The Unfolded Protein Response Reveals eIF2α Phosphorylation as a Critical Factor for Direct MHC Class I Antigen Presentation

Nagamine, Brandy; Godil, Jamila; Dolan, Brian P.

doi:10.4049/immunohorizons.2100012

“…CHIKV nsP2 interferes with numerous cellular functions, including host cell transcription by promoting degradation of RPB1 [ 15 ], type 1 IFN responses by nuclear export of STAT1 [ 17 ] and inhibition of Jak/STAT phosphorylation [ 18 , 49 ], and the unfolded protein response (UPR) [ 19 ], all of which can be important for MHC-I antigen presentation [ 50 , 51 , 52 , 53 ]. Given this, we hypothesized that CHIKV nsP2 impairs MHC-I antigen presentation in CHIKV-infected cells.…”

Section: Resultsmentioning

confidence: 99%

Chikungunya virus infection disrupts MHC-I antigen presentation via nonstructural protein 2

Ware,

Parks,

da Silva

et al. 2024

PLoS Pathog

2

0

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Infection by chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes severe polyarthralgia and polymyalgia, which can last in some people for months to years. Chronic CHIKV disease signs and symptoms are associated with the persistence of viral nucleic acid and antigen in tissues. Like humans and nonhuman primates, CHIKV infection in mice results in the development of robust adaptive antiviral immune responses. Despite this, joint tissue fibroblasts survive CHIKV infection and can support persistent viral replication, suggesting that they escape immune surveillance. Here, using a recombinant CHIKV strain encoding the fluorescent protein VENUS with an embedded CD8+ T cell epitope, SIINFEKL, we observed a marked loss of both MHC class I (MHC-I) surface expression and antigen presentation by CHIKV-infected joint tissue fibroblasts. Both in vivo and ex vivo infected joint tissue fibroblasts displayed reduced cell surface levels of H2-Kb and H2-Db MHC-I proteins while maintaining similar levels of other cell surface proteins. Mutations within the methyl transferase-like domain of the CHIKV nonstructural protein 2 (nsP2) increased MHC-I cell surface expression and antigen presentation efficiency by CHIKV-infected cells. Moreover, expression of WT nsP2 alone, but not nsP2 with mutations in the methyltransferase-like domain, resulted in decreased MHC-I antigen presentation efficiency. MHC-I surface expression and antigen presentation was rescued by replacing VENUS-SIINFEKL with SIINFEKL tethered to β2-microglobulin in the CHIKV genome, which bypasses the requirement for peptide processing and TAP-mediated peptide transport into the endoplasmic reticulum. Collectively, this work suggests that CHIKV escapes the surveillance of antiviral CD8+ T cells, in part, by nsP2-mediated disruption of MHC-I antigen presentation.

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“…CHIKV nsP2 interferes with numerous cellular functions, including host cell transcription by promoting degradation of RPB1 (15), type 1 IFN responses by nuclear export of STAT1 (16) and inhibition of Jak/STAT phosphorylation (17, 48), and the unfolded protein response (UPR) (18), all of which can be important for MHC-I antigen presentation (49, 50, 51, 52). Given this, we hypothesized that CHIKV nsP2 impairs MHC-I antigen presentation in CHIKV-infected cells.…”

Section: Resultsmentioning

confidence: 99%

“…To assess MHC-I antigen presentation by physiologically relevant CHIKV target cells, we generated primary ex vivo ankle (EVA) cell cultures by adapting a previously established protocol in which single cell suspensions were generated from murine ankle tissue (41), depleted of erythrocytes and nonadherent cells, and then cultured in growth medium. 16) and inhibition of Jak/STAT phosphorylation (17,48), and the unfolded protein response (UPR) (18), all of which can be important for MHC-I antigen presentation (49,50,51,52). Given this, we hypothesized that CHIKV nsP2 impairs MHC-I antigen presentation in CHIKV-infected cells.…”

Section: Chikv Infected Primary Joint Tissue Fibroblasts Display Decr...mentioning

confidence: 99%

Chikungunya virus infection disrupts MHC-I antigen presentation via nonstructural protein 2

Ware,

Parks,

Morrison

2023

Preprint

1

0

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Infection by chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes severe polyarthralgia and polymyalgia, which can last in some people for months to years. Chronic CHIKV disease signs and symptoms are associated with the persistence of viral nucleic acid and antigen in tissues. Like humans and nonhuman primates, CHIKV infection in mice results in the development of robust adaptive antiviral immune responses. Despite this, joint tissue fibroblasts survive CHIKV infection and can support persistent viral replication, suggesting that they escape immune surveillance. Here, using a recombinant CHIKV strain encoding a chimeric protein of VENUS fused to a CD8+T cell epitope, SIINFEKL, we observed a marked loss of both MHC class I (MHC-I) surface expression and antigen presentation by CHIKV-infected joint tissue fibroblasts. Bothin vivoandex vivoinfected joint tissue fibroblasts displayed reduced cell surface levels of H2-Kband H2-DbMHC proteins while maintaining similar levels of other cell surface proteins. Mutations within the methyl transferase-like domain of the CHIKV nonstructural protein 2 (nsP2) increased MHC-I cell surface expression and antigen presentation efficiency by CHIKV-infected cells. Moreover, expression of WT nsP2 alone, but not nsP2 with mutations in the methyltransferase-like domain, resulted in decreased MHC-I antigen presentation efficiency. MHC-I surface expression and antigen presentation could be rescued by replacing VENUS-SIINFEKL with SIINFEKL tethered to β2-microglobulin in the CHIKV genome, which bypasses the need for peptide processing and TAP-mediated peptide transport into the endoplasmic reticulum. Collectively, this work suggests that CHIKV escapes the surveillance of antiviral CD8+T cells, in part, by nsP2-mediated disruption of MHC-I antigen presentation.AUTHOR SUMMARYArthritogenic alphaviruses, including chikungunya virus (CHIKV), are re-emerging global public health threats with no approved vaccines or antiviral therapies. Infection with these viruses causes debilitating musculoskeletal disease for months to years that is associated with the persistence of viral RNA and antigen. Prior studies using a mouse model found that CD8+T cells, which recognize viral peptides in the context of major histocompatibility class I (MHC-I) displayed on the surface of infected cells, have a limited role in the control and clearance of CHIKV infection in joint-associated tissues, suggesting that CHIKV infected cells evade these critical effectors of the anti-viral immune response. Here, we show that MHC-I antigen presentation is inefficient in CHIKV-infected joint tissue fibroblasts, and that a protein encoded by CHIKV and produced in infected cells, nonstructural protein 2 (nsP2), disrupts the surface display of MHC-I molecules and antigen recognition of infected cells by CD8+T cells. Our findings support a role for CHIKV nsP2 in the evasion of the CD8+T cell response and establishment of persistent infection.

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“…The Shield-controlled Recombinant Antigenic Protein (SCRAP) system is a powerful tool for measuring peptide presentation from three different forms of the same precursor protein: DRiPs, retirees, and normal protein turnover (or non-DRiP). The method for quantifying peptide presentation from this system has been used extensively and previously published (27)(28)(29)(30)(31)(32). Briefly, EL4/SCRAP-mCherry cells are washed in citric acid buffer and then re-cultured in either ethanol (negative control), Shield-1, or MG132 for six hours, stained with APC-coupled 25D-1.16 mAbs and analysed by flow cytometry.…”

Section: Antigen Presentation Assays In Stable Cell Linesmentioning

confidence: 99%

“…The use of Shield-1 to stabilize SCRAP-mCherry allows us to control the source of SIINFEKL peptides for MHC class I antigen presentation and independently measure peptide presentation from previously synthesized SCRAP-mCherry being "retired" by the cell, normal protein turnover based upon the predicted half-life of SCRAP-mCherry, and Defective Ribosomal Products (DRiPs) which is a form of SCRAP-mCherry which is inherently refractory to stabilization by Shield-1 treatment. This system has been used multiple times to identify which cellular components control presentation of peptides from their respective sources (27)(28)(29)(30)(31)(32).…”

Section: Presentation Of Peptides From Drips Is Dependent On Nedd8 Ac...mentioning

confidence: 99%

NEDD8 activity is important for direct antigen MHC class I antigen presentation

Vijayasimha

¹

,

Leestemaker-Palmer

²

,

Gibbs

³

et al. 2022

Preprint

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0

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Successful direct MHC class I antigen presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides which can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin-proteasome system, however other ubiquitin-like (UBL) proteins have also been implicated in protein degradation and direct antigen presentation. Here, we examine the role of neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) in direct antigen presentation. NEDD8 is the UBL with highest similarity to ubiquitin and fusion of NEDD8 to the amino-terminus of a target protein can lead to the target proteins degradation. We find that appending NEDD8 to the N-terminus of the model antigen ovalbumin resulted in degradation by both the proteasome and autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1 (NUB1), resulted in peptide presentation. When directly compare to ubiquitin, NEDD8-fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924, inhibited the presentation of peptides from Defective Ribosomal Products (DRiPs) derived from a model antigen. These results demonstrate that NEDD8 activity in the cell is important for direct antigen presentation, but not by directly targeting proteins for degradation.

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The Unfolded Protein Response Reveals eIF2α Phosphorylation as a Critical Factor for Direct MHC Class I Antigen Presentation

Cited by 6 publications

References 62 publications

Chikungunya virus infection disrupts MHC-I antigen presentation via nonstructural protein 2

Chikungunya virus infection disrupts MHC-I antigen presentation via nonstructural protein 2

Chikungunya virus infection disrupts MHC-I antigen presentation via nonstructural protein 2

NEDD8 activity is important for direct antigen MHC class I antigen presentation

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