Rotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4's appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.

Rotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4’s appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.

Rotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4’s appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.

Rotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4's appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.

Rotavirus (RV) nonstructural protein 4 (NSP4) is a multifunctional glycoprotein that induces secretory diarrhea in mouse pups in the absence of other viral proteins. The intracellular transport route(s) and functional mechanism(s) of NSP4 are poorly understood; however, the recent association of the enterotoxin with cellular caveolin-1 may provide a link between NSP4 transport and function. To determine if NSP4 traffics to a specific subset of lipid rafts at the plasma membrane (PM), we isolated caveolae from a PM-enriched fraction with a new method that yielded endoplasmic reticulum (ER)-free caveolae membranes with a unique membrane structure and composition. Comparison of these caveolae with other detergent- and non-detergent-extracted membranes revealed that each caveolae/raft fraction contained caveolae markers; however, only our PM caveolae fraction mimicked the membrane structure and sterol exchange dynamics of intact PM without ER or non-raft PM contaminants. When these PM caveolae were isolated from RV-infected cells, full-length, high-mannose glycosylated NSP4 was present. Confocal imaging showed association of NSP4 with caveolin-1 moving from perinuclear and cytoplasmic sites toward the PM as the infection progressed. Fluorescent imaging also indicated exposure of the NSP4 Cterminus at the exofacial PM surface without transport of the enterotoxin through the Golgi apparatus. Surface-specific biotinylation was used to confirm NSP4 exposure at the surface of infected MDCK cells and to determine that the exposed protein was fulllength and high-mannose glycosylated. This study presents an ER contaminant-free PM caveolae isolation methodology, identifies the presence of full-length, high-mannose glycosylated NSP4 in both PM caveolae and exposed at the cell surface, and confirms the Golgi-bypassing nature of NSP4 ER to PM transport in RV-infected MDCK cells.

Rotavirus NSP4, the first described viral enterotoxin, localizes to the plasma membrane of infected cells, possibly through interaction with caveolin-1. A direct interaction between NSP4 and caveolin-1, the structural protein of caveolae, was shown by yeast two-hybrid, peptide binding, and FRET analyses. To dissect the precise NSP4 binding domain to caveolin-1, mutants were prepared by altering either the charged or hydrophobic face of the NSP4 C-terminal amphipathic alpha-helix and examined for binding to caveolin-1. Replacing six charged residues with alanine (FLNSP4Ala) disrupted the charged face, while the hydrophobic face was disrupted by replacing selected hydrophobic residues with charged amino acids (aa) (FLNSP4HydroMut). In yeast two-hybrid and peptide binding assays, FLNSP4Ala retained its binding capacity, whereas FLNSP4HydroMut failed to bind caveolin-1. Mutants were generated with an Nterminal truncated clone (NSP446-175), which removed the hydrophobic domains and aided in yeast-two hybrid assays. These mutants exhibited the same binding pattern as FLNSP4 confirming that the N-terminus of NSP4 lacks the caveolin-1 binding site and NSP446-175 is sufficient for binding. Seven additional mutants were prepared from NSP4HydroMut in which individually charged residues were reverted to the original hydrophobic aa or were replaced with alanine. Analyses of the interaction of these revertants with caveolin-1 localized the NSP4 binding domain to one critical hydrophobic aa (L116) and one or two additional aa (I113, L127, and/or L134) on the hydrophobic face. Those mutants that bound caveolin-1 bound both the N- and C-terminal caveolin-1 peptides, but lacked binding to a centrally located peptide. These data suggest conformational and hydrophobic constraints play a role in the NSP4-caveolin-1 association. The mutant NSP4 molecules also were evaluated for transport to the plasma membrane. Mammalian cells were transfected with FLNSP4, FLNSP41-175Ala, and NSP41-175HydroMut plasmid DNA, surface biotinylated, and examined by IFA or Western blot for NSP4 expression. Epifluorescence revealed FLNSP4 and FLNSP4Ala were exposed on the cell surface in the absence of other viral proteins, whereas NSP4HydroMut remained intracellular. Further, NSP4-transfected cells displayed an intracellular association of with caveolin-1 or the caveolin-1 chaperone complex proteins. These data indicate NSP4 interacts with caveolin-1 in the absence of other viral proteins.

Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular calcium through multiple mechanisms that promote abnormal ion transport and subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially interact with model membranes mimicking caveolae in lipid composition and radius of curvature. Our laboratory has recently reported the colocalization and coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae. Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least one terminus were capable of binding the NSP4 bait. An in vitro binding assay confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1 residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates NSP4 signaling and/or intracellular trafficking.

The rotavirus NSP4 protein is the first described viral enterotoxin. Abundant data from our laboratory reveals that NSP4 binds both the N- and C-termini of caveolin- 1 (aa2-31 and 161-178, respectively). Yeast two-hybrid and peptide binding analysis mapped the caveolin-1 binding site to three hydrophobic residues within the amphipathic a-helix, enterotoxic peptide domain (aa114-135). The research studies herein utilized peptides to investigate the interaction between NSP4 and caveolin-1. Peptides were synthesized corresponding to the amphipathic a-helix and caveolin-1 binding domain of NSP4 (aa112-140) and to the N- (aa2-20 and 19-40) and C- (161-178) termini of caveolin-1, and were utilized in structural and functional studies. Fluorescence binding assays revealed that NSP4 (aa112-140) binds to the N-terminus (aa19-40) of caveolin-1 with a stronger affinity than the C-terminus (aa161-178). In addition, this assay further delineated the NSP4 binding domain on caveolin-1 to aa19-40. Secondary structural changes following NSP4-caveolin-1 peptide-peptide interactions were investigated by circular dichroism analysis. Changes in a-helix formation were observed only upon interaction of the NSP4112-140 peptide with the C-terminal caveolin-1 peptide (C-Cav161- 178). The NSP4112-140 peptide contains a potential cholesterol recognition amino acid consensus (CRAC) sequence. Therefore this peptide was examined for cholesterol binding. Results of the binding assay revealed NSP4 binds cholesterol with a Kd of 7.67 +/- 1.49nM and this interaction occurs via aa112-140. Mutation of amino acid residues within the CRAC motif resulted in weaker binding affinities between each of the corresponding mutant peptides and cholesterol. NSP4 peptides containing mutations within the hydrophobic and charged faces of the amphipathic a-helix, enterotoxic peptide and caveolin-1 binding domain of NSP4 were examined for changes in secondary structure as well as diarrhea induction in mouse pups. Circular dichroism analysis revealed that mutation of hydrophobic residues resulted in a decrease in a-helix formation, whereas mutation of acidic and basic charged residues caused little to no change in a-helical content. When tested for diarrhea induction in mouse pups, the peptides containing mutations of either the hydrophobic or basic charged residues did not cause diarrhea. Taken together, the results of this research suggest a complex interplay between NSP4 secondary structure, caveolin-1 and cholesterol binding and diarrheagenic function.

Rotavirus is the leading cause of severe infantile diarrhea worldwide. Most related deaths occur in infants from developing countries. Current vaccines are expensive and not readily available throughout the world. Chloroplast transformation technology can be utilized to generate genetically modified plants that produce large quantities of therapeutic proteins and vaccine antigens within their leaves. Plants that are used as bioreactors for vaccine antigens are economically advantageous because they eliminate the need for purification steps and are cheaper to transport. A genetically modified crop could potentially be grown near an endemic area and harvested as needed. There are many influencing factors for transgene expression levels within plant leaves that must be taken into account prior to their harvest. In this work, we seek to determine the optimal expression of CTB-NSP4 in two different cultivars of tobacco plant that have been previously generated by the Daniell lab. The fusion protein, CTBNSP4, is hoped to confer resistance to both rotavirus and cholera. We will determine how the expression of the protein is affected by different variables such as the lighting conditions during harvest and the relative age of leaf at the time of harvest. This knowledge can be used to raise the productivity of the genetically modified plants, further decreasing the cost. Additionally, as unprocessed leaf cannot be used directly for oral delivery due to an unknown concentration of the vaccine antigen, quantification is an important barrier to overcome. Low cost vaccines can be prepared after optimization of dosage and stability. This project seeks to substantiate and quantify genetically modified tobacco plants producing the rotavirus and cholera vaccine antigens.

Made available in DSpace on 2014-07-29T15:26:22Z (GMT). No. of bitstreams: 1 Tese Talissa de Morais Tavares.pdf: 2094396 bytes, checksum: cca589203914a2c676b55bcf55e62bbc (MD5) Previous issue date: 2008-04-28 / Group A rotaviruses are the major cause of gastroenteritis in children throughout the world. Epidemiological surveys and molecular analysis of rotavirus strains are required for gastroenteritis control and prevention. Studies using VP6, an important immunogenic structural protein, and NSP4, a transmembrane nonstructural glycoprotein which is critical to rotavirus morphogenesis and pathogenesis, have been performed. In this study, 330 rotavirus-positive fecal samples previously obtained from children with or without diarrhea, between 1987 and 2003, in three cities of Central West Region of Brazil (Goiânia, Brasília and Campo Grande), were characterized for VP6- and NSP4-encoding genes. The VP6 and NSP4 genes were amplified by reverse transcription- polymerase chain reaction followed by sequencing and phylogenetic analysis. Detection rates of 84.8% and 78.5% were observed for VP6 and NSP4 genes, respectively. Two distinct genotypes could be recognized for NSP4 (A and B). It was observed that the G9P[6] samples were associated with genotype A, whereas the G1P[6], G1P[8], G2P[8], G3P[8], G4P[8] and G9P[8] samples were associated with genotype B. The analysis of VP6 gene allowed genogrouping of samples in two clusters, genogroups I and II. The G2P[4], G3P[4] and G9P[6] samples were identified as genogroup I, whereas the G1P[6], G1P[8], G2P[8], G4P[6], G4P[8] and G9P[8] samples were identified as genogroup II. In addition, it was showed that samples identified as VP6 genogroup I were associated with NSP4 genotype A and samples identified as VP6 genogroup II were associated with NSP4 genotype B. This investigation described different genetic groups representing diversity of group A rotavirus samples circulating in the Central West Region of Brazil. / Os rotavírus do grupo A são considerados como os principais agentes de gastroenterite em crianças em todo o mundo. Investigações de vigilância epidemiológica e molecular são importantes para o controle e prevenção da doença. Nesse sentido, destacam-se os estudos utilizando VP6, proteína estrutural que se tem mostrado como a mais imunogênica, e NSP4, uma glicoproteína não estrutural transmembrana envolvida na morfogênese e patogênese viral. No presente estudo, 330 amostras de rotavírus coletadas de 1987 a 2003, provenientes de espécimes fecais de crianças com ou sem diarréia, em três cidades da Região Centro-Oeste do Brasil (Goiânia, Brasília e Campo Grande), foram caracterizadas em relação aos genes que codificam as proteínas VP6 e NSP4. Inicialmente, foi feita a amplificação dos genes de VP6 e NSP4 pela reação em cadeia pela polimerase pós-transcrição reversa, seguida do seqüenciamento genômico e de análise filogenética. Os genes de VP6 e NSP4 foram detectados em 84,8% e 78,5% das amostras, respectivamente. Dois genótipos de NSP4 foram identificados (A e B). Foi observado que as amostras G9P[6] associaram-se ao genótipo A e as amostras G1P[6], G1P[8], G2P[8], G3P[8], G4P[8] e G9P[8] associaram-se ao genótipo B. A análise do gene VP6 permitiu a identificação de dois genogrupos distintos (I e II). As amostras G2P[4], G3P[4] e G9P[6] foram caracterizadas como genogrupo I, enquanto as amostras G1P[6], G1P[8], G2P[8], G4P[6], G4P[8] e G9P[8] foram caracterizadas como genogrupo II. Ainda foi evidenciado que as amostras genogrupo I de VP6 estavam associadas com genótipo A de NSP4, e as amostras genogrupo II de VP6 estavam associadas com genótipo B de NSP4. A presente investigação identificou diferentes variantes genéticas, mostrando a diversidade dos rotavírus do grupo A circulantes na Região Centro-Oeste do Brasil.