Figure 1.
Induction of IFNβ is blocked during OSU infection, and the block is mediated by NSP1.
(A) Subcellular localization of IRF3 in MA104 cells infected with three pfu/cell of the indicated virus strain was determined six hours post-infection (hpi) by IF microscopy (40×, NA 0.75). Fixed cells were stained with anti-IRF3 monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Nuclei were visualized by DAPI staining. (B) Induction of IRF3-regulated genes was analyzed by RT-PCR using RNA collected eight hpi from MA104 cells infected with three pfu/cell of the indicated virus strain. Actin mRNA was used as a template loading control. (C) 293-TLR3 cells were co-transfected with pN-NSP1 (NCDV) or pO-NSP1 (OSU), Renilla luciferase reporter plasmid phRL-CMV (Promega) and IFNβ-Luc. Forty hours post-transfection, the cells were treated with 100 µg/mL polyI:C , and lysates were prepared eight hours post-treatment. Reporter activity was measured with the Dual Luciferase Assay System (Promega). Data are represented as fold-inhibition of IFNβ reporter induction normalized to cells transfected with empty expression vector. Error bars indicate the SEM.
Figure 2.
Inhibition of NFκB activation in OSU infected cells is mediated by NSP1.
(A) MA104 cells were infected with 10 pfu/cell of the indicated virus strain, and cell lysates were prepared six hpi. NFκB subunit p50 binding to κB sites was determined by the TransAM p50 ELISA. Data are represented as the mean OD450 nm value from three independent experiments. Error bars indicated the SEM. p-values were determined by unpaired Student's t-test (asterisk = p<0.01). (B) 293-TLR3 cells were transfected with pN-NSP1 (NCDV) or pO-NSP1 (OSU), Renilla luciferase reporter plasmid phRL-CMV, and pNFκB-Luc Cis. Forty hours post-transfection, cells were treated with 100 µg/mL polyI:C for eight hours, and then reporter activity in the lysates was measured with the Dual Luciferase Assay System. Data are represented as fold inhibition of NFκB reporter induction normalized to cells transfected with the empty expression vector. Error bars indicated the SEM.
Figure 3.
NFκB subunit p65 is stable in rotavirus infected cells.
(A) MA104 cells were infected with three pfu/cell of the indicated virus strain. Lysates were prepared at 2, 4, 6, 8, and 10 hpi and the abundance of p65 was determined by immunoblot using anti-p65 antibody. Blots were probed with anti-GAPDH as a loading control. (B) The subcellular localization of p65 in MA104 cells infected with three pfu/cell of the indicated virus strain was determined at six hpi by confocal microscopy (63×, NA 1.40). Cells were stained with anti-p65 antibody, followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG.
Figure 4.
p65 activation and nuclear translocation.
MA104 cells were infected with A5-16, OSU or NCDV at an moi of three pfu/cell. Six hours post-infection, nuclear and cytoplasmic fractions were separated with the nuclear extract kit following the manufacturer's instructions (Active Motif). (A) p65 activation measured by p65 TransAm ELISA. Error bars are the standard error of the mean. (B) Nuclear fractions were probed with anti-p65 antibody, anti-laminA/C (nuclear, BD Biosciences), and anti- GAPDH (cytoplasmic) antibodies.
Figure 5.
IκBα is stable in cells infected with strains that encode NSP1.
(A) MA104 cells were infected with three pfu/cell of the indicated virus strain and lysates were prepared every two hours for ten hours. Immunoblots were probed with anti-IκBα antibody. All blots were probed with anti-GAPDH antibody as a loading control. (B) IκBα levels were quantified by densitometry and are plotted as the ratio of IκBα to GAPDH, normalized to mock infected control. Error bars are the standard error of the mean. (C) Immunblot of lysates in (A) probed with anti-p-IκBα antibody (D) MA104 cells were infected with three pfu/cell of the indicated virus strain. Eight hours post-infection, cells were treated with 50 ng/mL TNFα for 15 minutes. Immunoblots were probed with anti-IκBα or anti-p-IκBα, and anti-GAPDH antibodies. IκBα is not detectable in A5-16 infected cells because it is degraded by six hours post-infection (see panel A).
Figure 6.
Rotavirus strains that express NSP1 induce proteasome-mediated degradation of β-TrCP.
(A) 293 cells were transfected with Flag-tagged β-TrCP (pFlag-βTrCP) and then infected with three pfu/cell of the indicated virus strain. Cell lysates were prepared eight hpi, and immunoblots were probed with anti-Flag antibody (top panel); anti-Flag and anti-NCDV NSP1 antibody (middle panel); or anti-Flag and anti-OSU NSP1 (bottom panel, Input). In the bottom right panel, the lysate from β-TrCP expressing, OSU infected cells was subjected to immunoprecipitation with anti-Flag M2 Agarose Affinity Resin, and blots were probed with anti-Flag antibody or anti-OSU NSP1 antibody (B) The same experiment as described in (A), except that proteasome inhibitor MG132 was added at the time of virus infection to final concentration of 10 µM.
Figure 7.
NSP1 expressed in isolation binds to and induces degradation of β-TrCP.
293 cells were co-transfected with pFlag-β-TrCP and either pN-NSP1 or pO-NSP1. Forty hours post-transfection, MG132 was added to the media to a final concentration of 10 µM and the cells were incubated an additional eight hours. 10% of each sample was set aside as input and the remaining lysate was subjected to immunoprecipitation with anti-FLAG M2 Agarose Affinity Resin (Sigma). Immunoblots were probed with anti-Flag and anti-myc (NSP1 detection) antibodies.