Mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection

Abstract

Infection with many mammalian orthoreovirus (MRV) strains results in shutoff of host, but not viral, protein synthesis via protein kinase R (PKR) activation and phosphorylation of translation initiation factor eIF2alpha. Following inhibition of protein synthesis, cellular mRNAs localize to discrete structures in the cytoplasm called stress granules (SGs), where they are held in a translationally inactive state. We examined MRV-infected cells to characterize SG formation in response to MRV infection. We found that SGs formed at early times following infection (2 to 6 h postinfection) in a manner dependent on phosphorylation of eIF2alpha. MRV induced SG formation in all four eIF2alpha kinase knockout cell lines, suggesting that at least two kinases are involved in induction of SGs. Inhibitors of MRV disassembly prevented MRV-induced SG formation, indicating that viral uncoating is a required step for SG formation. Neither inactivation of MRV virions by UV light nor treatment of MRV-infected cells with the translational inhibitor puromycin prevented SG formation, suggesting that viral transcription and translation are not required for SG formation. Viral cores were found to colocalize with SGs; however, cores from UV-inactivated virions did not associate with SGs, suggesting that viral core particles are recruited into SGs in a process that requires the synthesis of viral mRNA. These results demonstrate that MRV particles induce SGs in a step following viral disassembly but preceding viral mRNA transcription and that core particles are themselves recruited to SGs, suggesting that the cellular stress response may play a role in the MRV replication cycle.

FIG. 1.

Infection with MRV induces SG formation at early times p.i. HeLa (first row), MEF (second row), CV-1 (third row), and DU-145 (fourth row) cells were infected with MRV T3D^C virions (1,000 PFU/cell). At 4 h p.i., cells were fixed and immunostained with rabbit anti-MRV core polyclonal antiserum (left column) and mouse monoclonal antibody against G3BP (first row, right column), goat anti-TIA-1 (second row, right column), or goat anti-TIAR (third and fourth rows, right column) polyclonal antibodies, followed by Alexa 594-conjugated donkey anti-rabbit IgG and Alexa 488-conjugated donkey anti-mouse IgG or donkey anti-goat IgG. More than 200 infected cells were counted on each slide, and the percentage of infected cells containing SG-like granules at the time of fixation is indicated. Scale bars = 10 μm.

FIG. 2.

Infection with MRV ISVPs induces SG formation in a dose-dependent manner. (A) HeLa cells were infected with T1L ISVPs (top row), T2J ISVPs (middle row) or T3D^C ISVPs (bottom row) (1,000 PFU/cell). At 2 h p.i., cells were fixed and immunostained with rabbit anti-MRV core antiserum (left column) and goat polyclonal anti-TIAR antibodies (right column), followed by Alexa 594-conjugated donkey anti-rabbit IgG and Alexa 488-conjugated donkey anti-goat IgG. Scale bars = 10 μm. (B) Cells were infected with MRV ISVPs (T1L, T2J, and T3D^C) at 10, 100, or 1,000 PFU/cell and fixed and immunostained as in panel A. More than 200 infected cells were counted in each treatment to calculate the percentage of infected cells containing SG-like granules at the time of fixation.

FIG. 3.

MRV-induced SGs contain translation initiation factors eIF3, eIF4G, and eIF4E but not P body component DCP1a. HeLa cells were infected with T1L ISVPs (1,000 PFU/cell) for 2 h and then fixed and immunostained with mouse anti-λ2 monoclonal antibody 7F4 (Core) (left column); goat anti-TIA-1 or goat anti-TIAR antibodies (second column); and rabbit anti-eIF3, eIF4G, and eIF4E antibodies or DCP1a antisera (third column); followed by Alexa 594-conjugated donkey anti-mouse IgG, Alexa 488-conjugated donkey anti-goat IgG, and Alexa 350-conjugated donkey anti-rabbit IgG. Merged images are shown in the right column. Scale bars = 10 μm.

FIG. 4.

Ammonium chloride and E-64 prevent MRV induction of SGs. HeLa cells were pretreated with 2 mM ammonium chloride (NH₄Cl) (first row) or 2 mM E-64 (second row) or left untreated (third row) for 4 h and then infected with MRV T3D^C virions (1,000 PFU/cell). At 4 h p.i., cells were fixed and immunostained with rabbit anti-MRV core polyclonal antisera (first, second, and third rows, left column) and goat anti-TIAR polyclonal antibodies (first, second, and third rows, right column), followed by Alexa 594-conjugated donkey anti-rabbit IgG and Alexa 488-conjugated donkey anti-goat IgG. Uninfected HeLa cells were treated with 2 mM ammonium chloride (fourth row) or 2 mM E-64 (fifth row), incubated (fourth and fifth rows, left column) or additionally treated for 1 h with 0.5 mM SA (fourth and fifth rows, right column), and then fixed and stained with goat anti-TIAR polyclonal antibodies followed by Alexa 594-conjugated donkey anti-rabbit IgG. Scale bars = 10 μm.

FIG. 5.

UV-inactivated MRV virions induce SGs. (A) HeLa cells were infected with untreated (top row) or UV-inactivated (bottom row) T3D^C virions (1,000 PFU/cell). Cells were fixed at 4 h p.i. and immunostained with rabbit anti-MRV core polyclonal antiserum (left column) and goat anti-TIAR antibodies (right column), followed by Alexa 594-conjugated donkey anti-rabbit IgG and Alexa 488-conjugated donkey anti-goat IgG. (B) HeLa cells were infected, fixed, and immunostained as in panel A at 2, 4, 6, 12, and 24 h p.i. The percentage of infected cells containing SGs was calculated at each time point as described in Materials and Methods.

FIG. 6.

Puromycin inhibits viral translation but does not prevent MRV induction of SGs. (A) HeLa cells were untreated (first row) or pretreated with 0.1 mg/ml puromycin (second row) for 1 h and then infected with MRV T3D^C virions (1,000 PFU/cell) for 1 h. New medium containing 0.1 mg/ml puromycin was added to cells following infection (second row). At 10 h p.i., cells were fixed and immunostained with rabbit anti-MRV core polyclonal antisera (first and second rows, left column) and goat anti-TIAR polyclonal antibodies (first and second rows, right column), followed by Alexa 594-conjugated donkey anti-rabbit IgG and Alexa 488-conjugated donkey anti-goat IgG. Uninfected HeLa cells were treated with 0.1 mg/ml puromycin (third row, left and right columns), incubated (left column) or treated with 0.5 mM SA for 1 h (right column) after 9 h puromycin treatment, and then fixed and stained with goat anti-TIAR polyclonal antibodies followed by Alexa 488-conjugated donkey anti-goat IgG. Following immunostaining, the percentage of infected cells containing SGs (first and second rows) or total cells containing SGs (third row) was quantified as described in Materials and Methods. Percentages of SG-containing cells are indicated. Scale bars = 10 μm. (B) HeLa cells were infected with MRV T3D^C virions (1,000 PFU/cell) with or without 0.1 mg/ml puromycin (Pm) as indicated, and at 4 and 10 h p.i., cells were lysed, and proteins were separated by SDS-PAGE, followed by immunoblotting using anti-μNS polyclonal antiserum or anti-β-actin polyclonal antibodies. Proteins were detected using HRP-conjugated goat anti-rabbit IgG, followed by chemiluminescence imaging.

FIG. 7.

eIF2α phosphorylation correlates with, and is required for, MRV induction of SGs. (A) HeLa cells were infected with untreated or UV-inactivated T3D^C virions (1,000 PFU/cell), and cells were harvested at 0, 2, 4, and 6 h p.i. Following cell lysis, proteins were separated by SDS-PAGE and immunoblotted using rabbit anti-μNS polyclonal antiserum, rabbit anti-eIF2α polyclonal antibodies, and rabbit anti-phospho-eIF2α polyclonal antibodies. Proteins were detected and quantified using HRP-conjugated goat anti-rabbit antibodies, followed by chemiluminescence imaging. Levels of phosphorylated eIF2α relative to total eIF2α at each time point were calculated and normalized to the level seen at time zero and are indicated below the panel for each time point. (B) MEF^S51S/S51S (S/S) (first row) and MEF^S51A/S51A (A/A) (second row) cells were infected with MRV T3D^C virions (1,000 PFU/cell). At 4 h p.i., cells were fixed and immunostained with rabbit anti-MRV core antiserum (left column) and goat anti-TIAR polyclonal antibodies (right column), followed by Alexa 594-conjugated donkey anti-rabbit IgG and Alexa 488-conjugated donkey anti-goat IgG. Scale bars = 10 μm.

FIG. 8.

MRV induces SG formation in eIF2α kinase knockout cell lines. GCN^−/− (first row), PERK^−/− (second row), PKR^−/− (third row), or HRI^−/− (fourth row) cells were infected with T3D^C virions (1,000 PFU/cell) (left and middle columns) or mock infected (right column). At 4 h p.i., cells were fixed and immunostained with rabbit anti-MRV core antiserum (left column) and goat anti-TIAR polyclonal antibodies (middle and right columns), followed by Alexa 594-conjugated donkey anti-rabbit IgG and Alexa 488-conjugated donkey anti-goat IgG. More than 200 infected cells were counted on each slide, and the percentage of infected cells containing SG-like granules at the time of fixation is indicated. Scale bars = 10 μm.

FIG. 9.

MRV core particles colocalize with SGs in a manner dependent on viral gene expression. (A) HeLa cells were infected with MRV T1L ISVPs (1,000 PFU/cell). At 2 h p.i., cells were fixed and stained with rabbit anti-MRV core antiserum (left) and goat anti-TIAR antibodies (middle), followed by Alexa 594-conjugated donkey anti-rabbit IgG and Alexa 488-conjugated donkey anti-goat IgG. A merged image is shown (right). Confocal images were taken at 0.1-μm-slice intervals using a Leica SP5 X confocal microscope. The boxed regions in each image were amplified and are shown in insets in the merged image. (B) HeLa cells were infected with UV-inactivated T3D^C virions (1,000 PFU/cell). At 4 h p.i., cells were fixed and stained with rabbit anti-MRV core antiserum (left) and goat anti-TIAR antibodies (middle). A merged image is shown (right). The boxed region in the merged image was amplified and is shown in the inset. (C) HeLa cells were pretreated with 0.1 mg/ml puromycin for 1 h, incubated with T3D^C virions (1,000 PFU/cell) for 1 h, and then retreated with puromycin for an additional 10 h, at which point cells were fixed and stained with rabbit anti-MRV core antiserum (left) and goat anti-TIAR polyclonal antibodies (middle). A merged image is shown (right). The boxed region in the merged image was amplified and is shown in the inset.