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. 2006 Jun;80(12):6061-71.
doi: 10.1128/JVI.02167-05.

Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated With Viroplasms

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Free PMC article

Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated With Viroplasms

Z Berkova et al. J Virol. .
Free PMC article

Abstract

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.

Figures

FIG. 1.
FIG. 1.
Expression of NSP4-EGFP in inducible HEK 293/NSP4-EGFP cells. (A) Schematic diagram of NSP4-EGFP fusion protein; (B) autoradiograph of radiolabeled NSP4-EGFP immunoprecipitated from doxycycline-induced cells using anti-NSP4 120-147 and anti-GFP antibody; (C) autoradiograph of radiolabeled NSP4-EGFP immunoprecipitated with anti-NSP4 120-147 antibody without and with treatment with 500 units of Endo H for 1 h at 37°C; (D) autoradiograph of radiolabeled NSP4-EGFP precipitated with anti-NSP4 120-147 antibody at indicated times postinduction as described in Materials and Methods. *, fully glycosylated NSP4-EGFP.
FIG. 2.
FIG. 2.
All expressed NSP4-EGFP is recognized by rabbit anti-NSP4 120-147 antibody, and there is no intracellular NSP4 protein not fused with EGFP. HEK 293/NSP4-EGFP cells were induced for 24 h, fixed, and stained with rabbit anti-NSP4 120-147 antibody and antirabbit Alexa 594-conjugated secondary antibody as described in Materials and Methods. Stained cells were then observed by confocal microscopy. (A) NSP4-EGFP fluorescence; (B) immunofluorescence with anti-NSP4 120-147 antibody staining; (C) merged image of A and B. Bar = 15 μm.
FIG. 3.
FIG. 3.
Expressed NSP4-EGFP fusion protein is initially localized in the ER of HEK 293 cells. Cells were induced for 24 h, fixed, and stained with mouse monoclonal anti-PDI antibody (B, C) or rabbit anti-calnexin antibody (E, F) and corresponding Alexa 594-conjugated secondary antibody as described in Materials and Methods. Stained cells were then observed by confocal microscopy. (A, D) NSP4-EGFP fluorescence; (B) immunostaining of PDI; (C) merged image of A and B; (E) immunostaining of calnexin; (F) merged image of D and E. Arrows indicate NSP4-EGFP-positive, calnexin-negative vesicles in the vicinity of the ER. Bars = 10 μm.
FIG. 4.
FIG. 4.
Only a portion of vesicular NSP4-EGFP is localized within the ER-Golgi intermediate compartment, and no NSP4-EGFP enters the Golgi apparatus in HEK 293 cells. Cells were fixed 24 h postinduction and stained with mouse monoclonal antibody to ERGIC-53 or to giantin, followed by the Alexa 594-conjugated anti-mouse secondary antibody. Cells were then observed by confocal microscopy. (A, D) NSP4-EGFP fluorescence; (B) ERGIC-53 immunostaining; (C) merged image of A and B; (E) Golgi marker giantin staining; (F) merged image of D and E. Bars = 10 μm.
FIG. 5.
FIG. 5.
NSP4-EGFP vesicles do not align along microtubules in HEK 293 cells. Cells were induced for 24 h, and 1 h prior to fixation and permeabilization the cells were kept at 4°C to induce coalescence of microtubules into MTOC. Cells were stained with mouse monoclonal antibody to β-tubulin, followed by the Alexa 594-conjugated antimouse secondary antibody. Cells were then observed by confocal microscopy. (A) NSP4-EGFP fluorescence; (B) β-tubulin immunostaining; (C) merged image of A and B. Bar = 10 μm.
FIG. 6.
FIG. 6.
A population of NSP4-EGFP fusion protein localizes in the ER or ERGIC compartment in HEK 293 cells. Cells were induced for 24 h, fixed, and stained with rabbit anti-calnexin and mouse monoclonal anti-ERGIC-53 antibodies, followed with Alexa 568-conjugated anti-mouse and Alexa 647-conjugated anti-rabbit antibodies. Cells were then observed by confocal microscopy. (A) NSP4-EGFP fluorescence; (B) immunostaining of ERGIC-53; (C) immunostaining of calnexin; (D) merged image of A, B, C; (E) merged image of A and B; (F) merged image of A and C. Bar = 10 μm.
FIG. 7.
FIG. 7.
NSP4-EGFP does not localize in endosomes or lysosomes of HEK 293 cells. Fixed and permeabilized cells, 24 h postinduction, were stained with mouse anti-Rab9 or rabbit anti-β-galactosidase antibody and the corresponding Alexa 594-conjugated secondary antibody. (A, D) NSP4-EGFP fluorescence; (B) lysosomal β-galactosidase staining; (C) merged image of A and B; (E) endosomal marker Rab9 staining; (F) merged image of D and E. Bars = 10 μm.
FIG. 8.
FIG. 8.
NSP4-EGFP is localized in the vicinity of, but not in, the plasma membrane of HEK 293 cells. Twenty-four hours postinduction, cells were fixed with 4% formaldehyde, incubated with Texas Red-conjugated wheat germ agglutinin without permeabilization to label glycoproteins in the plasma membrane, and observed by confocal microscopy. (A) NSP4-EGFP fluorescence; (B) plasma membrane staining; (C) merged image of A and B. (D, E, F) cell from images A, B, and C with magnification ×2 to show detail. Bars = 10 μm.
FIG. 9.
FIG. 9.
NSP4-EGFP colocalizes with the autophagosomal marker LC3 in HEK 293 cells. Cells were fixed and permeabilized 24 h postinduction and stained with rabbit anti-LC3 antibody and the Alexa 594-conjugated anti-rabbit secondary antibody. (A) NSP4-EGFP fluorescence; (B) LC3 staining; (C) merged image of A and B. The asterisks mark NSP4-EGFP-negative cells showing a diffuse cytoplasmic staining of LC3. HEK 293 cells were also transiently transfected with a plasmid expressing the LC3-GFP fusion protein and 48 h later with the plasmid expressing NSP4 fused to monomeric red fluorescent protein (Ds2Red). Fixed cells were then observed by confocal microscopy. (D) LC3-GFP fluorescence; (E) NSP4-DsRed fluorescence; (F) merged image of D and E. Panels D, E, and F illustrate two LC3-GFP- and NSP4-DsRed-positive cells acquired separately. Bars = 20 μm and 10 μm, respectively.
FIG. 10.
FIG. 10.
NSP4- and LC3-positive vesicles associate with viroplasms in rotavirus-infected MA104 cells. MA104 cells were infected with rotavirus strain SA114F at a MOI of 10 and fixed 7 h postinfection. Permeabilized cells were stained with mouse anti-NSP4 114-135 antibody, rabbit anti-LC3 antibody, guinea pig anti-NSP5 antibody, and the corresponding secondary antibodies: Alexa 488-anti-mouse antibody, Alexa 568 anti-guinea pig antibody, and Alexa-647 anti-rabbit antibody. Cells were then observed by confocal microscopy, and acquired images of fluorescence were pseudocolored green, red, and blue, respectively. (A) NSP4 staining; (B) LC3 staining; (C) merged staining of NSP4 and LC3; (D) NSP5 staining; (E) NSP4, NSP5, and LC3 staining; (F) higher (×2.5) magnification of E to illustrate the localization of NSP4 and LC3 relative to NSP5. Bars = 10 μm.
FIG. 11.
FIG. 11.
Limited colocalization of NSP4 with ERGIC-53 and extensive colocalization of NSP4 with LC3 in rotavirus-infected Cos-7 cells. COS-7 cells were infected with rotavirus strain SA114F at a MOI of 10 and fixed 12 h postinfection. Permeabilized cells were stained with rabbit anti-NSP4 120-147 antibody, mouse anti-ERGIC-53 antibody, and the corresponding secondary antibodies: Alexa 488-antimouse antibody and Alexa 594 anti-rabbit antibody. Cells were then observed by confocal microscopy. (A) ERGIC-53 staining; (B) NSP4 staining; (C) merged staining of NSP4 and ERGIC-53. (Bar = 10 μm). Cos-7 cells were transiently transfected 48 h prior to infection to express LC3-GFP. Permeabilized cells were stained with rabbit anti-NSP4 120-147 antibody and Alexa 594-conjugated anti-rabbit antibody. (D) LC3-GFP fluorescence; (E) NSP4 staining; (F) merged images of D and E. Bar = 5 μm.
FIG. 12.
FIG. 12.
NSP4-EGFP presence in vesicular structures in HEK 293 cells requires elevated levels of intracellular calcium. Cells were induced and grown for 24 h in calcium-free medium (see Materials and Methods). To normalize extracellular calcium, 2 mM calcium chloride was added to the bath solution at 0 min. (A) Live cells were observed by confocal microscopy, and images were acquired at 0, 1, 5, and 10 min; (B) changes of intracellular calcium upon addition of 2 mM calcium chloride into bathing solution were measured in Fura-2-loaded cells as described in Materials and Methods.

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