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, 81 (24), 13315-24

Type I Interferon Production During Herpes Simplex Virus Infection Is Controlled by Cell-Type-Specific Viral Recognition Through Toll-like Receptor 9, the Mitochondrial Antiviral Signaling Protein Pathway, and Novel Recognition Systems

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Type I Interferon Production During Herpes Simplex Virus Infection Is Controlled by Cell-Type-Specific Viral Recognition Through Toll-like Receptor 9, the Mitochondrial Antiviral Signaling Protein Pathway, and Novel Recognition Systems

Simon B Rasmussen et al. J Virol.

Abstract

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-alpha/beta) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-alpha/beta production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.

Figures

FIG. 1.
FIG. 1.
The pDC-TLR9 pathway is responsible for early but not for late IFN-α/β production during HSV infection in vivo. (A to H) C57BL/6 or TLR9−/− mice were infected i.p. with 2 × 107 PFU of HSV-1 or HSV-2 (five mice per group). At the indicated time points p.i., sera were harvested (A to F), or spleen cDCs and pDCs were isolated and cultured for ex vivo cytokine expression for 24 h (G to H). The levels of IFN-α/β were measured by bioassay. Similar results were obtained in three to four independent experiments. (I to L) WT mice (129sv in panels I and J; C57BL/6 in panels K and L), IFNAR−/− mice, and TLR9−/− mice were infected i.p. with 106 PFU of HSV-2 (five mice per group). At 2 days p.i., livers and spleens were harvested, and the viral loads in the organs were determined by plaque assay. Similar results were obtained in three independent experiments. (M) Mice treated as in panels I to L were monitored for 6 days. The mice were sacrificed when they displayed symptoms irreversibly associated with death. Error bars indicate the standard errors of the mean (SEM). *, P < 0.05.
FIG. 2.
FIG. 2.
Cell-type-specific requirements for TLR9 and virus replication for induction of IFN-α/β by HSV-2 in vitro. HSV-2 was treated with UV light for the indicated time intervals before being subjected to plaque assay (A) or used for infection of Vero cells for 5 h at an MOI of 1 (B) before harvesting of total RNA and detection of ICP27 and β-actin by RT-PCR. Similar results were obtained in two independent experiments. (C to J) Cells were harvested from WT or TLR9−/− mice and cultured with medium alone or in the presence of 3 × 106 PFU of HSV-2/ml or an equivalent amount of virus UV inactivated for 4 min. Supernatants were harvested 24 h p.i., and the levels of IFN-α/β were determined by bioassay. Similar results were obtained in three independent experiments. (K to N) Splenic pDCs, cDCs, and macrophages, as well as MEFs, were cultured and infected with 105 PFU of HSV-2/ml. Supernatants were harvested 6, 24, and 48 h p.i., and the viral load was measured by plaque assay. Similar results were obtained in two independent experiments. Error bars indicate the SEM.
FIG. 3.
FIG. 3.
IFN-α/β expression in vivo involves at least three mechanisms with differential requirements for TLR9 and virus replication. C57BL/6 or TLR9−/− mice were infected i.p. with 2 × 107 PFU of infectious or inactivated HSV-1 (A and C) or HSV-2 (B and D) (five mice per group). After 8 h (A and B) or 16 h (C and D) of treatment, serum was harvested, and the levels of IFN-α/β were measured by bioassay. (E and F) Splenic cDCs and macrophages were harvested from TLR9−/− mice infected as described above for 16 h. The cells were cultured for 24 h, and the ex vivo production of IFN was measured by bioassay. Similar results were obtained in three independent experiments. Error bars indicate the SEM. *, P < 0.05.
FIG. 4.
FIG. 4.
HSV-induced expression of IFN-α/β is dependent on virion DNA and displays a differential requirement for viral entry. (A) Thioglycolate-activated macrophages were harvested from BALB/c mice and treated with 3 × 106 PFU of HSV-1 or ΔgL/ml for 24 h. IL-12p40 was measured by ELISA. Similar results were obtained in three independent experiments. (B and C) Genomic DNA or total lysates of the preparations of the indicated viruses were analyzed for content of DNA and gD protein, respectively (upper panels). Nuclear extracts were prepared from Vero cells treated for 3 h with the indicated virus at an MOI of 12. VP16 was detected in the extracts by Western blotting (lower panel). Similar results were obtained in three independent experiments. (D to I) Splenic pDCs, cDCs, and macrophages, together with MEFs from C57BL/6 and TLR9−/− mice, were cultured and treated for 20 h with 3 × 106 PFU of HSV-1, ΔgL, or ΔUL15 viruses/ml as indicated. The supernatants were harvested, and the IFN-α/β levels were measured. Similar results were obtained in three to four independent experiments. (J) C57BL/6 and TLR9−/− mice were injected i.p. with 2 × 107 PFU of HSV-1, ΔgL, or ΔUL15 viruses (five mice per group). After 16 h, sera were harvested, and the type I IFN levels were measured. Similar results were obtained in three independent experiments. Error bars indicate the SEM.
FIG. 5.
FIG. 5.
MEFs use the MAVS pathway to induce type I IFN production in response to HSV infection. (A and B) MEFs from C57BL/6 or MAVS−/− mice were seeded and infected with 3 × 106 PFU of HSV-1 or HSV-2/ml. After 24 and 4 h, supernatants and total RNA were harvested for measurement of the IFN-α/β bioactivity (A) and mRNA (B), respectively. (C) MEFs from C57BL/6 or MAVS−/− mice were seeded and transfected with calf thymus-derived DNA using Lipofectamine 2000. Twenty-four hours later the supernatants were harvested for measurement of the IFN-α/β bioactivity. (D) MEFs from C57BL/6 mice were seeded and infected with 3 × 106 PFU/ml of the 17+ or KOS strains of HSV-1 and the indicated mutants. After 24 h, the supernatants were harvested, and the IFN-α/β levels were measured. For all data in this figure, similar results were obtained in three independent experiments. Error bars indicate the SEM.

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