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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

Virus Internalization in Wild-Type MEFs.Wild-type MEFs were infected with MuPyV and then fixed at the indicated times post infection (30 min, 3hrs). (A) Slides were stained for cell surface VP1 (red), and then permeabilized and stained for total VP1, showing both cell surface and intracellular VP1 (green). (B) At 30 mins post-infection line scan analysis shows that MEFs exhibit similar staining for cell surface and total VP1, indicating minimal virus internalization. (C) At 3 hrs post-infection line scan analysis shows that MEFs exhibit abundant intracellular VP1 staining (green only), indicating internalized virus.
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ppat.1005175.g004: Virus Internalization in Wild-Type MEFs.Wild-type MEFs were infected with MuPyV and then fixed at the indicated times post infection (30 min, 3hrs). (A) Slides were stained for cell surface VP1 (red), and then permeabilized and stained for total VP1, showing both cell surface and intracellular VP1 (green). (B) At 30 mins post-infection line scan analysis shows that MEFs exhibit similar staining for cell surface and total VP1, indicating minimal virus internalization. (C) At 3 hrs post-infection line scan analysis shows that MEFs exhibit abundant intracellular VP1 staining (green only), indicating internalized virus.

Mentions: Using the B4St8 ganglioside KO MEFs we determined whether gangliosides are required for virus entry. Wild-type MEFs and B4St8 KO MEFs were infected with MuPyV (RA, 50 PFU/cell) and then fixed at the indicated times post-infection (30 min, 3 hrs). At 30 mins post-infection in wild-type MEFs line scan analysis showed that MEFs exhibit similar staining for cell surface (shown in red) and total VP1 (shown in green), indicating minimal virus internalization at this early time (Fig 4B). Similar results were seen in B4St8 KO MEFs at 30 min post infection (Fig 5B). At 3 hrs post-infection in wild-type MEFs line scan analysis showed that MEFs had abundant intracellular VP1 staining (green only), indicating a large fraction of internalized virus (Fig 4C). B4St8 ganglioside KO-MEFs also displayed high levels of internalized virus as shown by line scan analysis (green only) (Fig 5C). These data demonstrate that gangliosides are not required for virus entry into MEFs, and virus can enter cells through non-ganglioside mediated pathways. Given the complete resistance of these B4St8 KO cells to MuPyV infection (Table 1), it can be assumed that virus uptake via these alternative routes proceeds along a non-infectious or ‘dead end’ pathway. While the presence of glycoproteins such as α4β1 integrin have been observed to enhance infection [18, 19], gangliosides are required for virus uptake along infectious pathways.


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)

Virus Internalization in Wild-Type MEFs.Wild-type MEFs were infected with MuPyV and then fixed at the indicated times post infection (30 min, 3hrs). (A) Slides were stained for cell surface VP1 (red), and then permeabilized and stained for total VP1, showing both cell surface and intracellular VP1 (green). (B) At 30 mins post-infection line scan analysis shows that MEFs exhibit similar staining for cell surface and total VP1, indicating minimal virus internalization. (C) At 3 hrs post-infection line scan analysis shows that MEFs exhibit abundant intracellular VP1 staining (green only), indicating internalized virus.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608836&req=5

ppat.1005175.g004: Virus Internalization in Wild-Type MEFs.Wild-type MEFs were infected with MuPyV and then fixed at the indicated times post infection (30 min, 3hrs). (A) Slides were stained for cell surface VP1 (red), and then permeabilized and stained for total VP1, showing both cell surface and intracellular VP1 (green). (B) At 30 mins post-infection line scan analysis shows that MEFs exhibit similar staining for cell surface and total VP1, indicating minimal virus internalization. (C) At 3 hrs post-infection line scan analysis shows that MEFs exhibit abundant intracellular VP1 staining (green only), indicating internalized virus.
Mentions: Using the B4St8 ganglioside KO MEFs we determined whether gangliosides are required for virus entry. Wild-type MEFs and B4St8 KO MEFs were infected with MuPyV (RA, 50 PFU/cell) and then fixed at the indicated times post-infection (30 min, 3 hrs). At 30 mins post-infection in wild-type MEFs line scan analysis showed that MEFs exhibit similar staining for cell surface (shown in red) and total VP1 (shown in green), indicating minimal virus internalization at this early time (Fig 4B). Similar results were seen in B4St8 KO MEFs at 30 min post infection (Fig 5B). At 3 hrs post-infection in wild-type MEFs line scan analysis showed that MEFs had abundant intracellular VP1 staining (green only), indicating a large fraction of internalized virus (Fig 4C). B4St8 ganglioside KO-MEFs also displayed high levels of internalized virus as shown by line scan analysis (green only) (Fig 5C). These data demonstrate that gangliosides are not required for virus entry into MEFs, and virus can enter cells through non-ganglioside mediated pathways. Given the complete resistance of these B4St8 KO cells to MuPyV infection (Table 1), it can be assumed that virus uptake via these alternative routes proceeds along a non-infectious or ‘dead end’ pathway. While the presence of glycoproteins such as α4β1 integrin have been observed to enhance infection [18, 19], gangliosides are required for virus uptake along infectious pathways.

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