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The virion host shut-off (vhs) protein blocks a TLR-independent pathway of herpes simplex virus type 1 recognition in human and mouse dendritic cells.

Cotter CR, Nguyen ML, Yount JS, López CB, Blaho JA, Moran TM - PLoS ONE (2010)

Bottom Line: These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein.This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs.Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology Institute, Mount Sinai School of Medicine, New York, New York, USA.

ABSTRACT
Molecular pathways underlying the activation of dendritic cells (DCs) in response to Herpes Simplex Virus type 1 (HSV-1) are poorly understood. Removal of the HSV virion host shut-off (vhs) protein relieves a block to DC activation observed during wild-type infection. In this study, we utilized a potent DC stimulatory HSV-1 recombinant virus lacking vhs as a tool to investigate the mechanisms involved in the activation of DCs by HSV-1. We report that the release of pro-inflammatory cytokines by conventional DC (cDC) during HSV-1 infection is triggered by both virus replication-dependent and replication-independent pathways. Interestingly, while vhs is capable of inhibiting the release of cytokines during infection of human and mouse cDCs, the secretion of cytokines by plasmacytoid DC (pDC) is not affected by vhs. These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein. Using cDCs isolated from mice deficient in both the TLR adaptor protein MyD88 and TLR3, we show that HSV-1 and the vhs-deleted virus can activate cDCs independently of TLR signaling. In addition, virion-associated vhs fails to block cDC activation in response to treatment with TLR agonists, but it efficiently blocked cDC activation triggered by the paramyxoviruses Sendai Virus (SeV) and Newcastle Disease Virus (NDV). This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs. Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.

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Virion-associated vhs blocks the activation of cDCs and can influence the phenotype of CD4+ T-cells during allogeneic in vitro co-culture.(a.) hu-cDCs were infected with infectious (live) and UV inactivated KOS or vhs- at an MOI of 5. At 24 hpi, media from cultures were collected and assayed for secreted IL-6, IL-12p70, and IP-10 using multiplex ELISA. Error bars represent the difference between duplicate assays. (b.) hu-cDCs were infected with KOS and vhs- viruses at an MOI of 5; at 24 hpi, cells were harvested, and cell surface expression of hu-CD86 was measured by Flow Cytometry. (c.) hu-cDCs were infected with KOS and vhs- viruses at an MOI of 5. After a 1-hour infection, virus was removed and the hu-cDCs were co-cultured in a 1∶5 ratio with either naïve CD4+ or CD8+ T-cells. At days 3, 4, and 5, cells were harvested and the supernatants were analyzed for secreted IFN-γ by ELISA. Error bars represent the difference between duplicate assays. ** denotes a p-value <0.005. (d.) Co-cultures of cDCs and CD4+ T-cells were harvested at day 4 and assayed for secreted IL-2 (Th1 cytokine), IL-4 and IL-5 (Th2 cytokines) by Multiplex ELISA. * denotes a p-value <0.05 (e). Co-cultures of cDCs and CD4+ T-cells were harvested at day 4. RNA was isolated from cell pellets and subject to qRT-PCR to analyze the relative expression of Th1-associated genes relative to normalized housekeeping genes. * denotes a p-value <0.05; ** denotes a p-value <0.005.
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pone-0008684-g002: Virion-associated vhs blocks the activation of cDCs and can influence the phenotype of CD4+ T-cells during allogeneic in vitro co-culture.(a.) hu-cDCs were infected with infectious (live) and UV inactivated KOS or vhs- at an MOI of 5. At 24 hpi, media from cultures were collected and assayed for secreted IL-6, IL-12p70, and IP-10 using multiplex ELISA. Error bars represent the difference between duplicate assays. (b.) hu-cDCs were infected with KOS and vhs- viruses at an MOI of 5; at 24 hpi, cells were harvested, and cell surface expression of hu-CD86 was measured by Flow Cytometry. (c.) hu-cDCs were infected with KOS and vhs- viruses at an MOI of 5. After a 1-hour infection, virus was removed and the hu-cDCs were co-cultured in a 1∶5 ratio with either naïve CD4+ or CD8+ T-cells. At days 3, 4, and 5, cells were harvested and the supernatants were analyzed for secreted IFN-γ by ELISA. Error bars represent the difference between duplicate assays. ** denotes a p-value <0.005. (d.) Co-cultures of cDCs and CD4+ T-cells were harvested at day 4 and assayed for secreted IL-2 (Th1 cytokine), IL-4 and IL-5 (Th2 cytokines) by Multiplex ELISA. * denotes a p-value <0.05 (e). Co-cultures of cDCs and CD4+ T-cells were harvested at day 4. RNA was isolated from cell pellets and subject to qRT-PCR to analyze the relative expression of Th1-associated genes relative to normalized housekeeping genes. * denotes a p-value <0.05; ** denotes a p-value <0.005.

Mentions: Consistent with a prior study using a highly mutated replication-defective HSV virus, the induction of pro-inflammatory cytokines (IL-6 and TNF-α) in response to HSV-1 was blocked by vhs (Figures 2a and S3) [31]. Interestingly, the vhs protein inhibited both the virus replication independent triggering of IL-6 and TNF-α (UV inactivated virus) and the replication dependent induction of IL-12, highlighting that the activation of cDCs in response to HSV-1 occurs after sensing viral replication dependent and independent events. The production of the chemokine IP-10 was not affected by vhs. A detailed kinetic analysis revealed that the block to IL-6 and TNF-α production occurs as early as 6 hpi, whereas IL-12 release is blocked by 12 hpi (Figure S2b). This block to cytokine release is also observed at the level of mRNA expression (Figure S2c). Lastly, we also observed elevated cell surface expression of the co-stimulatory molecule CD86 in the vhs- infected cultures at 24 hpi (Figure 2b).


The virion host shut-off (vhs) protein blocks a TLR-independent pathway of herpes simplex virus type 1 recognition in human and mouse dendritic cells.

Cotter CR, Nguyen ML, Yount JS, López CB, Blaho JA, Moran TM - PLoS ONE (2010)

Virion-associated vhs blocks the activation of cDCs and can influence the phenotype of CD4+ T-cells during allogeneic in vitro co-culture.(a.) hu-cDCs were infected with infectious (live) and UV inactivated KOS or vhs- at an MOI of 5. At 24 hpi, media from cultures were collected and assayed for secreted IL-6, IL-12p70, and IP-10 using multiplex ELISA. Error bars represent the difference between duplicate assays. (b.) hu-cDCs were infected with KOS and vhs- viruses at an MOI of 5; at 24 hpi, cells were harvested, and cell surface expression of hu-CD86 was measured by Flow Cytometry. (c.) hu-cDCs were infected with KOS and vhs- viruses at an MOI of 5. After a 1-hour infection, virus was removed and the hu-cDCs were co-cultured in a 1∶5 ratio with either naïve CD4+ or CD8+ T-cells. At days 3, 4, and 5, cells were harvested and the supernatants were analyzed for secreted IFN-γ by ELISA. Error bars represent the difference between duplicate assays. ** denotes a p-value <0.005. (d.) Co-cultures of cDCs and CD4+ T-cells were harvested at day 4 and assayed for secreted IL-2 (Th1 cytokine), IL-4 and IL-5 (Th2 cytokines) by Multiplex ELISA. * denotes a p-value <0.05 (e). Co-cultures of cDCs and CD4+ T-cells were harvested at day 4. RNA was isolated from cell pellets and subject to qRT-PCR to analyze the relative expression of Th1-associated genes relative to normalized housekeeping genes. * denotes a p-value <0.05; ** denotes a p-value <0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2823768&req=5

pone-0008684-g002: Virion-associated vhs blocks the activation of cDCs and can influence the phenotype of CD4+ T-cells during allogeneic in vitro co-culture.(a.) hu-cDCs were infected with infectious (live) and UV inactivated KOS or vhs- at an MOI of 5. At 24 hpi, media from cultures were collected and assayed for secreted IL-6, IL-12p70, and IP-10 using multiplex ELISA. Error bars represent the difference between duplicate assays. (b.) hu-cDCs were infected with KOS and vhs- viruses at an MOI of 5; at 24 hpi, cells were harvested, and cell surface expression of hu-CD86 was measured by Flow Cytometry. (c.) hu-cDCs were infected with KOS and vhs- viruses at an MOI of 5. After a 1-hour infection, virus was removed and the hu-cDCs were co-cultured in a 1∶5 ratio with either naïve CD4+ or CD8+ T-cells. At days 3, 4, and 5, cells were harvested and the supernatants were analyzed for secreted IFN-γ by ELISA. Error bars represent the difference between duplicate assays. ** denotes a p-value <0.005. (d.) Co-cultures of cDCs and CD4+ T-cells were harvested at day 4 and assayed for secreted IL-2 (Th1 cytokine), IL-4 and IL-5 (Th2 cytokines) by Multiplex ELISA. * denotes a p-value <0.05 (e). Co-cultures of cDCs and CD4+ T-cells were harvested at day 4. RNA was isolated from cell pellets and subject to qRT-PCR to analyze the relative expression of Th1-associated genes relative to normalized housekeeping genes. * denotes a p-value <0.05; ** denotes a p-value <0.005.
Mentions: Consistent with a prior study using a highly mutated replication-defective HSV virus, the induction of pro-inflammatory cytokines (IL-6 and TNF-α) in response to HSV-1 was blocked by vhs (Figures 2a and S3) [31]. Interestingly, the vhs protein inhibited both the virus replication independent triggering of IL-6 and TNF-α (UV inactivated virus) and the replication dependent induction of IL-12, highlighting that the activation of cDCs in response to HSV-1 occurs after sensing viral replication dependent and independent events. The production of the chemokine IP-10 was not affected by vhs. A detailed kinetic analysis revealed that the block to IL-6 and TNF-α production occurs as early as 6 hpi, whereas IL-12 release is blocked by 12 hpi (Figure S2b). This block to cytokine release is also observed at the level of mRNA expression (Figure S2c). Lastly, we also observed elevated cell surface expression of the co-stimulatory molecule CD86 in the vhs- infected cultures at 24 hpi (Figure 2b).

Bottom Line: These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein.This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs.Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology Institute, Mount Sinai School of Medicine, New York, New York, USA.

ABSTRACT
Molecular pathways underlying the activation of dendritic cells (DCs) in response to Herpes Simplex Virus type 1 (HSV-1) are poorly understood. Removal of the HSV virion host shut-off (vhs) protein relieves a block to DC activation observed during wild-type infection. In this study, we utilized a potent DC stimulatory HSV-1 recombinant virus lacking vhs as a tool to investigate the mechanisms involved in the activation of DCs by HSV-1. We report that the release of pro-inflammatory cytokines by conventional DC (cDC) during HSV-1 infection is triggered by both virus replication-dependent and replication-independent pathways. Interestingly, while vhs is capable of inhibiting the release of cytokines during infection of human and mouse cDCs, the secretion of cytokines by plasmacytoid DC (pDC) is not affected by vhs. These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein. Using cDCs isolated from mice deficient in both the TLR adaptor protein MyD88 and TLR3, we show that HSV-1 and the vhs-deleted virus can activate cDCs independently of TLR signaling. In addition, virion-associated vhs fails to block cDC activation in response to treatment with TLR agonists, but it efficiently blocked cDC activation triggered by the paramyxoviruses Sendai Virus (SeV) and Newcastle Disease Virus (NDV). This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs. Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.

Show MeSH
Related in: MedlinePlus