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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-12-7454 |
| Date | 2009 December 1900 |
| Creators | Schroeder, Megan Elizabeth |
| Contributors | Ball, Judith M. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | application/pdf |
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