Feline Calicivirus Infection Disrupts Assembly of Cytoplasmic Stress Granules and Induces G3BP1 Cleavage

Abstract

In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6(Pro) Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6(Pro)-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway.

Importance: Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6(Pro) This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.

FIG 1

eIF2α phosphorylation during FCV infection. CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1 for 2 or 6 h as indicated. As a control, CRFK cells were treated with 0.5 mM sodium arsenite (SA; +) for 30 min or mock treated (−). Following treatments, cell extracts were prepared and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated on the left side of the panel. p-eIF2α, phospho-eIF2α.

FIG 2

SG accumulation during FCV infection. (A) CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1. Infected cells, with (+) or without (−) sodium arsenite (SA) treatment, were fixed at the times indicated postinfection and stained with either mouse monoclonal antibodies specific for G3BP1 and rabbit polyclonal antiserum specific for FCV NS6/7 (A) or mouse polyclonal antibodies specific for VP1 and rabbit polyclonal antiserum specific for eIF4G (B). This was followed by staining with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 555-conjugated donkey anti-mouse IgG secondary antibodies. Nuclei were stained with To-Pro-3. Stained cells were examined by fluorescence microscopy, and representative images are shown. (C) The percentage of cells containing SGs out of the total number of cells was calculated, and the means of and standard deviations of three experimental replicates are shown. Results were analyzed by one-way ANOVA with Bonferroni corrections: ***, P < 0.001 (GraphPad Prism, version 6).

FIG 3

SG accumulation following arsenite treatment in FCV-infected cells. (A) CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1 for 6 h. Following infection, cells were untreated (−) or treated (+) with 0.5 mM sodium arsenite (SA) for 30 min. The cells were fixed and stained with mouse monoclonal antibodies specific for G3BP1 and rabbit polyclonal antiserum specific for FCV NS6/7. This was followed by staining with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 555-conjugated donkey anti-mouse IgG secondary antibodies. Nuclei were stained with To-Pro-3. Stained cells were examined by fluorescence microscopy, and representative images are shown. (B) The percentage of cells containing SGs out of the total number of cells was calculated, and the means and standard deviations of three experimental replicates are shown. Results were analyzed by one-way ANOVA with Bonferroni corrections: **, P < 0.01; ns not significant (GraphPad Prism, version 6). (C) CRFK cells were infected with FCV or UV-inactivated FCV at an MOI of 1. The cells were incubated for 12 h, and the viral titer was estimated by a TCID₅₀ assay. Three separate experiments were analyzed by standard t test (**, P < 0.01; GraphPad Prism, version 6). −FCV, mock infection; +FCV(UV), infection with UV-inactivated FCV.

FIG 4

SG accumulation following hydrogen peroxide treatment in FCV-infected cells. (A) CRFK cells were mock infected or infected with FCV Urbana at an MOI of 1 for 6 h and treated with 1 mM hydrogen peroxide (H₂O₂; +) for 1 h or mock treated (−). Following treatments, cell extracts were prepared and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated on the left side of the panels. (B) CRFK cells were mock infected (−FCV) or infected with FCV Urbana (+FCV) at an MOI of 1 for 6 h. Following infection, cells were treated with 1 mM H₂O₂ for 1 h. The cells were fixed and stained with mouse monoclonal antibodies specific for G3BP1 and rabbit polyclonal antiserum specific for FCV NS6/7. This was followed by staining with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 555-conjugated donkey anti-mouse IgG secondary antibodies. Nuclei were stained with To-Pro-3. Stained cells were examined by fluorescence microscopy, and representative images are shown. (C) The percentage of cells containing SGs out of the total number of cells was calculated, and the means and standard deviations of three experimental replicates are shown. Results were analyzed by one-way ANOVA with Bonferroni corrections: ***, P < 0.001 (GraphPad Prism, version 6).

FIG 5

G3BP1 cleavage during FCV infection. CRFK (A) and FEA (B) cells were mock infected (−) or infected (+) with FCV Urbana at an MOI of 1 for 6 h. Cell extracts were prepared and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated to the right of the panels. The arrows denote the positions of cleavage products, and the molecular mass standards (kilodaltons) are shown on the left.

FIG 6

G3BP1 and G3BP2 cleavage by FCV NS6^Pro. (A) EGFP, EGFP-FCV NS6^Pro, EGFP-MNV NS6 ^Pro, or EGFP-PV 3C ^Pro was expressed in 293T cells. Cell extracts were prepared and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated to the right of the panels. The arrows denote the positions of cleavage products, and the molecular mass standards (kilodaltons) are shown on the left. (B) EGFP, EGFP-FCV NS6 ^Pro, EGFP-MNV NS6 ^Pro, or EGFP-PV 3C ^Pro was coexpressed with Flag-G3BP1 or Flag-G3BP2 in 293T cells, as indicated. Cell extracts were prepared and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated to the right of the panels. The arrows denote the positions of cleavage products, and the molecular mass standards (kilodaltons) are shown on the left. (C) Comparison of the C-terminal sequences of human, feline, and murine G3BP1 proteins. The positions of the viral proteinase cleavage sites, identified in this study and previously, are indicated above the sequences boxed in black. The positions where G3BP1 alanine mutants have been introduced are highlighted in black and gray. (D) EGFP, EGFP-FCV NS6 ^Pro, or EGFP-PV 3C^Pro was coexpressed with wild-type (wt) or mutant Flag-G3BP1 in 293T cells, as indicated. Cell extracts were prepared and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated to the right of the panels. The arrows denote the positions of cleavage products, and the molecular mass standards (kilodaltons) are shown on the left. H. sapiens, Homo sapiens; F. catus, Felis catus; M. musculus, Mus musculus.

FIG 7

FCV NS6^Pro-mediated G3BP1 cleavage impairs SG assembly. (A) HeLa-R19-G3BP1^KO cells were cotransfected with a Flag-G3BP1 or Flag-G3BP1-E405A expression plasmid together with an EGFP or EGFP-FCV-NS6^Pro expression plasmid and treated with 50 μM sodium arsenite for 1 h. Cells were then fixed and stained with goat polyclonal antibodies specific for eIF3η and mouse monoclonal specific for G3BP1. This was followed by staining with species-matched Alexa Fluor-conjugated secondary antibodies. Nuclei were stained with DAPI. Stained cells were examined by fluorescence microscopy, and representative images are shown. (B) Cell extracts were prepared from HeLa (wt) and HeLa-R19-G3BP1^KO (G3BP1^KO) cells and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated on the right side of the panels. The molecular mass standards (kilodaltons) are shown on the left.

FIG 8

SG accumulation during MNV infection. (A) J774 cells were mock infected or infected with MNV at an MOI of 10 and untreated (−) or treated (+) with 0.5 mM sodium arsenite (SA) for 45 min. Cell extracts were prepared and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated on the left side of the panels. The molecular mass standards (kilodaltons) are shown on the right. (B) J774 cells were mock infected or infected with MNV1 at an MOI of 10. Following infection cells were untreated or treated with 0.5 mM SA for 45 min. Cells were fixed at the times indicated postinfection and stained with rabbit polyclonal antibodies specific for NS3 and mouse monoclonal antibodies specific for G3BP1. This was followed by staining with species-matched Alexa Fluor-conjugated secondary antibodies.