Full-length, glycosylated NSP4 is localized to plasma membrane caveolae by a novel raft isolation technique.
Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.
Storey
Stephen M
SM
Department of Pathobiology, TVMC, Texas A and M University, TAMU 4467, College Station, TX 77843-4467, USA.
Gibbons
Thomas F
TF
Williams
Cecelia V
CV
Parr
Rebecca D
RD
Schroeder
Friedhelm
F
Ball
Judith M
JM
D000818:Animals; D021941:Caveolae / Q000378:metabolism* / Q000648:ultrastructure / Q000821:virology; D002460:Cell Line; D004285:Dogs; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D019073:HT29 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism* / Q000821:virology; D017830:Octoxynol; D012401:Rotavirus / Q000378:metabolism; D014118:Toxins, Biological / Q000302:isolation & purification / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000302:isolation & purification / Q000378:metabolism*
D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D017830:Octoxynol