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Ganglioside and Non-ganglioside Mediated Host Responses to the Mouse Polyomavirus.

You J, O'Hara SD, Velupillai P, Castle S, Levery S, Garcea RL, Benjamin T - PLoS Pathog. (2015)

Bottom Line: Specificity is determined by recognition of carbohydrate moieties on the ganglioside by the major viral capsid protein VP1.Ganglioside-deficient fibroblasts responded rapidly to virus exposure with a transient induction of c-fos as an early manifestation of a mitogenic response.Thus, while gangliosides are essential for infection in the animal, gangliosides are not required for mitogenic responses and innate immune responses to the virus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Gangliosides serve as receptors for internalization and infection by members of the polyomavirus family. Specificity is determined by recognition of carbohydrate moieties on the ganglioside by the major viral capsid protein VP1. For the mouse polyomavirus (MuPyV), gangliosides with terminal sialic acids in specific linkages are essential. Although many biochemical and cell culture experiments have implicated gangliosides as MuPyV receptions, the role of gangliosides in the MuPyV-infected mouse has not been investigated. Here we report results of studies using ganglioside-deficient mice and derived cell lines. Knockout mice lacking complex gangliosides were completely resistant to the cytolytic and pathogenic effects of the virus. Embryo fibroblasts from these mice were likewise resistant to infection, and supplementation with specific gangliosides restored infectibility. Although lacking receptors for viral infection, cells from ganglioside-deficient mice retained the ability to respond to the virus. Ganglioside-deficient fibroblasts responded rapidly to virus exposure with a transient induction of c-fos as an early manifestation of a mitogenic response. Additionally, splenocytes from ganglioside-deficient mice responded to MuPyV by secretion of IL-12, previously recognized as a key mediator of the innate immune response. Thus, while gangliosides are essential for infection in the animal, gangliosides are not required for mitogenic responses and innate immune responses to the virus.

No MeSH data available.


Related in: MedlinePlus

A. GT1a and GD1b restore infectibility to B4St8 MEFs. GT1a, GD1a, and GD1b supplementation of B4St8 KO MEFs rescued RA infection in a dose responsive manner (0.5μM to 2μM). GM1 supplementation did not rescue infection of B4St8 KO MEFs. B. Virus binding to wild-type, B4St8, and GD1a-supplemented B4St8 MEFs. WT, B4St8, and GD1a-supplemented B4St8 KO MEFs were assayed for MuPyV binding by flow cytometry. Cells were starved overnight in serum free media with or without 5μM GD1a. Cells were then infected with MuPyV (NG59RA, MOI 10) at 4°C. After 30 mins at 4°C, bound virus was detected by VP1 staining. WT, B4St8, and GD1a-supplemented B4ST8 KO MEFs had a 6 to 8 fold increase in VP1 staining compared to uninfected cells (shown in grey). GD1a-supplemented B4St8 KO MEFs showed a similar VP1 binding pattern as WT MEFs and B4St8 KO MEFs showed lower levels of VP1 accumulation on the cell surface. The x-axis is VP1 staining and the y-axis is normalized cell counts, each sample contained >10,000 cells.
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ppat.1005175.g003: A. GT1a and GD1b restore infectibility to B4St8 MEFs. GT1a, GD1a, and GD1b supplementation of B4St8 KO MEFs rescued RA infection in a dose responsive manner (0.5μM to 2μM). GM1 supplementation did not rescue infection of B4St8 KO MEFs. B. Virus binding to wild-type, B4St8, and GD1a-supplemented B4St8 MEFs. WT, B4St8, and GD1a-supplemented B4St8 KO MEFs were assayed for MuPyV binding by flow cytometry. Cells were starved overnight in serum free media with or without 5μM GD1a. Cells were then infected with MuPyV (NG59RA, MOI 10) at 4°C. After 30 mins at 4°C, bound virus was detected by VP1 staining. WT, B4St8, and GD1a-supplemented B4ST8 KO MEFs had a 6 to 8 fold increase in VP1 staining compared to uninfected cells (shown in grey). GD1a-supplemented B4St8 KO MEFs showed a similar VP1 binding pattern as WT MEFs and B4St8 KO MEFs showed lower levels of VP1 accumulation on the cell surface. The x-axis is VP1 staining and the y-axis is normalized cell counts, each sample contained >10,000 cells.

Mentions: GD1a and GT1b have previously been shown to confer susceptibility to MuPyV infection when added to ganglioside-deficient rat and mouse cell lines [2, 28, 29]. We sought to extend these results using our B4St8 KO cells, which are genetically defined and have known ganglioside composition. GD1a was used as a positive control, and GM1, the SV40 receptor, as a negative control to confirm previous results. We then tested the ability of additional gangliosides, GT1a and GD1b, to confer susceptibility using the RA strain of virus. GT1a had been suggested as a possible receptor based on co-crystallization with MuPyV VP1 [26]. Cells were pre-incubated with 0.5 to 2.0 μM gangliosides in serum-free medium for 16 hrs, then infected and scored for T-antigen expression 24 hrs post-infection. GT1a conferred infectibility slightly more efficiently than GD1a, a result consistent with in vitro affinity studies [26], and GD1b conferred low levels of infectibility, much less efficiently than GT1a or GD1a (Fig 3A).


Ganglioside and Non-ganglioside Mediated Host Responses to the Mouse Polyomavirus.

You J, O'Hara SD, Velupillai P, Castle S, Levery S, Garcea RL, Benjamin T - PLoS Pathog. (2015)

A. GT1a and GD1b restore infectibility to B4St8 MEFs. GT1a, GD1a, and GD1b supplementation of B4St8 KO MEFs rescued RA infection in a dose responsive manner (0.5μM to 2μM). GM1 supplementation did not rescue infection of B4St8 KO MEFs. B. Virus binding to wild-type, B4St8, and GD1a-supplemented B4St8 MEFs. WT, B4St8, and GD1a-supplemented B4St8 KO MEFs were assayed for MuPyV binding by flow cytometry. Cells were starved overnight in serum free media with or without 5μM GD1a. Cells were then infected with MuPyV (NG59RA, MOI 10) at 4°C. After 30 mins at 4°C, bound virus was detected by VP1 staining. WT, B4St8, and GD1a-supplemented B4ST8 KO MEFs had a 6 to 8 fold increase in VP1 staining compared to uninfected cells (shown in grey). GD1a-supplemented B4St8 KO MEFs showed a similar VP1 binding pattern as WT MEFs and B4St8 KO MEFs showed lower levels of VP1 accumulation on the cell surface. The x-axis is VP1 staining and the y-axis is normalized cell counts, each sample contained >10,000 cells.
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ppat.1005175.g003: A. GT1a and GD1b restore infectibility to B4St8 MEFs. GT1a, GD1a, and GD1b supplementation of B4St8 KO MEFs rescued RA infection in a dose responsive manner (0.5μM to 2μM). GM1 supplementation did not rescue infection of B4St8 KO MEFs. B. Virus binding to wild-type, B4St8, and GD1a-supplemented B4St8 MEFs. WT, B4St8, and GD1a-supplemented B4St8 KO MEFs were assayed for MuPyV binding by flow cytometry. Cells were starved overnight in serum free media with or without 5μM GD1a. Cells were then infected with MuPyV (NG59RA, MOI 10) at 4°C. After 30 mins at 4°C, bound virus was detected by VP1 staining. WT, B4St8, and GD1a-supplemented B4ST8 KO MEFs had a 6 to 8 fold increase in VP1 staining compared to uninfected cells (shown in grey). GD1a-supplemented B4St8 KO MEFs showed a similar VP1 binding pattern as WT MEFs and B4St8 KO MEFs showed lower levels of VP1 accumulation on the cell surface. The x-axis is VP1 staining and the y-axis is normalized cell counts, each sample contained >10,000 cells.
Mentions: GD1a and GT1b have previously been shown to confer susceptibility to MuPyV infection when added to ganglioside-deficient rat and mouse cell lines [2, 28, 29]. We sought to extend these results using our B4St8 KO cells, which are genetically defined and have known ganglioside composition. GD1a was used as a positive control, and GM1, the SV40 receptor, as a negative control to confirm previous results. We then tested the ability of additional gangliosides, GT1a and GD1b, to confer susceptibility using the RA strain of virus. GT1a had been suggested as a possible receptor based on co-crystallization with MuPyV VP1 [26]. Cells were pre-incubated with 0.5 to 2.0 μM gangliosides in serum-free medium for 16 hrs, then infected and scored for T-antigen expression 24 hrs post-infection. GT1a conferred infectibility slightly more efficiently than GD1a, a result consistent with in vitro affinity studies [26], and GD1b conferred low levels of infectibility, much less efficiently than GT1a or GD1a (Fig 3A).

Bottom Line: Specificity is determined by recognition of carbohydrate moieties on the ganglioside by the major viral capsid protein VP1.Ganglioside-deficient fibroblasts responded rapidly to virus exposure with a transient induction of c-fos as an early manifestation of a mitogenic response.Thus, while gangliosides are essential for infection in the animal, gangliosides are not required for mitogenic responses and innate immune responses to the virus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Gangliosides serve as receptors for internalization and infection by members of the polyomavirus family. Specificity is determined by recognition of carbohydrate moieties on the ganglioside by the major viral capsid protein VP1. For the mouse polyomavirus (MuPyV), gangliosides with terminal sialic acids in specific linkages are essential. Although many biochemical and cell culture experiments have implicated gangliosides as MuPyV receptions, the role of gangliosides in the MuPyV-infected mouse has not been investigated. Here we report results of studies using ganglioside-deficient mice and derived cell lines. Knockout mice lacking complex gangliosides were completely resistant to the cytolytic and pathogenic effects of the virus. Embryo fibroblasts from these mice were likewise resistant to infection, and supplementation with specific gangliosides restored infectibility. Although lacking receptors for viral infection, cells from ganglioside-deficient mice retained the ability to respond to the virus. Ganglioside-deficient fibroblasts responded rapidly to virus exposure with a transient induction of c-fos as an early manifestation of a mitogenic response. Additionally, splenocytes from ganglioside-deficient mice responded to MuPyV by secretion of IL-12, previously recognized as a key mediator of the innate immune response. Thus, while gangliosides are essential for infection in the animal, gangliosides are not required for mitogenic responses and innate immune responses to the virus.

No MeSH data available.


Related in: MedlinePlus