Rotavirus NSP4 Research Articles

The structural basis of IRF-3 recruitment, activation, and targeting in innate immunity

Abstract Type I interferons (IFNs) are key cytokines mediating innate antiviral immunity. cGAS, RLRs, and TLRs recognize microbial DNA, RNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of IRF-3. STING, MAVS, and TRIF mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, while residues upstream of the motif confer specificity. The structure of IRF-3 phosphomimetic mutant S386/396E bound to CBP reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 employs a pLxIS motif to target IRF-3 for degradation. However, phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune response.

Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases.

The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity. The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.

Challenging the Roles of NSP3 and Untranslated Regions in Rotavirus mRNA Translation.

Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex for rotavirus mRNAs. To further explore the effects of NSP3 and untranslated regions (UTRs) on rotavirus mRNAs translation, we used a quantitative in vivo assay with simultaneous cytoplasmic NSP3 expression (wild-type or deletion mutant) and electroporated rotavirus-like and standard synthetic mRNAs. This assay shows that the last four GACC nucleotides of viral mRNA are essential for efficient translation and that both the NSP3 eIF4G- and RNA-binding domains are required. We also show efficient translation of rotavirus-like mRNAs even with a 5’UTR as short as 5 nucleotides, while more than eleven nucleotides are required for the 3’UTR. Despite the weak requirement for a long 5’UTR, a good AUG environment remains a requirement for rotavirus mRNAs translation.

Rotavirus-induced miR-142-5p elicits proviral milieu by targeting non-canonical transforming growth factor beta signalling and apoptosis in cells.

MicroRNA (miRNA) expression is significantly influenced by viral infection, because of either host antiviral defences or proviral factors resulting in the modulation of viral propagation. This study was undertaken to identify and analyse the significance of cellular miRNAs during rotavirus (SA11 or KU) infection. Sixteen differentially regulated miRNAs were identified during rotavirus infection of which hsa-miR-142-5p was up-regulated and validated by quantitative polymerase chain reaction. Exogenous expression of miR-142-5p inhibitor resulted in a significant reduction of viral titer indicating proviral role of miR-142-5p. Functional studies of hsa-miR-142-5p identified its role in transforming growth factor beta (TGFβ) signalling as TGFβ receptor 2 and SMAD3 were degraded during both hsa-miR-142-5p overexpression and rotavirus infection. TGFβ is induced during rotavirus infection, which may promote apoptosis by activation of non-canonical pathways in HT29 cells. However, up-regulated miR-142-5p resulted in the inhibition of TGFβ-induced apoptosis suggesting its anti-apoptotic function. Rotavirus NSP5 was identified as a regulator of miR-142-5p expression. Concurrently, NSP5-HT29 cells showed inhibition of TGFβ-induced apoptosis and epithelial to mesenchymal transition by blocking non-canonical pathways. Overall, the study identified proviral function of hsa-miR-142-5p during rotavirus infection. In addition, modulation of TGFβ-induced non-canonical signalling in microsatellite stable colon cancer cells can be exploited for cancer therapeutics.

Diagnostic Study on NSP5 of Human Rotavirus in Najaf Governorate

Objectives: Human rotavirus is the common cause of diarrhea-related illness and death among infants and young children, can lead to severe and sometimes lethal dehydration . Methodology: This study deals with the prevalence of rotavirus during a period extending of March to December 2014 . A total of 120 samples of diarrheal samples from patients up to 5 years age, who were admitted for medically care unit in the hospital of pediatrics and maternity of Al-Najaf governorate were investigated by latex agglutination, based on specific monoclonal antibodies directed against VP4 and VP7 of human rotavirus then the detection by PCR technique and isolation of the virus on these culture. Results: Rota virus was detected in 80 cases (66.7%) of the total cases investigated by latex agglutination test, while PCR positivity was recorded in 85 cases (70.8%). Rota virus infection was also detected in healthy children who were involved as control group; 1/50(2%) detected by Latex agglutination assay versus 3/50 (6%) detected by NSP5 gene PCR assay. From the total of ten isolates that selected to propagate via cell culture using rhabdomyosarcoma (RD) cells, confirmation of the detection was accomplished by 8 isolates (80 %). Conclusions: We concluded that polymerase chain reaction is sensitive and specific technique for the detection of NSP5 coding gene and RD cell line is successful for the propagation of rotavirus. Recommendations: we recommended to achieve future therapeutic studies depending on this study conclusions.Key words: RD cell culture, rotavirus NSP5, real-time PCR.

Rotavirus disrupts cytoplasmic P bodies during infection

Cytoplasmic Processing bodies (P bodies), the RNA-protein aggregation foci of translationally stalled and potentially decaying mRNA, have been reported to be differentially modulated by viruses. Rotavirus, the causative agent of acute infantile gastroenteritis is a double stranded RNA virus which completes its entire life-cycle exclusively in host cell cytoplasm. In this study, the fate of P bodies was investigated upon rotavirus infection. It was found that P bodies get disrupted during rotavirus infection. The disruption occurred by more than one different mechanism where deadenylating P body component Pan3 was degraded by rotavirus NSP1 and exonuclease XRN1 along with the decapping enzyme hDCP1a were relocalized from cytoplasm to nucleus. Overall the study highlights decay and subcellular relocalization of P body components as novel mechanisms by which rotavirus subverts cellular antiviral responses.

Rotavirus NSP4 protein as a viral biotoxin selectively promotes cytotoxicity

Rotavirus NSP4 protein as a viral biotoxin selectively promotes cytotoxicity

Rotavirus NSP3 Is a Translational Surrogate of the Poly(A) Binding Protein-Poly(A) Complex

Through its interaction with the 5' translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5'-capped and 3'-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3' GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3' end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery.

Solar and temperature treatments affect the ability of human rotavirus wa to bind to host cells and synthesize viral RNA.

Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation.

CD8+ T lymphocyte response against extrahepatic biliary epithelium is activated by epitopes within NSP4 in experimental biliary atresia.

Interferon (IFN)-γ-driven and CD8+ T cell-dependent inflammatory injury to extrahepatic biliary epithelium (EHBE) is likely to be involved in the development of biliary atresia (BA). We previously showed that viral protein NSP4 is the pathogenic immunogen that causes biliary injury in BA. In this study, NSP4 or four synthetic NSP4 (NSP4(157-170), NSP4(144-152), NSP4(93-110), NSP4(24-32)) identified by computer analysis as candidate CD8+ T cell epitopes were injected into neonatal mice. The pathogenic NSP4 epitopes were confirmed by studying extrahepatic bile duct injury, IFN-γ release and CD8+ T cell response against EHBE. The results revealed, at 7 days postinjection, inoculation of glutathione S-transferase (GST)-NSP4 caused EHBE injury and BA in neonatal mice. At 7 or 14 days postinject, inoculation of GST-NSP4, NSP4(144-152), or NSP4(157-170) 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 rotavirus (RRV)-infected or naive EHBE cells. Furthermore, depletion of CD8+ T cells in mice abrogated the elevation of GST-NSP4-induced serum IFN-γ. Lastly, parenteral immunization of mouse dams with GST-NSP4, NSP4(144-152), or NSP4(157-170) decreased the incidence of RRV-induced BA in their offspring. Overall, this study reports the CD8+ T cell response against EHBE is activated by epitopes within rotavirus NSP4 in experimental BA. Neonatal passive immunization by maternal vaccination against NSP4(144-152) or NSP4(157-170) is effective in protecting neonates from developing RRV-related BA.

Characterization of the NSP4 gene of group A human rotavirus G1P[8] strains circulating in Sapporo, Japan from 1987 to 2000

The genetic diversity of the NSP4 gene of rotavirus G1P[8] strains obtained in Sapporo was analyzed, Japan from 1987 to 2000. Sixty-four strains, which were distributed across the whole study period, were included. All G1P[8] NSP4 genes detected in this study belonged to genotype E1, which divided into at least three lineages. The Sapporo rotavirus G1P[8] isolates were found in each lineage. The mean estimated substitution rate was 1.40 × 10(-3) nucleotide substitutions per site per year, which was comparable to that of the G1P[8] VP7 gene. Comparison of the deduced NSP4 amino acid sequences showed genetic diversity at the center of antigenic site II, but not in the enterotoxic domain. This report represents the first investigation of the genetic diversity and evolution of group A rotavirus NSP4 genes in Asia.

232: Inhibition of IFN-mediated STAT1 activation in intestinal epithelial cells by NSP1 – The rotavirus-encoded inhibitor of IFN induction

Rotavirus (RV) replication in intestinal epithelial cells (IECs) in vivo is robust despite the vigorous activation of local early host interferon (IFN) responses. Using single cell microfluidics qRT-PCR coupled with flow sorting, we recently demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo and, using genetic approaches, associated this function with the RV NSP1 protein. Paradoxically, RV also induces robust type I IFNs in the intestinal hemaotopoietic compartment in a relatively replication-independent manner raising the possibility that RV replication and spread in IECs occurs despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. However, viral factor(s) responsible for the possible inhibition of STAT1 function are unknown. Here we report the ability of RV to mediate direct and remote inhibition of IFN-mediated STAT1 activation in IECs and identify the RV NSP1 protein as a direct inhibitor of the pathway. Infection of human IECs with RV strains that inhibit IFN induction by two distinct strategies targeting either NF-kB or IRF3 both resulted in effective regulation of IFN secretion. At early times during infection, neither type of RV strain could effectively inhibit the activation of STAT1 in response to exogenously added IFN. However, at later times in infection, both types of RV could inhibit IFN-mediated STAT1 activation, and by single cell analysis this blockage occurred in both infected and bystander cell populations. We found that the rotavirus NSP1 protein, when expressed by itself, is an efficient inhibitor of STAT1 activation. Analysis of NSP1 determinants in the inhibition of IFN induction and signaling pathways revealed that these properties are encoded on discrete domains. These findings explain previously observed remote regulation of the host IFN response in the intestine and uncover a novel function of NSP1 as a STAT1 antagonist.

Rotavirus NSP1 Mediates Degradation of Interferon Regulatory Factors through Targeting of the Dimerization Domain

Rotavirus nonstructural protein NSP1 can inhibit expression of interferon (IFN) and IFN-stimulated gene products by inducing proteasome-mediated degradation of IFN-regulatory factors (IRFs), including IRF3, IRF5, and IRF7. All IRF proteins share an N-terminal DNA-binding domain (DBD), and IRF3, IRF5, and IRF7 contain a similar C-proximal IRF association domain (IAD) that mediates IRF dimerization. An autoinhibitory domain (ID) at the extreme C terminus interacts with the IAD, burying residues necessary for IRF dimerization. Phosphorylation of serine/threonine residues in the ID induces charge repulsions that unmask the IAD, enabling IRF dimerization and subsequent nuclear translocation. To define the region of IRF proteins targeted for degradation by NSP1, we generated IRF3 and IRF7 truncation mutants and transiently expressed each with simian SA11-4F NSP1. These assays indicated that the IAD represented a necessary and sufficient target for degradation. Because NSP1 did not mediate degradation of truncated forms of the IAD, NSP1 likely requires a structurally intact IAD for recognition and targeting of IRF proteins. IRF9, which contains an IAD-like region that directs interactions with signal inducer and activator of transcription (STAT) proteins, was also targeted for degradation by NSP1, while IRF1, which lacks an IAD, was not. Analysis of mutant forms of IRF3 unable to undergo dimerization or that were constitutively dimeric showed that both were targeted for degradation by NSP1. These results indicate that SA11-4F NSP1 can induce degradation of inactive and activated forms of IAD-containing IRF proteins (IRF3 to IRF9), allowing a multipronged attack on IFN-based pathways that promote antiviral innate and adaptive immune responses.

Molecular Characterization of the Porcine Group A Rotavirus NSP2 and NSP5/6 Genes from São Paulo State, Brazil, in 2011/12

Rotaviruses are responsible for the acute diarrhea in various mammalian and avian species. The nonstructural proteins NSP2 and NSP5 are involved in the rotavirus replication and the formation of viroplasm, cytoplasmic inclusion bodies within which new viral particles morphogenesis and viral RNA replication occur. There are few studies on the genetic diversity of those proteins; thus this study aims at characterizing the diversity of rotavirus based on NSP2 and NSP5 genes in rotaviruses circulating in Brazilian pig farms. For this purpose, 63 fecal samples from pig farms located in six different cities in the São Paulo State, Brazil, were screened by nested RT-PCR. Seven strains had the partial nucleotide sequencing for NSP2, whereas in six, the total sequencing for NSP5. All were characterized as genotype H1 and N1. The nucleotide identity of NSP2 genes ranged from 100% to 86.4% and the amino acid identity from 100% to 91.5%. For NSP5, the nucleotide identity was from 100% to 95.1% and the amino acid identity from 100% to 97.4%. It is concluded that the genotypes of the strains circulating in the region of study are in agreement with those reported in the literature for swine and that there is the possibility of interaction between human and animal rotaviruses.

Molecular Mechanism behind Rotavirus NSP1-Mediated PI3 Kinase Activation: Interaction between NSP1 and the p85 Subunit of PI3 Kinase

Our previous study had reported on the interaction of rotavirus NSP1 with cellular phosphoinositide 3-kinase (PI3K) during activation of the PI3K pathway (P. Bagchi et al., J. Virol. 84:6834-6845, 2010). In this study, we have analyzed the molecular mechanism behind this interaction. Results showed that this interaction is direct and that both α and β isomers of the PI3K regulatory subunit p85 and full-length NSP1 are important for this interaction, which results in efficient activation of the PI3K/Akt pathway during rotavirus infection.

Novel NSP4 genotype in a camel G10P[15] rotavirus strain

In this study a Kuwaiti camel rotavirus strain, RVA/Camel-wt/KUW/s21/2010/G10P[15], is characterized by sequencing and phylogenetic analysis. The strain had multiple genes with high nucleotide sequence similarities to ovine and bovine strains (VP2, ≤ 96%; NSP2 and NSP5, ≤ 97%, NSP3, ≤ 94%), or, to porcine strains (VP1, ≤ 89%). Other genes had moderate sequence similarities (VP4, ≤ 87%; VP6, ≤ 81%; VP7, ≤ 82%) with reference strains from ruminants. The NSP4 gene shared limited sequence identity (≤ 71%) with other mammalian and avian rotavirus NSP4 types, and was designated a novel genotype, E15. This study demonstrates genetic diversity in the outer capsid and some backbone genes of an old-world camelid rotavirus strain and uncovers its common evolutionary roots with strains from other ruminants.

Evidence of discordant genetic linkage in the VP4, VP6, VP7 and NSP4 encoding genes of rotavirus strains from adolescent and adult patients with acute gastroenteritis

The vast diversity within rotavirus strains circulating in the developing countries continues to be a major challenge for the efficacy of currently used preset rotavirus vaccines. The sequence analysis and phylogeny of multiple genes of rotavirus strains enable identification of reassortant strains and their human or animal origin. The objective of this study was to monitor the genetic linkage between the rotavirus VP4(P), VP6(I), VP7(G) and NSP4(E) encoding genes. The G, P, I and E genotypes of a total of 80 rotavirus strains isolated from adolescent and adult cases of acute gastroenteritis at the two time points [1993–1996 (n=67) and 2004–2007 (n=13)] were determined by nucleotide sequencing and phylogenetic analysis. The rotavirus strains from the 1990s and 2000s revealed common combinations of genotypes (G1–P[8]–I1–E1, G2–P[4]–I2–E2, G3–P[8]–I1–E1 and G4–P[8]–I1–E1) in 47.8% and 30.8%, unusual combinations of the same genotypes (G2–P[8]–I2–E2, G9–P[6]–I1–E1, G9–P[6]–I1–E2, G9–P[6]–I2–E1 and G4–P[4]–I1–E2, G1–P[4]–I2–E1, G9–P[4]–I1–E1) in 7.5% and 23% and mixed infections of different G and P genotypes in 31.3% and 46.2%, respectively. Discordance in the association of I with E, G with I and E and P with I and E genotypes was found to be contributed respectively by 23.8–38.5%, 40.3–69.8% and 49.3–61.5% of the rotavirus strains at the two time points.The data suggest relatively high occurrence of intergenogroup reassortment in circulating rotavirus strains emphasizing the need for continuous surveillance and whole genome sequence based characterization of rotavirus strains for better understanding of their evolution and ecology.

Dissecting the Ca2+ entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells

Rotavirus infection modifies Ca2+ homeostasis provoking an increase in Ca2+ permeation, cytoplasmic Ca2+ concentration ([Ca2+]cyto), total Ca2+ pools and, a decrease of Ca2+ response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca2+ homeostasis is not yet known. It has been proposed that the increase in [Ca2+]cyto is the result of Ca2+ release from intracellular stores, thereby activating store-operated Ca2+ entry (SOCE). We studied the mechanisms involved in the changes of Ca2+ permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca2+ stores induced by thapsigargin or ATP was not able to elicit Ca2+ entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca2+]cyto, is permeable to Mn2+ and inhibited by La3+ and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca2+ entry is small and not modified by infection. The plasma membrane permeability to Ca2+ in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na+/Ca2+ exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca2+ entry.

Antigenemia, RNAemia, and innate immunity in children with acute rotavirus diarrhea

Antigenemia is commonly detected in children with acute rotavirus diarrhea, but the prevalence of viremia has not been clearly defined. We examined antigenemia in plasma and RNAemia in peripheral blood mononuclear cells (PBMC) of children with acute diarrhea by EIA, RT-PCR, and Southern hybridization, using primers and a probe specific to rotavirus NSP4 gene. We detected the presence of rotavirus antigen in 33.3% and almost full-length NSP4 gene in 70.8% of the acute-phase plasma and PBMC, respectively. In contrast, antigenemia and RNAemia were detected in 0% and 4.2% of the convalescent-phase plasma and PBMC, respectively, which were similar to antigenemia (0%) and RNAemia (7.7%) in healthy controls. We demonstrated an increase in the proportions of activated myeloid dendritic cells (mDC) and activated plasmacytoid DC (pDC) in acute-phase PBMC of patients when compared to those in convalescent phase of patients and in PBMC of healthy controls. The activation of mDC peaked on days 2-4 after illness onset, and the activation of acute-phase pDC appeared to correlate with levels of antigenemia. High prevalence of NSP4 gene in acute-phase PBMC indicates possible rotavirus replication in white blood cells, and extraintestinal spread and the activation of DC may have implications for the prevention of rotavirus disease in children.

Elucidation of the Rotavirus NSP4-Caveolin-1 and -Cholesterol Interactions Using Synthetic Peptides

Rotavirus (RV) NSP4, the first described viral enterotoxin, is a multifunctional glycoprotein that contributes to viral pathogenesis, morphogenesis, and replication. NSP4 binds both termini of caveolin-1 and is isolated from caveolae fractions that are rich in anionic phospholipids and cholesterol. These interactions indicate that cholesterol/caveolin-1 plays a role in NSP4 transport to the cell surface, which is essential to its enterotoxic activity. Synthetic peptides were utilized to identify target(s) of intervention by exploring the NSP4-caveolin-1 and -cholesterol interactions. NSP4112–140 that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus (CRAC) motif and both termini of caveolin-1 (N-caveolin-12–20, 19–40 and C-caveolin-1161–180) were synthesized. Direct fluorescence-binding assays were employed to determine binding affinities of the NSP4-caveolin-1 peptides and cholesterol. Intracellular cholesterol alteration revealed a redistribution of NSP4 and disintegration of viroplasms. These data further imply interruption of NSP4112–140-N-caveolin-119–40 and cholesterol interactions may block NSP4 intracellular transport, hence enterotoxicity.