Defining the Role of Stress Granules in Innate Immune Suppression by the Herpes Simplex Virus 1 Endoribonuclease VHS

Abstract

In response to virus-induced shutoff host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), a substrate of the double-stranded RNA (dsRNA)-activated kinase protein kinase R (PKR). The detection of innate immune sensors and effectors like PKR at SGs suggests a role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus 1 (HSV-1), the virus-encoded virion host shutoff (VHS) endoribonuclease is required to restrict interferon production, PKR activation, and SG formation, although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (ΔVHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for beta interferon mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING signaling downstream of cGMP-AMP synthase (cGAS)-dependent DNA sensing is required for beta interferon induction. In contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2α-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation, and beta interferon to address this question. We show that dsRNA is localized to SGs and that SGs can function to promote PKR activation in the context of a DNA virus infection, but we find no evidence to support their importance for interferon induction during HSV-1 infection.

Keywords: DNA sensing; HSV-1 replication; PKR; VHS; antiviral immunity; stress granules; virion host shutoff.

FIG 1

dsRNA accumulates in ΔVHS-infected cells and partially localizes to stress granules. NHDFs were mock infected or infected with WT HSV-1 (F-strain) or ΔVHS (R2621; F-strain) at an MOI of 5. At 18 hpi, the cells were fixed and analyzed by immunofluorescence for dsRNA using viral protein ICP4 (A) or the J2 monoclonal antibody (B) (green) and the SG marker G3BP1 (red). Nuclei are stained by DAPI (blue). Scale bars, 50 μm.

FIG 2

Kinetics of induction of IFN-β and ISG mRNA, TBK1 and PKR activation, and SG assembly during ΔVHS infection. NHDFs were infected with ΔVHS at an MOI of 5 and collected at 3-h intervals. Uninfected (UI) cells were collected at 0 hpi. (A) mRNA levels of IFN-β and ISGs IFIT2 and MDA5 were analyzed by qRT-PCR from total RNA and normalized to 18S. The means ± the standard errors of the mean (SEM) of three independent experiments are plotted. (B) TBK1 (S172) and PKR (T446) phosphorylation were analyzed by immunoblotting of whole-cell lysates. (C) SG induction was quantified by counting the number of cells containing two or more G3BP1-stained SGs. More than 100 cells were counted for each time point, and the means ± the SEM of three independent experiments are plotted.

FIG 3

IFN-β and ISG induction is not dependent on viral DNA synthesis. NHDFs were mock infected or infected with ΔVHS and treated with 300 μg/ml PAA or a vehicle control at 1 hpi. (A) PKR phosphorylation and ISG protein (MDA5, RIG-I) and late viral protein (gB, ICP5) accumulation were analyzed by immunoblotting of whole-cell lysates collected at 12 hpi. (B) IFN-β mRNA levels were analyzed by qRT-PCR from total RNA collected at 6 hpi and normalized to 18S. Means ± the SEM of three independent experiments are plotted. ns, P > 0.05 (Student t test compared to control). (C) SG induction and dsRNA accumulation were visualized at 12 hpi by immunofluorescence for G3BP1 (red) and dsRNA (green) with nuclei stained by DAPI (blue). Scale bar, 50 μm.

FIG 4

IFN-β and ISG induction is dependent on DNA sensing. NHDFs were transfected with siRNAs targeting dsRNA- and DNA-sensing pathway components or a nonsilencing control siRNA. At 3 days posttransfection, the cells were infected with ΔVHS at an MOI of 5. (A) Total RNA was isolated at 6 hpi and subject to qRT-PCR for IFN-β and normalized to 18S. The means ± the SEM of three independent experiments is shown. *, P < 0.05; **, P < 0.01 (Student t test compared to control). ns, nonsignificance. (B) ISG protein accumulation (IFIT2, MDA5, and RIG-I) and siRNA-mediated knockdown efficiency were analyzed by immunoblotting whole-cell lysates collected at 12 hpi. (C) Knockdown of IFI16 was verified by immunoblotting of uninfected cells 3 days after siRNA transfection. (D) Knockdown of cGAS was verified by qRT-PCR in uninfected cells 3 days after siRNA transfection. Means ± the SEM of three independent experiments are plotted. (E and F) NHDFs were transfected with siRNAs targeting STING, MAVS, or a nonsilencing control siRNA. At 3 days posttransfection, the cells were harvested and the siRNA-mediated knockdown efficiency was analyzed by immunoblotting whole-cell lysates (E) or cells infected with ΔVHS at an MOI of 0.001 (F). At 48 hpi, cultures were collected, and infectious virus was quantified by titering on Vero cells. Means ± the SEM of three independent experiments are plotted. **, P < 0.01 (Student t test compared to control).

FIG 5

SG inhibitor ISRIB prevents PKR activation in ΔVHS-infected cells. (A) NHDFs were treated with 0.5 mM sodium arsenite and 200 nM ISRIB (+) or vehicle control (−) for 30 min before immunofluorescence staining for the SG marker G3BP1 (green). (B and C) NHDFs were infected with WT HSV-1 or ΔVHS at an MOI of 5, and infection was allowed to proceed in the presence of 200 nM ISRIB (+) or vehicle control (−) for 15 h. (B) SG induction was examined in ΔVHS cells by immunofluorescence staining for G3BP1 (green) with nuclei stained by DAPI (blue). Scale bar, 50 μm. (C) PKR phosphorylation, ISG accumulation (MDA5), viral protein accumulation (ICP0), and phosphorylated and total eIF2α were analyzed by immunoblotting whole-cell lysates.

FIG 6

SG assembly is required for full PKR activation in ΔVHS-infected cells. NHDFs were transfected with siRNAs targeting SG scaffold proteins G3BP1 or TIA1 or a nonsilencing control siRNA. At 3 days posttransfection, the cells were infected with WT (F-strain) HSV-1 or ΔVHS at an MOI of 5, and infection was allowed to proceed for 18 h. (A) SG induction was quantified in cells transfected with control and G3BP1 (#1) and TIA1 (#1) targeting siRNAs by counting the number of cells containing two or more HuR-stained SGs. More than 100 cells were counted for each condition, and the means ± the SEM of three independent experiments are shown. *, P < 0.05 (Student t test compared to control). (B and C) PKR phosphorylation and knockdown efficiency were analyzed by immunoblotting of whole-cell lysates.