; VP7:4. Oral administration of this reassortant to colostrum-deprived gnotobiotic newborn pigs induced a high level of neutralizing antibodies not only to Gottfried VP7 but also to OSU VP3, thus demonstrating that VP3 is as potent an immunogen as VP7 in inducing neutralizing antibodies during experimental oral infection. Gnotobiotic piglets infected previously with the reassortant were completely resistant to oral challenge with the virulent Gottfried strain (VP3:4; VP7:4), as indicated by failure of symptoms to develop and lack of virus shedding. Similarly, prior infection with the reassortant induced almost complete protection against diarrhea and significant restriction of virus replication after oral challenge with the virulent OSU strain (VP3:5; VP7:5). Thus, it appears that (i) the immune system of the piglet responds equally well to two rotavirus outer capsid proteins, VP3 and VP7, during primary enteric rotavirus infection; (ii) antibody to VP3 and antibody to VP7 are each associated with resistance to diarrhea; and (iii) infection with a reassortant rotavirus bearing VP3 and VP7 neutralization antigens derived from two viruses of different serotype induces immunity to both parental viruses. The relevance of these findings to the development of effective reassortant rotavirus vaccines is discussed.     

Expression and purification of polyhistidine-tagged rotavirus NSP4 proteins in insect cells

The rotavirus nonstructural NSP4 protein, a transmembrane endoplasmic reticulum-specific glycoprotein, has been described as the first viral enterotoxin. Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induces diarrhea in young mice. NSP4 has a membrane-destabilizing activity and causes an increase in intracellular calcium levels and chloride secretion by a calcium-dependent signalling pathway in eucaryotic cells. In this study, four recombinant baculoviruses were generated expressing the rotavirus NSP4 glycoprotein from the human strains Wa and Ito, the porcine strain OSU, and the simian strain SA11, which belong to two different NSP4 genotypes, A and B. The recombinant glycoproteins, expressed as polyhistidine-tagged molecules, were analyzed by Western blotting and immunoprecipitation. Newborn mice responded with diarrhea after inoculation with each of the recombinant NSP4 proteins.     

Permissive Replication of Homologous Murine Rotavirus in the Mouse Intestine Is Primarily Regulated by VP4 and NSP1

Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, nonstructural protein 1 (NSP1), and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts, and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000- to 10,000-fold greater than that of a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 reduced murine RV infectivity only slightly; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

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 alpha-helix located between aa 73 and 85 (AAH73-85) at the N terminus of DeltaN72, 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, AAH73-85, 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.     

Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectin

Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established.     

人轮状病毒NSP4蛋白的表达及其致小鼠腹泻作用的初步研究

研究人轮状病毒非结构蛋白NSP4在轮状病毒致病性中的作用。分离得到我国人轮状病毒97SZ8株,以谷胱甘肽S-转移酶融合蛋白的形式在大肠杆菌BL-21中表达NSN蛋白C端86-175氨基酸并用G1utathione SepharoseTM 4B亲和纯化。将纯化蛋白分别以0．4nmol和1．5nmol的剂量腹腔注射新生Balb／C乳鼠,记录腹泻发生和体重变化情况。当注射0．4nmol GST-NSP4重组蛋白时,有1只小鼠发生-过性腹泻(1／6),给予1．5nmol重组蛋白时,实验组所有乳鼠都先后出现了腹泻,存在一定的剂量依赖性。本研究初步在新生小鼠建立了一种人轮状病毒腹泻动物模型,该模型有望在人轮状病毒的致腹泻机理、治疗和预防研究中发挥重要作用。     

轮状病毒疫苗研究进展及其转基因植物疫苗的开发前景

轮状病毒是目前婴幼儿秋冬季腹泻的最主要病原物,作者通过轮状病毒减毒株疫苗,亚单位疫苗和DNA疫苗研究进展的综合分析,指出了减毒株疫苗在实际应用中存在的弊端,论述了开发新型轮状病毒疫苗,特别是转基因植物疫苗的必要性和可行性。     

Antigenic analysis of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13

In order to analyze the antigenic structure of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13, 25 monoclonal antibodies (MAbs) against NSP4 expressed in Escherichia coli were produced. All MAbs reacted with NSP4 on Western blotting, indicating that they recognized sequential epitopes. To determine the antigenic sites (ASs) recognized by the produced MAbs, seven truncated NSP4s were expressed in E. coli. Western blotting analysis showed that there are at least four major ASs on PO-13 NSP4, designated as AS I located in amino acids (aa) 151 to 169, AS II (aa 136 to 150), AS III (aa 112 to 133) and AS IV (aa 1 to 24). Two MAbs reacted exclusively with AS III encompassing the region that has been reported to be an enterotoxin domain. MAbs against ASs II, III and IV reacted with all avian rotaviruses tested by indirect immunofluorescent antibody assays. MAbs against AS I reacted with turkey strains, Ty-1 and Ty-3, but not with a chicken strain, Ch-1. Nine of 11 MAbs against AS II cross-reacted with NSP4 of mammalian rotavirus strains with different NSP4 genotypes. These results suggest that AS II on NSP4 is widely conserved among a variety of rotaviruses.     

Group A Rotavirus Infection and Age-Dependent Diarrheal Disease in Rats: a New Animal Model To Study the Pathophysiology of Rotavirus Infection

Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (EC(wt)) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not > or =21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naIve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine EC(wt) rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.     

Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins vp3 and vp7.

Monoclonal antibodies directed against two rotavirus surface proteins (vp3 and vp7) as well as a rotavirus inner capsid protein (vp6) were tested for their ability to protect suckling mice against virulent rotavirus challenge. Monoclonal antibodies to two distinct epitopes of vp7 of simian rotavirus strain RRV neutralized RRV in vitro and passively protected suckling mice against RRV challenge. A monoclonal antibody directed against vp3 of porcine rotavirus strain OSU neutralized three distinct serotypes in vitro (OSU, RRV, and UK) and passively protected suckling mice against OSU, RRV, and UK virus-induced diarrhea. The role of vp3 in eliciting protection against heterotypic rotavirus challenge should be considered when developing a vaccine with cloned rotavirus genes. Alternatively, immunization with a reassortant rotavirus containing vp3 and vp7 from two antigenically distinct rotavirus parents might protect against diarrhea induced by two or more rotavirus serotypes.     

人轮状病毒NSP4基因变异与功能关系的初步研究

在比较我国人A组轮状病毒一般腹泻患者分离株和重症患者分离株非结构蛋白(NSP)4 cDNA序列时发现,两者在可能与致病性有关的区域(aa131～146)内存在着显著的差异.为进一步探讨这种变异是否与毒力改变有关,利用杆状病毒表达载体在昆虫细胞Sf9中表达两种毒株的NSP4,通过激光扫描共聚焦显微镜初步观察了它对细胞内钙离子浓度的影响.结果表明:两种来源的NSP4均可使细胞内钙离子浓度明显升高,在48h时大致升高3.1～3.4倍,96h时升高5.6～5.8倍,但两种毒株之间的差别并不明显.研究证实,人轮状病毒NSP4与以往报道的动物轮状病毒NSP4一样,可以引起细胞内钙离子增高,即可能与病毒的致病性有关.但重症腹泻毒株SZ1 NSP4第131～146位氨基酸位点出现的变异并未提高其毒力.轮状病毒的毒力改变可能与其它因素有关.     

我国人轮状病毒不同基因型NSP4的致病性研究

对我国轮状病毒流行株NSP4基因变异特点的分析表明,NSP4基因主要可分为Wa组和Kun组,在Wa组内可形成三个亚组,形成了4种NSP4基因型。为了进一步阐明人轮状病毒流行株NSP4基因变异与其致病性变化是否存在联系,我们首先利用杆状病毒载体对NSP4蛋白进行表达,获得了对应4种不同NSP4基因型的重组杆状病毒rvBac97B6,rvBac97S34,rvBac97S36和rvBac97SZ8。用这些病毒感染Sf9细胞后,检测细胞内Ca2 浓度的变化,发现与野生型杆状病毒感染细胞相比,重组病毒感染细胞内的Ca2 浓度显著升高,但各个重组病毒之间无显著性差异。在此基础上,我们进一步在E.coli中分别表达纯化了代表Wa和Kun基因分组的97S34和97SZ8流行株的NSP4。分别用纯化的重组NSP4蛋白攻击乳鼠后,发现不同基因型的NSP4蛋白的致腹泻活性没有明显差异,这种作用可被NSP4抗体拮抗,但这种拮抗作用存在基因型特异性。上述结果表明人轮状病毒流行株NSP4氨基酸序列间的变异并没有使其钙调节及致腹泻能力产生改变,在致腹泻作用中发挥关键作用(或决定性作用)的氨基酸位点在不同NSP4基因型间可能是相对保守的。针对NSP4抗体的有效性也为新型轮状病毒疫苗和药物研究提供了线索。     

Direct inhibitory effect of rotavirus NSP4(114-135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of D-glucose and of L-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na(+)-D-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na(+)-L-leucine symport about equally and partially via a different transport mechanism, in that Na(+) behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.     

Mechanisms controlling virulence thresholds of mixed viral populations

The propensity of RNA viruses to revert attenuating mutations contributes to disease and complicates vaccine development. Despite the presence of virulent revertant viruses in some live-attenuated vaccines, disease from vaccination is rare. This suggests that in mixed viral populations, attenuated viruses may limit the pathogenesis of virulent viruses, thus establishing a virulence threshold. Here we examined virulence thresholds using mixtures of virulent and attenuated viruses in a transgenic mouse model of poliovirus infection. We determined that a 1,000-fold excess of the attenuated Sabin strain of poliovirus was protective against disease induced by the virulent Mahoney strain. Protection was induced locally, and inactivated virus conferred protection. Treatment with a poliovirus receptor-blocking antibody phenocopied the protective effect of inactivated viruses in vitro and in vivo, suggesting that one mechanism controlling virulence thresholds may be competition for a viral receptor. Additionally, the type I interferon response reduces poliovirus pathogenesis; therefore, we examined virulence thresholds in mice lacking the alpha/beta interferon receptor. We found that the attenuated virus was virulent in immunodeficient mice due to the enhanced replication and reversion of attenuating mutations. Therefore, while the type I interferon response limits the virulence of the attenuated strain by reducing replication, protection from disease conferred by the attenuated strain in immunocompetent mice can occur independently of replication. Our results identified mechanisms controlling the virulence of mixed viral populations and indicate that live-attenuated vaccines containing virulent virus may be safe, as long as virulent viruses are present at levels below a critical threshold.