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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-3258 |
| Date | 15 May 2009 |
| Creators | Williams, Cecelia V. |
| Contributors | Ball, Judith M. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | electronic, application/pdf, born digital |
Page generated in 0.0018 seconds