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Open Access

Peer-reviewed

Research Article

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

  • Brian C. Ware,

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America

    ⨯
  • M. Guston Parks,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America

    ⨯
  • Mariana O. L. da Silva,

    Roles Investigation, Writing – review & editing

    Affiliations Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America, Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    ⨯
  • Thomas E. Morrison

    Roles Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing

    thomas.morrison@cuanschutz.edu

    Affiliation Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America

    ⨯

Abstract

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.

Author summary

Arthritogenic 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 antiviral 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.

Citation: Ware BC, Parks MG, da Silva MOL, Morrison TE (2024) Chikungunya virus infection disrupts MHC-I antigen presentation via nonstructural protein 2. PLoS Pathog 20(3): e1011794. https://doi.org/10.1371/journal.ppat.1011794

Editor: Gorben P. Pijlman, Wageningen University, NETHERLANDS

Received: November 2, 2023; Accepted: March 4, 2024; Published: March 14, 2024

Copyright: © 2024 Ware et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by Public Health Service grants R01 AI141436 (T.E.M.) and R01 AI148144 (T.E.M.) from the National Institute of Allergy and Infectious Diseases (https://www.niaid.nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Since its re-emergence in 2004 in the Indian Ocean region [1,2], chikungunya virus (CHIKV) has spread to numerous subtropical and tropical regions of Asia [3], the Caribbean [4], the Americas [2] and Europe [5] causing large-scale mosquito-borne epidemics that challenge public health systems. In the Americas, over three million CHIKV cases in 45 countries have been documented, and the global spread of CHIKV places approximately 1.3 billion people at risk for infection [6]. CHIKV infection causes an acute debilitating febrile illness accompanied by high fever, severe myalgia, and arthralgia. Musculoskeletal tissue pain and inflammation can become chronic in up to two thirds of patients [7,8,9]. Due to the increasing incidence of CHIKV disease, the unpredictable nature of its outbreaks, and the lack of approved vaccines or antiviral medications, it remains critical to elucidate CHIKV pathogenesis and immune restriction and evasion mechanisms.

CHIKV is one of ~32 Togaviridae family members [10] and is comprised of an 11.8 kilobase, positive-sense, single-stranded RNA genome that contains two open reading frames encoding nonstructural and structural polyproteins [11]. After an alphavirus particle binds a host cell, the virion is internalized by clathrin-mediated endocytosis. Upon fusion of the viral and endosomal membranes, the virion disassembles, and viral RNA (vRNA) is released into the cytoplasm. The vRNA is translated by host ribosomes to generate the nonstructural polyprotein P123 or P1234 via read-through of the opal termination codon in nsP3 [11]. The protease activity of nonstructural protein 2 (nsP2) cleaves P1234 into P123 and nsP4, thus allowing for formation of a replication complex that synthesizes full-length negative-strand RNA from the template viral genome. Subsequent processing of P123 into nsP1, nsP2, and nsP3 results in formation of a replicase that synthesizes positive sense genomic vRNA as well as a subgenomic mRNA that is translated into the viral structural polyprotein. Though nsP1-4 are synthesized at equal molar ratios in their polyprotein form, only a single nsP2 for every twelve nsP1s is found within the replicase complex [12,13], suggesting nsP2 has auxiliary functions apart from the replicase. Indeed, nsP2 has been reported to enter nuclei and export STAT1 [14], suppress host mRNA transcription through the induction of RPB1 ubiquitination and degradation [15], induce translational shutoff by phosphorylation of eEF2 [16], disrupt type I interferon (IFN) signaling [17,18], and suppress the unfolded protein response (UPR) [19].

CD8+ T cells are key executors of the adaptive immune response against many viral infections, including some arbovirus infections [20,21,22,23,24,25]. Their capacity to surveil, recognize, and lyse virus-infected cells is well documented [26,27,28,29]. Early innate immune responses, including inflammatory cytokines [30,31], and the presence of viral peptides displayed by major histocompatibility complex class I (MHC-I) molecules on cell surfaces dictate the breadth and duration of the CD8+ T cell response. Importantly, CD8+ T cells scan for the presence of viral peptide-loaded MHC-I (pMHC-I) complexes on the surface of infected cells via their T cell receptor (TCR) [32,33]. Ligation of the TCR leads to activation and release of cytotoxic effectors such as perforin 1 and granzyme B and the subsequent destruction of the infected target cell [34]. Numerous viruses have evolved mechanisms to subvert this crucial immune surveillance system through downregulation, suppression, or elimination of pMHC-I complexes from the surface of infected cells, thereby evading CD8+ T cell-mediated clearance [35]. Although this paradigm has been extensively illustrated for DNA viruses such as herpesviruses and poxviruses [36], some RNA viruses also have evolved mechanisms for interfering with CD8+ T cell recognition. For example, the human immunodeficiency virus 1 Nef protein diverts pMHC-I complexes to the protein degradation pathway [37,38], and infection with influenza A and B viruses also suppresses surface MHC-I expression [39]. SARS-CoV-2 infection was shown to disrupt MHC-I cell surface expression [40,41], in part, by disrupting nuclear transport of NLRC5 [41], the principal MHC-I transcription factor.

Recently, we observed that during CHIKV infection, CD8+ T cells accumulate in joint-associated tissue and lyse exogenously peptide-loaded splenocytes that had been adoptively transferred into the tissue. Despite this finding, CHIKV-specific CD8+ T cells appear to have little to no impact on the clearance of CHIKV-infected cells, as the viral burden in joint tissues of WT and Cd8α-/- mice are similar throughout the course of infection, suggesting that CHIKV-infected cells evade the CD8+ T cell response [42].

Here, using a recombinant chimeric protein composed of the reporter VENUS fused to a well characterized CD8+ T cell epitope (ovalbumin 257–264; SIINFEKL), embedded in the CHIKV genome, we observed a marked loss of both MHC-I cell surface expression and antigen presentation by CHIKV-infected joint tissue fibroblasts. Mutations within the methyl transferase-like (MTL) domain of the CHIKV nsP2 restored MHC-I cell surface expression and antigen presentation efficiency by CHIKV-infected cells. In contrast to cell surface MHC-I expression, intracellular MHC-I levels were unaffected by CHIKV infection. Moreover, MHC-I antigen presentation by CHIKV-infected cells was rescued by replacing the VENUS-SIINFEKL in the CHIKV genome with SIINFEKL tethered to β2-microglobulin (β2m), which obviates the need for peptide processing and TAP-mediated peptide transport into the endoplasmic reticulum (ER). Finally, we find that expression of wild-type (WT) nsP2 alone, but not nsP2 with mutations in the MTL domain, is sufficient to disrupt MHC-I antigen presentation efficiency in primary joint tissue fibroblasts. In contrast, expression of nsP2 with mutations known to abrogate helicase or protease activity disrupts MHC-I antigen presentation similar to WT nsP2, suggesting a specific role for the MTL domain in disrupting MHC-I antigen presentation. Collectively, this work suggests that CHIKV-infected cells escape surveillance by antiviral CD8+ T cells, in part, by nsP2-mediated disruption of MHC-I antigen presentation.

Results

CHIKV-infected primary joint tissue fibroblasts display decreased MHC-I cell surface expression

To determine if virus-encoded peptides are presented by MHC-I on CHIKV-infected cells, a recombinant CHIKV strain (CHIKV-VENKL) was engineered to express the chimeric protein VENUS-SIINFEKL (VENKL) wherein VENUS, a fluorescent reporter protein [43], and the CD8+ T cell peptide SIINFEKL from ovalbumin [44] were encoded between the capsid autoproteolysis domain [45] and an engineered 2a ribosomal skip site in-frame in the structural open reading frame of the CHIKV genome (Fig 1A), a design we have used previously [46]. Following the C-terminus of VENUS, SIINFEKL is flanked by the amino acid sequences LEQLE and TEW, which are important for efficient peptide processing and MHC-I loading of SIINFEKL [47]. This design allows for the measurement of MHC-I antigen presentation efficiency because we can measure SIINFEKL presentation as a function of source protein (i.e., VENUS) across all cells on a per-cell basis [47]. 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 [42], depleted of erythrocytes and nonadherent cells, and then cultured in growth medium.

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Fig 1. CHIKV-infected primary joint tissue fibroblasts display decreased MHC-I cell surface expression.

(A) Schematic of the recombinant CHIKV-VENKL. The coding sequence for the VENUS-SIINFEKL chimeric protein was inserted in-frame into the structural ORF of the CHIKV genome with known cleavage motifs flanking SIINFEKL. (B-E) EVA cells were inoculated with PBS (mock) or 106 PFU CHIKV-VENKL. (B) At 24 hpi, the percentages of CD45- (open circles) and CD45+ (open squares) cells among mock-inoculated cells and VENUS+ cells were determined. (C) The magnitude and frequency of expression of CD29 on CD45- cells was determined by flow cytometry. (D) Representative flow cytometry plots of H2-Kb and VENUS expressing cells from mock (black) and CHIKV-VENKL-infected cell populations (VENUS+, red; VENUS-, grey). (E) Quantification of surface expression and frequency of H2-Kb and H2-Db from mock and CHIKV-VENKL-infected EVA cells. Data are pooled (B), or representative (C, E) from 3–4 independent experiments. P values were determined by unpaired student’s t-test (B) or one-way ANOVA with Tukey’s test for multiple comparisons (C,E). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.g001

After 24 hours (h) in culture, EVA cells were assessed for cellular composition, the capacity to support CHIKV infection, and MHC-I cell surface expression by flow cytometry. More than 95% of EVA cells were CD45- (Figs 1B and S1A) and within that population most cells (99%) expressed the fibroblast marker CD29 (Figs 1C and S1A), a marker of ankle tissue fibroblasts shown previously to support CHIKV infection in vivo [48]. In addition, 75–80% of the CD45- cells had detectable H2-Kb and H2-Db MHC-I molecules on the cell surface (Fig 1D–1E). To evaluate their capacity to support CHIKV infection, EVA cells were mock-inoculated or inoculated with 106 plaque forming units (PFU), a multiplicity of infection (MOI) of ~0.67 FFU/cell, of CHIKV-VENKL and at 24 h post-inoculation (hpi) cells were evaluated for VENUS expression as an indicator of productive CHIKV infection. Consistent with prior studies [48], most CHIKV-infected cells were CD45- and expressed CD29 on their surface (Figs 1B, 1C, and S1A). Next, we assessed surface levels of MHC-I on CHIKV-infected VENUS+ cells and control cells. VENUS+CD45- EVA cells showed marked reduction of H2-Db and H2-Kb cell surface expression compared with VENUS-CD45- EVA cells exposed to CHIKV, but not mock-infected CD45- EVA cells, as measured by geometric mean fluorescence intensity (gMFI) (Fig 1D–1E). The percentage of VENUS+CD45- EVA cells that displayed cell surface H2-Kb and H2-Db was reduced compared with mock-infected CD45- EVA cells (Fig 1D–1E). In contrast, the percentage of VENUS+CD45- EVA cells that expressed cell surface CD29 was unchanged compared with control cells (Fig 1C). To assess the extent to which VENUS expression correlated with cell surface levels of MHC-I, cells were stratified into gates based on the intensity of VENUS expression (S1B Fig) and then assessed for H2-Kb and H2-Db expression. As VENUS expression increased, H2-Kb and H2-Db expression decreased (S1C Fig), suggesting cells with more CHIKV infection express less cell surface MHC-I. Collectively, these data suggest that CHIKV infection suppresses cell surface MHC-I expression but not expression of other cell surface proteins.

Determinants in nsP2 promote impairment of MHC-I antigen presentation by CHIKV-infected cells

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. In prior studies, Akhrymuk et al. demonstrated that mutations in the MTL domain of CHIKV nsP2 at ATL674-676QMS (QMS) abrogate nsP2-mediated inhibition of cellular transcription and type I IFN responses, as well as the cytopathic effects induced by CHIKV infection, without compromising CHIKV replication in vitro [54]. We therefore incorporated the QMS mutations into CHIKV-VENKL (CHIKVQMS-VENKL) (Fig 2A) and confirmed that murine fibroblasts supported comparable viral replication of CHIKV-VENKL and CHIKVQMS-VENKL, particularly in the absence of type I IFN signaling (Fig 2B). EVA cells were inoculated with 106 PFU of CHIKV-VENKL or CHIKVQMS-VENKL and at 24 hpi cells were evaluated by flow cytometry for VENUS and cell surface H2-Kb (Figs 2C and S2). In addition, we quantified cell surface SIINFEKL-loaded H2-Kb (H2-Kb-SIINFEKL) using the 25D1.15 T cell receptor mimic monoclonal antibody [55]. CHIKV-VENKL and CHIKVQMS-VENKL both infected CD45- EVA cells, albeit with modestly different efficiencies (Figs 2D and S2). The surface level of CD29 was similar on cells infected with either CHIKV-VENKL or CHIKVQMS-VENKL (Figs 2E and S2). In contrast, the surface level of H2-Kb on cells infected with CHIKV-VENKL was reduced compared with cells infected with CHIKVQMS-VENKL (Fig 2F). Moreover, the percentage of VENUS+ cells that were double positive for both H2-Kb-SIINFEKL (i.e., viral peptide-loaded MHC-I) and H2-Kb was significantly lower among cells infected with CHIKV-VENKL compared with CHIKVQMS-VENKL, and these cells showed markedly lower pMHC-I presentation efficiency (Fig 2G–2H). These data indicate that MHC-I presentation of viral peptides is less efficient in CHIKV-infected cells and that this can be improved by mutations in nsP2.

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Fig 2. Determinants in nsP2 promote impairment of MHC-I antigen presentation by CHIKV-infected cells.

(A) Schematic depicting the location of nsP2 V-loop mutations (QMS; blue) compared with wildtype nsP2 (ATL; red). (B) WT and Ifnar1-/- murine embryonic fibroblasts (MEFs) were inoculated with CHIKV-VENKL or CHIKVQMS-VENKL at an MOI of 0.1 FFU/cell. At 0 (input), 1, 24, 48, and 72 hpi, the amount of infectious virus present in culture supernatants was quantified by focus formation assay. (C-H) EVA cells were mock-inoculated or inoculated with CHIKV-VENKL or CHIKVQMS-VENKL. At 24 hpi, cells were assessed for cell surface expression of H2-Kb, and SIINFEKL-loaded H2-Kb (H2-Kb-SIINFEKL) by flow cytometry. (C) Representative flow cytometry plots depicting H2-Kb-SIINFEKL and H2-Kb cell surface expression on live, CD45- and live, CD45-, VENUS+ cells from mock-, CHIKV-VENKL-, and CHIKVQMS-VENKL-inoculated EVA cells. (D) Percentage of VENUS+ cells among live, CD45-, EVA cells. (E) CD29 cell surface expression on live, CD45-, VENUS+ EVA cells. (F) Quantification of H2-Db and H2-Kb surface expression on live, CD45-, VENUS+ EVA cells. (G) Percentage of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells on live, CD45-, VENUS+ EVA cells. (H) pMHC-I presentation efficiency on live, CD45-, VENUS+ EVA cells. Data are representative of 4 experiments (n = 12). P values were determined by unpaired student’s t-test (D-H). **, P<0.01; ***, P<0.001; ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.g002

To further assess whether CHIKV infection interferes with functional MHC-I antigen presentation, and validate our direct assessment of cell surface H2-Kb-SIINFEKL detected by the 25D1.16 antibody, we developed a co-culture system utilizing highly specific and sensitive transgenic OT-I CD8+ T cells, which upregulate cell surface expression of CD69 and CD25 upon recognition of and activation by H2-Kb-SIINFEKL complexes [56]. To determine if OT-I CD8+ T cells are activated by CHIKV-infected cells, EVA cells were inoculated with mock inoculum or 106 PFU of CHIKV-VENKL or CHIKVQMS-VENKL. At 24 hpi, all groups of cells were washed and co-cultured for 6 h with a 1:1 mixture of 106 purified OT-I CD8+ T cells and 106 bystander CD8+ T cells with >95% purity and minimal background activation prior to addition (S3A–S3C Fig). Consistent with the low level of cell surface H2-Kb-SIINFEKL detected on CHIKV-VENKL-infected EVA cells (Fig 2C and 2G), OT-I CD8+ T cells co-cultured with CHIKV-VENKL-infected EVA cells showed significantly lower fractions of CD69 and CD25 double positive cells (Fig 3A and 3B), and surface expression of CD69 (Fig 3C) and CD25 (Fig 3D) than CHIKVQMS-VENKL-infected EVA cells. OT-I CD8+ T cells co-cultured with CHIKV-VENKL infected EVA cells had increased cell surface CD69 expression compared with bystander CD8+ T cells (Fig 3A and 3C), however, few of these cells expressed CD25 (Fig 3A) indicating that their activation was not specific to TCR ligation. These data suggest that CHIKV-infected cells are inefficiently recognized by antigen-specific CD8+ T cells and that this evasion is promoted by determinants in nsP2. In addition, these findings demonstrate that the lower signal detected by the H2-Kb-SIINFEKL antibody on CHIKV-VENKL-infected cells functionally impacts antigen presentation to CD8+ T cells.

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Fig 3. WT CHIKV-infected cells inefficiently activate CD8+ T cells in vitro.

(A-D) EVA cells were mock-inoculated or inoculated with 106 PFU of CHIKV-VENKL or CHIKVQMS-VENKL. At 24 hpi, EVA cells were cocultured with a 1:1 mixture of 106 bystander CD8+ T cells and 106 OT-I SIINFEKL-specific CD8+ T cells for 6 h. Bystander CD8+ T cells were distinguished from OT-I CD8+ T cells by gating on CD8+, KbOVA257-264 tetramer positive or negative cells, and both populations were assessed for the dual expression of CD69 and CD25 by flow cytometry. (A) Representative flow cytometry plots. (B) Quantification of CD25+CD69+ double positive cell frequencies for bystander (black) and OT-I (colored) CD8+ T cells. (C) Cell surface expression of CD69 (gMFI). (D) Cell surface expression of CD25 (gMFI). Data are representative of two independent experiments (n = 6). P values were determined by paired student’s t-test. ***, P<0.001; ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.g003

MHC-I antigen presentation is inefficient in CHIKV-infected cells in vivo

To evaluate whether CHIKV-infected cells display poor MHC-I antigen presentation in vivo, WT C57BL/6 mice were inoculated in the left-rear footpad with 103 PFU of either CHIKV-VENKL or CHIKVQMS-VENKL and ankle-associated cells were evaluated by flow cytometry for VENUS, as an indicator of infection (Figs 4A and S4), and cell surface CD45, CD29, CD44, H2-Kb and H2-Kb-SIINFEKL expression on day 1, 2, and 5 post inoculation. Like EVA cells infected ex vivo, 85–95% of VENUS+ cells in both CHIKV-VENKL and CHIKVQMS-VENKL infected mice were CD45-CD29+ (Figs 4B and S4), indicating that most cells infected by either virus are joint tissue-associated fibroblasts. However, fewer infected cells were detected at both 1 and 2 dpi in mice inoculated with CHIKVQMS-VENKL (Figs 4A and S4), suggesting replication of this virus is restricted. At 2 and 5 dpi, CHIKV-VENKL-infected cells had lower cell surface expression of H2-Kb, as determined by the ratio of H2-Kb gMFI on VENUS+ cells to VENUS- cells, compared with CHIKVQMS-VENKL-infected cells (Fig 4C). While CD44 (Fig 4D) and CD29 (Fig 4E) expression on CHIKV-VENKL- and CHIKVQMS-VENKL-infected cells eventually differ by 5 dpi, at 1 and 2 dpi cell surface expression of both molecules was comparable. In comparison with CHIKVQMS-VENKL-infected cells, a lower percentage of CHIKV-VENKL-infected cells displayed H2-Kb-SIINFEKL on their surface across all time points examined, resulting in reduced pMHC-I presentation efficiency (Fig 4F–4H).

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Fig 4. CHIKV-infected cells inefficiently display MHC-I antigen in vivo.

(A-H) WT C57BL/6 mice were inoculated with 103 PFU of CHIKV-VENKL (red) or CHIKVQMS-VENKL (blue). At 1, 2, and 5 dpi, total cells were isolated from the ipsilateral foot and ankle and evaluated by flow cytometry. (A) Frequency of VENUS+ cells among live, CD45- cells. (B) Frequency of CD29+ cells among live, CD45-, VENUS+ cells at 2 dpi. (C-E) Cell surface expression of H2-Kb, CD44, and CD29 among live, CD45-, VENUS+ cells normalized to VENUS- cells. (F) Representative flow cytometry plots of total H2-Kb and H2-Kb-SIINFEKL staining. (G) Quantification of the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells among live, CD45-, VENUS+ cells. (H) pMHC-I presentation efficiency among live, CD45-, VENUS+ cells. Data are representative of 3 experiments (n = 12) (A-B) or pooled from 3–4 independent experiments (C-H) (n = 8–13). P values were determined by paired student’s t-test (B) or by one-way ANOVA with Tukey’s multiple comparison test (A, C-E, G-H). *, P<0.05; ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.g004

Cell surface, but not intracellular, MHC-I expression is reduced in CHIKV-infected cells

CHIKV nsP2 impairs cellular transcription in vitro [57,58] which could explain the poor pMHC-I presentation efficiency by CHIKV-infected cells and the increase in pMHC-I presentation by cells infected with CHIKV encoding the QMS mutations in nsP2. To investigate whether the suppression of cell surface MHC-I expression in CHIKV-infected cells is due to a reduction in total cell-associated MHC-I, we developed a flow cytometry-based intracellular staining protocol for H2-Kb to allow for concurrent assessment of cell surface and cell-associated MHC-I on single EVA cells (Fig 5A). The intracellular quantity of H2-Kb and percentage of cells expressing intracellular H2-Kb was similar for CHIKV-VENKL- and CHIKVQMS-VENKL-infected cells. In contrast, fewer CHIKV-VENKL-infected cells displayed H2-Kb on their surface, and those that did had reduced expression levels (Fig 5B and 5C). These data suggest that CHIKV infection does not downregulate expression of H2-Kb by infected cells, but instead disrupts a step of the MHC-I antigen presentation pathway required for the display of mature pMHC-I on the cell surface.

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Fig 5. Cell surface, but not intracellular, MHC-I expression is reduced in CHIKV-infected cells.

(A) WT murine embryonic fibroblasts (MEFs) stably overexpressing H2-Kb (H2-Kb+) and H2-Kb-/- MEFs were analyzed for intracellular and cell surface expression of H2-Kb by flow cytometry (ICS = intracellular stain, Surf. = surface stain). (B-C) EVA cells were mock-inoculated or inoculated with 106 PFU of CHIKV-VENKL or CHIKVQMS-VENKL. At 24 hpi, cells were analyzed for intracellular (ICS) and cell surface (Surf.) expression of H2-Kb by flow cytometry. (B) Representative flow cytometry histograms and (C) quantification of the magnitude (gMFI) and frequency (%) of intracellular (ICS) and cell surface (Surf.) H2-Kb expression on CHIKV-infected cells (VENUS+) and VENUS- cells. Data are representative of 2 independent experiments (n = 6). P values were determined by unpaired student’s t-test. ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.g005

Bypassing peptide processing and ER transport restores MHC-I antigen presentation by CHIKV-infected cells

Given our findings that CHIKV-infected cells inefficiently present pMHC-I, and that intracellular expression of H2-Kb is normal during CHIKV-VENKL infection, we sought to determine the step(s) at which MHC-I maturation is disrupted in CHIKV-infected cells. Mature MHC-I complexes are composed of a membrane anchored heavy chain (e.g., H2-Kb), in association with beta-2-microgobulin (β2m), bound to an 8–10 amino acid peptide. β2m and peptide of proper length are required for the heavy chain of MHC-I to traffic to the cell surface [59], and the absence of an appropriate peptide destabilizes MHC-I during transit and at the cell surface [60,61]. Prior studies found that the covalent linkage of an antigenic peptide to β2m via a flexible linker allows for assembly of mature, peptide-loaded MHC-I complexes independent of peptide processing, transporter associated with antigen processing (TAP)-mediated peptide translocation into the ER lumen, and the peptide loading complex (PLC) [62,63,64]. To evaluate if CHIKV-infected cells are deficient for these early steps in MHC-I antigen presentation, the VENKL coding sequence in CHIKV-VENKL was replaced with SIINFEKL-GSSGSSGS-β2m (CHIKV-SIINFEKLβ2m) (Fig 6A). EVA cells were mock-inoculated or inoculated with CHIKV-VENKL, CHIKVQMS-VENKL, or CHIKV-SIINFEKLβ2m. At 24 hpi, CHIKV-infected cells were stained with an anti-E2 monoclonal antibody (CHK-11), which identified CHIKV-infected cells as efficiently as detection of virus-encoded VENUS (S5 Fig), and virus-infected cells were evaluated for cell surface H2-Kb and H2-Kb-SIINFEKL expression. In comparison with CHIKV-VENKL-infected EVA cells, an increased percentage of CHIKV-SIINFEKLβ2m-infected cells displayed H2-Kb-SIINFEKL on the cell surface, resulting in improved pMHC-I presentation efficiency that was elevated compared with CHIKVQMS-VENKL-infected cells (Fig 6B and 6C). Likewise, the inoculation of mice with CHIKV-SIINFEKLβ2m elicited an increased percentage of antigen presenting CHIKV-infected cells and increased pMHC-I presentation efficiency compared to CHIKV-VENKL at 2 dpi, even above that seen by cells infected with CHIKVQMS-VENKL (Fig 6D and 6E). These findings suggest CHIKV infection disables MHC-I antigen presentation at a step prior to β2m-peptide stabilization of the heavy chain within the ER.

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Fig 6. Bypassing peptide processing and ER transport restores MHC-I antigen presentation by CHIKV-infected cells.

(A) Schematic of the recombinant CHIKV-SIINFEKLβ2m viral genome. The coding sequence for the β2mSIINFEKL chimeric protein was inserted into the CHIKV genome in-frame in the viral structural ORF similar to the VENKL coding sequence. (B-C) EVA cells were mock-inoculated or inoculated with CHIKV-VENKL, CHIKVQMS-VENKL, or CHIKV-SIINFEKLβ2m. At 24 hpi, live, CD45-, E2 (CHIKV)+ cells were assessed for cell surface expression of H2-Kb and SIINFEKL-loaded H2-Kb (H2-Kb-SIINFEKL) by flow cytometry. (B) Representative flow cytometry plots depicting the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells among live, CD45-, CHIKV+ cells. (C) Quantification of the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells (left) and pMHC-I presentation efficiency (right). (D-E) At 24 hpi, ankle cells from C57BL/6 mice mock-inoculated or inoculated with 103 PFU CHIKV-VENKL, CHIKVQMS-VENKL, or CHIKV-SIINFEKLβ2m were assessed by flow cytometry. (D) Representative flow cytometry plots depicting the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells among live, CD45-, CHIKV+ cells. (E) Quantification of the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells (left) and pMHC-I presentation efficiency (right) among live, CD45-, CHIKV+ cells. Data are representative of 2 independent experiments (n = 8). P values were determined by one-way ANOVA with Tukey’s multiple comparison test. **, P<0.01; ***, P<0.001; ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.g006

To further demonstrate that presentation of viral antigen by MHC-I can occur more efficiently in CHIKV-infected cells when the peptide is linked to β2m, EVA cells were inoculated with mock inoculum or 106 PFU of CHIKV-VENKL or CHIKV-SIINFEKLβ2m. At 24 hpi, all groups of cells were washed and co-cultured for 6 h with a 1:1 mixture of 106 purified OT-I CD8+ T cells and 106 bystander CD8+ T cells. Like OT-I CD8+ T cells co-cultured with CHIKVQMS-VENKL-infected EVA cells (Fig 3), OT-I CD8+ T cells co-cultured with CHIKV-SIINFEKLβ2m-infected cells displayed increased expression of activation markers CD69 and CD25 compared with bystander levels (Fig 7A and 7B) and OT-I CD8+ T cells co-cultured with CHIKV-VENKL-infected EVA cells. Furthermore, these OT-I CD8+ T cells expressed elevated levels of IRF4, a transcription factor activated in response to TCR ligation strength [65], suggesting that specific engagement of CHIKV-infected cells with OT-I CD8+ T cells is restored when the virus-encoded peptide is directly linked with β2m. Notably, OT-I CD8+ T cells cocultured with CHIKV-SIINFEKLβ2m-, but not CHIKV-VENKL-infected EVA cells, showed a marked loss of CD8a expression (Fig 7B), which has been observed on activated CD8+ T cells [66], further suggesting that antigen-specific CD8+ T cells poorly engage cells infected with CHIKV-VENKL.

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Fig 7. CD8+ T cells are activated by CHIKV-infected cells when peptide processing and ER transport are bypassed.

(A-B) EVA cells were inoculated with 106 PFU CHIKV-VENKL or CHIKV-SIINFEKLβ2m. At 24 hpi, EVA cells were cocultured with a 1:1 mixture of 106 bystander and 106 OT-I SIINFEKL-specific CD8+ T cells for 6 h. Bystander CD8+ T cells were distinguished from OT-I CD8+ T cells by gating on CD8+, KbOVA257-264 tetramer positive or negative cells, and both populations were assessed for the expression of CD69, CD25, IRF4 and CD8 by flow cytometry. (A) Representative flow cytometry plots. (B) Quantification of CD25, CD69, IRF4 (intracellular), and CD8 cell surface expression (upper graphs) and frequency (lower graphs) for OT-I cells above bystander CD8+ T cells. Data are representative of two independent experiments (n = 6). P-values were determined by unpaired student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.g007

CHIKV nsP2 impairs antigen presentation efficiency

Our data shows that mutations in the MTL domain of nsP2 increased MHC-I viral antigen presentation by CHIKV-infected cells. Based on these data, we hypothesized that nsP2 expression alone would be sufficient to disrupt pMHC-I presentation efficiency. To test this idea, EVA cells were co-transfected with a CHIKV nsP2 expression vector along with an expression vector encoding VENKL. Control cells were co-transfected with a control vector plus VENKL. At 24 h post-transfection, ~20–30% of EVA cells were VENUS+ (S6A Fig). By Western blot analysis, we detected expression of both nsP2 and nsP2QMS, with nsP2QMS expression increased compared with nsP2 (Fig 8A). In cells transfected with nsP2, VENUS+CD45- EVA cells displayed a reduced percentage of cells double-positive for H2-Kb and H2-Kb-SIINFEKL, and decreased pMHC-I presentation efficiency (Fig 8B and 8C) compared with control cells. In contrast, EVA cells transfected with nsP2QMS and VENKL had a similar frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells and pMHC-I presentation efficiency as control cells (Fig 8B and 8C). These findings indicate that nsP2 expression alone can impair pMHC-I presentation efficiency similar to CHIKV-infected cells.

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Fig 8. Cells expressing nsP2 inefficiently present antigen and have decreased levels of cell surface MHC-I.

(A-F) EVA cells were co-transfected with the designated WT or mutant pCMV-nsP2 plasmid or an identical control plasmid lacking the methionine start codon for nsP2 (vector) and pCMV-VENKL. At 24 h post-transfection, cells were evaluated by flow cytometry and Western blot. (A, D) Western blot analysis of nsP2 and actin expression within transfected EVA cells (anti-nsP2, red, 90 kDa; anti-actin, green, 42 kDa). (B, E) Representative flow cytometry plots depicting the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells among live, CD45-, VENUS+ cells. (C, F) Quantification of the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells and pMHC-I presentation efficiency among live, CD45-, VENUS+ cells. Data are pooled from 3 independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. **, P<0.01; ***, P<0.001; ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.g008

Given that alphavirus nsP2 has additional functional domains (e.g., helicase and protease domains) in addition to the MTL domain, and that expression of nsP2 can inhibit host cell transcription and translation, we tested the capacity of other forms of nsP2 to impair pMHC-I presentation efficiency. As shown in Fig 8D–8F, we found that expression of nsP2 with a mutation in the protease active site (i.e., C478S) or the highly conserved lysine in the Walker A motif (i.e., K192A) results in lower levels of cell surface H-2Kb-SIINFEKL compared with control cells or cells expressing nsP2QMS and reduced pMHC-I presentation efficiency to the same extent as WT nsP2. Importantly, all forms of nsP2 were expressed to similar levels (Fig 8D) and cells from all groups showed similar levels of VENUS expression (S6B Fig). Notably, the K192A mutation has been previously reported to abrogate RBP1 degradation and eEF2 phosphorylation by nsP2 which are required for inhibition of cellular transcription [15] and translation [16], respectively. These findings suggest that nsP2 interferes with MHC-I antigen presentation by a mechanism that is independent of effects on host cell transcription or translation, and nsP2 protease activity.

Discussion

Replication of arthritogenic alphaviruses in musculoskeletal tissues of mice, nonhuman primates, and humans leads to the recruitment and infiltration of inflammatory cells [67,68,69,70,71,72]. Some of these cellular infiltrates, such as Ly6Chi monocytes, contribute to control of alphavirus infection [73, 74], whereas others, such as CD4+ T cells, mediate joint tissue pathology [75,76]. These tissue infiltrates also include CD8+ T cells [42], which are critical for the control and clearance of many viral infections. Remarkably, prior studies found that CD8+ T cells have little to no impact on CHIKV or RRV replication in joint-associated tissues [42,77]. However, CD8+ T cells in joint-associated tissues of CHIKV-infected mice were capable of lysing adoptively transferred splenocytes exogenously loaded with peptide [42], suggesting that CHIKV-infected cells in joint tissue escape CD8+ T cell surveillance. Thus, we investigated MHC-I antigen presentation by CHIKV-infected cells. Indeed, we found that joint-associated CHIKV-infected fibroblasts displayed reduced cell surface MHC-I expression and inefficiently presented a virus-encoded peptide in MHC-I both in vitro and in vivo. Moreover, CHIKV-infected fibroblasts were unable to activate viral antigen-specific CD8+ T cells, further indicating that MHC-I antigen presentation by CHIKV-infected cells is impaired.

Viruses have evolved numerous strategies to evade the MHC-I antigen presentation pathway, including disruption of MHC-I machinery by suppression of transcription [35, 36, 78, 79, 80], such as the suppression of NLRC5 function by the ORF6 protein of SARS-CoV-2 [41], highlighting the importance of CD8+ T cells in the elimination of virus-infected cells and control of virus infection. Infection of cells with CHIKV and other alphaviruses can inhibit host cell transcription [81]. Mechanistically, for CHIKV and several other alphaviruses, inhibition of transcription is mediated by translocation of the viral nsP2 protein into the nucleus where it promotes polyubiquitination and proteasomal degradation of RPB1 [15], the catalytic subunit of cellular DNA-dependent RNA polymerase II. Recently, CHIKV strains that lack the capacity to induce degradation of RPB1 but retain high levels of replication were developed–these strains encode specific mutations in the V-loop of the MTL domain of nsP2 [54]. We introduced these mutations into CHIKV-VENKL and found that cells infected with this virus retained cell surface expression of MHC-I, more efficiently presented MHC-I antigen, and better activated antigen-specific CD8+ T cells. Moreover, we found that transfection of primary murine ankle tissue fibroblasts with WT CHIKV nsP2, but not CHIKV nsP2 with mutations in the V-loop, was sufficient to diminish MHC-I surface expression and pMHC-I presentation efficiency. Thus, our studies identified a new role for nsP2 in immune evasion.

Our findings suggest that CHIKV nsP2 interferes with MHC-I antigen presentation by a mechanism that prevents display of mature peptide-loaded MHC-I on the cell surface (Fig 9). Prior studies by other groups found that MHC-I peptides that are covalently tethered to β2m are translocated into the ER by the β2m signal sequence, thus, bypassing the requirement for TAP-mediated transport [62]. In addition, this approach bypasses the need for peptide processing [64]. We engineered CHIKV to express SIINFEKL tethered to the N-terminus of β2m via a Gly/Ser linker [62] (CHIKV-SIINFEKLβ2m) and found that cells infected with this virus efficiently display SIINFEKL-loaded MHC-I on the cell surface. Furthermore, the capacity for viral expression of β2m-SIINFEKL to restore peptide-loaded MHC-I on the cell surface and promote activation of antigen-specific CD8+ T cells indicates that MHC-I trafficking is not altered by CHIKV-infected cells and further supports the idea that steps prior to MHC-I heavy chain stabilization and trafficking are disrupted. Mechanistically, these findings indicate that CHIKV infection and nsP2 expression disrupt MHC-I antigen presentation by altering peptide generation, peptide transport into the ER, or the peptide loading complex (PLC), and future studies are needed to resolve the precise mechanism of action. It is possible that nsP2 directly interacts with components of the antigen processing pathway to disrupt MHC-I antigen presentation. For example, ubiquinilin 4 (UBQLN4) directly interacts with nsP2 [82] and is known to target mis-localized membrane proteins to the proteasome [83]. Given the critical importance of the proteasome for the generation of CD8+ T cell epitopes [84], it is possible that the interaction between nsP2 and UBQLN4 leads to a reduction of pMHC-I antigenic epitopes for surveillance of CHIKV-infected cells by CD8+ T cells. Alternatively, in contrast to the MHC-I heavy chain, other components of the MHC-I antigen presentation pathway may be more sensitive to nsP2-mediated transcriptional or translational inhibition. For example, HCMV infection has been shown to disrupt transcription of tapasin more so than other components of the PLC [85]. However, as described below, our results suggest that nsP2 disrupts MHC-I antigen presentation by a mechanism distinct from the inhibition of host cell transcription or translation.

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Fig 9. Proposed model for CHIKV disruption of MHC-I antigen presentation.

CHIKV infection disables MHC-I antigen presentation at a step prior to β2m-peptide stabilization of the heavy chain within the ER. Figure made with Biorender.com.

https://doi.org/10.1371/journal.ppat.1011794.g009

Given that CHIKV infection and nsP2 expression can inhibit host cell transcription, and mutations in the nsP2 MTL domain that abrogate transcriptional inhibition [54] enhanced MHC-I antigen presentation by CHIKV-infected and nsP2-expressing cells, we developed a flow cytometry-based assay for quantifying cell-associated and cell surface MHC-I levels on individual cells to determine if CHIKV infection specifically alters cell surface levels of MHC-I or induces downregulation of total MHC-I expression. These studies revealed that while the cell surface level of MHC-I was decreased on CHIKV-infected cells, intracellular levels of MHC-I were unaffected, indicating that CHIKV infection does not result in a general loss of MHC-I expression due to inhibition of host cell transcription. That intracellular levels of MHC-I are unaffected by CHIKV infection is further supported by our studies utilizing CHIKV-SIINFEKLβ2m which demonstrated that nascent MHC-I heavy chains could be stabilized and shuttled to the cell surface by expression of the chimeric SIINFEKLβ2m protein. To further investigate a role for transcription or translation inhibition in nsP2-mediated impairment of MHC-I antigen presentation, we evaluated nsP2 with a mutation in the helicase domain (nsP2K192A) that abrogates nsP2-mediated RBP1 degradation and eEF2 phosphorylation required for inhibition of cellular transcription [15] and translation [16], respectively, for the capacity to impair pMHC-I antigen presentation efficiency. Remarkably, in contrast with expression of nsP2QMS, expression of either nsP2 or nsP2K192A similarly impaired pMHC-I antigen presentation, suggesting a role for the MTL domain in antagonism of MHC-I antigen presentation that is independent of effects on cellular transcription and/or translation. In addition to these findings, we also found that expression of nsP2 harboring a mutation in the protease site (nsP2C478A) disrupted pMHC-I antigen presentation efficiency, suggesting that protease activity is not required for nsP2-mediated disruption of MHC-I antigen presentation. In summary, we find that through an nsP2-mediated mechanism, CHIKV-infected joint tissue fibroblasts exhibit inefficient MHC-I presentation of virus-encoded peptides. These data provide one explanation for the limited role of CD8+ T cells in controlling CHIKV infection.

Materials and methods

Ethics statement

This study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. All animal experiments conducted at the University of Colorado Anschutz Medical Campus were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado School of Medicine (Assurance Number: A3269-01) under protocols 00026 and 00215. Experimental animals were humanely euthanized at defined endpoints by exposure to isoflurane vapors followed by bilateral thoracotomy.

Mice and mouse experiments

An animal biosafety level 3 laboratory (ABSL3) with biosafety cabinet was used for all mouse experiments. C57BL/6 mice (stock #000664) were obtained from The Jackson Laboratory. CD45.1+ OT-I (C57BL/6-Tg(TcraTcrb)1100Mjb/J) mice were bred and housed at the University of Colorado School of Medicine under specific pathogen-free conditions. CD45.1+ OT-1 mice were maintained as hemizygous animals and selected based on flow cytometry phenotyping of transgene positive CD8+ cells expressing both Vα2 and Vβ5.1/2 TCRs. All mice were used in experiments at 4 weeks of age. Mice were anesthetized with isoflurane vapors and subcutaneously inoculated with 103 PFU of CHIKV in the left rear footpad in 10 μL of PBS/1% FBS using a Hamilton syringe and 30G needle. Mice were euthanized by exposure to isoflurane vapors and bilateral thoracotomy, and then intracardially perfused with phosphate buffered saline (PBS) at the indicated time points.

Cells

BHK-21 cells (American Type Culture Collection, ATCC CCL-10) were cultured at 37°C in Minimum Essential Medium Alpha (MEMα) supplemented with 10% fetal bovine serum (FBS), 10% tryptose phosphate broth (TPB) and 100 U/mL of penicillin-streptomycin (P/S). Mouse embryonic fibroblasts (derived from WT and Ifnar1-/- C57BL/6 mice) were a gift from Michael Diamond (Washington University at St. Louis) and cultured in Dulbecco’s Modified Eagle medium (DMEM; HyClone 11965–084) supplemented with 10% FBS and 100 U/ml P/S (D:10). All cell lines were passaged when the monolayer was <90% confluent and fewer than 10 times. To generate primary ex vivo ankle (EVA) cells, 4-week-old C57BL/6 mice were euthanized, and ankle tissues dissected. A single-cell suspension was generated by horizontal shaking of ankle tissue dissections at 37°C for 2 h with 5 mm glass beads in digestion media containing D:10, 2.5 mg/mL collagenase type I (Worthington Biochemical), and 17 μg/mL DNase I (Roche). The cell suspension was filtered through a 100 μm sterile filter, washed with 1x PBS and centrifuged at 350 x g for 5 min at room temperature. The supernatant was decanted, and the cell pellet was resuspended in D:10. Approximately 1.5 x 106 ankle tissue cells were added to a single well of a 6-well plate in 4 mL of D:10 medium and cultured at 37°C for 24 h. EVA cells were transfected using Lipofectamine 3000 (L3000) according to the manufacturer’s instructions. Briefly, EVA cells were plated 24 h prior to direct addition of 50 μL containing: 0.2 μg of either pCMV, pCMV-VENKL, pCMV-nsP2, pCMV-nsP2K192A, pCMV-nsP2C478A or pCMV-nsP2QMS combined with 1.5 μL of L3000 and 2 μL of P3000 reagent in Opti-MEM. Twenty-four h later cells were harvested by EDTA dissociation, stained with designated antibodies, and assessed by flow cytometry. For virus infection studies, medium was aspirated, and cells were inoculated with 1 x 106 PFU of the indicated virus in 200 μL of in vitro diluent (PBS with 1% FBS, 1 mM Ca2+, and 1 mM Mg2+). After one hour with gentle rocking, the inoculum was removed, cells were washed with 1x PBS and then incubated for 16 h at 37°C in 4 mL D:10.

Viruses

CHIKV cDNA clones of AF15561 were generated as described previously [68,86]. The recombinant CHIKV-VENKL cDNA clone was constructed by insertion of a tandem sequence encoding the fluorescent GFP derivative, VENUS, linked to LEQLE-SIINFEKL-TEW followed by the 2A protease sequence of foot-and-mouth disease virus (FMDV2a) and inserted in-frame between capsid and E3 of CHIKV [46]. CHIKV-SIINFEKLβ2m was generated similarly by inserting SIINFEKL with a GSSGSSGS linker between the β2m signal sequence and transcriptional start site of human β2m. CHIKVQMS-VENKL was generated by site-directed mutagenesis of the sequence encoding amino acids 674ATL677 of the nsP2 using primers 5’ CACTAGGTCATACCTACCACTCATCTGTGGTAGACCCAgCTCTAGG-3’ and 5’-CCTAGAGCTGGGTCTACCACAGATGAGTGGTAGGTATGACCTAGTG-3’. The PCR amplicon was inserted into the WT vector. The complete viral genome sequence in all plasmids was confirmed by Sanger sequencing. Stocks of infectious CHIKV were generated from cDNA clones and titered by plaque assay on BHK-21 cells [68] or focus formation assay using Vero cells as previously described [87].

In vitro virus replication assay

Triplicate wells of MEFs were inoculated with virus at a MOI of 0.01 FFU/cell. Viruses were absorbed onto cells for 1 h at 37°C with gentle rocking. Cells were washed two times with 1 mL of 1x PBS, then 1 mL of D:10 was added to each well, and cells were incubated at 37°C. At the indicated time points, 100 μL samples of culture supernatants were collected, and an equal volume of D:10 was added to maintain a constant volume within each well. Samples were stored at −80°C before analysis by focus formation assay on Vero cells.

Focus formation assays

As previously described [87], Vero cells were seeded in 96-well plates and inoculated with 10-fold serial dilutions of samples for 2 h at 37°C. Cells were overlayed with 0.5% methylcellulose in MEM-alpha plus 10% FBS and incubated for 18 h at 37°C. Following fixation with 1% paraformaldehyde (PFA), CHIKV-infected cells were detected using CHK-11 monoclonal antibody [88] at 500 ng/mL, and foci were visualized with TrueBlue Peroxidase substrate (SeraCare 5510–0030) and counted using a CTL Biospot analyzer and Biospot software (Cellular Technology).

Flow cytometry

Single cell suspensions were generated either by trypsinizing cell monolayers from in vitro cultures or releasing cells from ankle tissue by mechanical and enzymatic digestion as previously described [42]. Single cell suspensions were blocked with anti-FcγRIII/II (2.4G2; BD Pharmingen) for 10 min at RT, stained with LIVE/DEAD Fixable Violet Dead Cell Stain (ThermoFisher) according to product instructions, then stained with indicated antibodies for 45 min on ice. Cells were washed 2x with FACS buffer (1% FBS, 2 uM EDTA, 20 mM HEPES in 1x PBS) and fixed by addition of 1% paraformaldehyde for 10 min at RT. For intracellular stains, fixed cells were incubated with addition of indicated antibodies in 0.1% saponin plus FACS buffer for 0.5–2 h at RT, washed 3x with 0.1% saponin plus FACS buffer and resuspended in FACS buffer. Samples were acquired on a BD LSR Fortessa cytometer (> 50,000 events) using FACSDiva software, or Cytek Aurora using Aurora software. Downstream analysis was performed using FlowJo software (Tree Star). Antibodies used: CD45 (30-F11), H2-Kb (AF6-88.5), H2-Kb-SIINFEKL (25D1.16), CD29 (TS2/16), CD44 (IM7) H2-Db (KH95), TCR (Vb8.2/8.3), TCR (Va2), CD8 (53–6.7), IL2Ra (7D4/CD25), IRF4 (3E4) and CD69 (H1.2F3).

Western blotting

EVA cells were trypsinized, washed twice with PBS then resuspended in cold NP-40 lysis buffer (1% NP-40, 50 mM Tris-Cl, 150 mM NaCl) with protease inhibitors (Cell Signaling, #5871) on ice for 30 min. Debris was removed by centrifugation at 17,000 x g and assessed for total protein content by Bradford assay (ThermoFisher, A55866). 10 ug of lysate was denatured by addition of 5% 2-mercaptoethanol and 0.1% SDS-containing sample buffer and heating at 95°C for 5 min. Samples were analyzed via 8% SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes by wet transfer and blocked in 10% milk in TBS containing 0.1% Tween (TBST). Anti-rabbit nsP2 (HLA1488, GeneTex) was used 1:500 overnight at 4°C in 5% milk TBST. Anti-mouse actin (mAbGEa, ThermoFisher) was used 1:5,000 1 h RT in 5% milk TBST. After washing in TBST, secondary antibodies, goat anti-mouse IR700 (Azurebio AC2135) and anti-rabbit IR800 (Azurebio AC2128), were used 1:20,000 1 h at RT in 5% milk TBST. After washing in TBST, blots were imaged using Azure spec 500 NIR fluorescent imager.

CD8+ T cell activation assays

Splenocytes from CD45.1+ OT-I mice were acquired as previously described [42]. CD8+ T cells were enriched by magnetic bead separation with negative selection following the StemCell protocol (StemCell, 19853) and only utilized with >90% purity. After enrichment, CD45.1+ OT-I CD8+ T cells were added to primary murine ankle cell cultures that had been previously infected for 24 h. After 6 h of co-culture, non-adherent cells were collected, stained with designated antibodies, and analyzed by flow cytometry.

Quantification and statistical analysis

Antigen presentation efficiency was calculated utilizing the Flowjo “derive parameter” function taking the mean fluorescence intensity of H2-Kb-SIINFEKL channel and dividing it by VENUS or E2 mean fluorescence intensity [47]. For statistical analysis, one-way analysis of variance (ANOVA) with multiple comparisons, and student’s t-test were performed on data using GraphPad Prism 10.02. P values of <0.05 were considered significant.

Supporting information

S1 Fig. Gating strategy for CHIKV-VENKL vs MOCK infection of EVA cells and correlation between MHC-I expression and productive infection.

(A-C) EVA cells were mock-inoculated or inoculated with 106 PFU CHIKV-VENKL and assessed for VENUS and MHC-I cell surface expression at 24 hpi by flow cytometry. (A) Flow cytometry plots demonstrating gating strategy for VENUS+ or VENUS- populations and CD29 expression. (B-C) Quantification and representative histograms of MHC-I cell surface expression stratified by VENUS expression [nil expression (grey), low (pink), medium (red), and high (dark red)]. Data are representative from 2 independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparison test. **, P<0.01; ***, P<0.001; ****, P<0.0001.

https://doi.org/10.1371/journal.ppat.1011794.s001

(EPS)

S2 Fig. Gating strategy for CHIKV-VENKL- vs CHIKVQMS-VENKL-infected EVA cells.

Representative flow cytometry plots demonstrating gating strategy for CD45-, CD29+, VENUS+ fibroblasts from EVA cell cultures. Cell surface expression of CD29 was assessed within singlets, CD45-, VENUS+ or VENUS- gates.

https://doi.org/10.1371/journal.ppat.1011794.s002

(EPS)

S3 Fig. OT-I CD8+ T cell enrichment efficiency, gating scheme, and baseline activation.

CD8+ T cells from naïve OT-I x CD45.1 mice were negatively selected for CD8+ cells by magnetic separation enrichment. (A) Frequency of CD8+ T cells before and after enrichment. (B) Gating scheme to differentiate bystander (KbOVA257-264 tetramer-) from OT-I (KbOVA257-264 tetramer+) CD8+ T cells. (C) Baseline activation of bystander (light grey; upper plot) and OT-I (black; lower plot) CD8+ T cells without coculture with EVA cells.

https://doi.org/10.1371/journal.ppat.1011794.s003

(EPS)

S4 Fig. Gating strategy for CHIKV-VENKL vs CHIKVQMS-VENKL infected ex vivo joint-associated fibroblasts.

Representative flow cytometry plots illustrating the gating strategy for CD45-, CD29+, VENUS+ fibroblasts from ex vivo joint-associated tissues. Cell surface expression of CD29, CD44 were assessed within live, singlet, CD45-, VENUS+ or VENUS- gates.

https://doi.org/10.1371/journal.ppat.1011794.s004

(EPS)

S5 Fig. E2 and VENUS are equally detectable in CHIKV-infected EVA cells at 24 hpi.

EVA cells were inoculated with 106 PFU of CHIKV-VENKL for 24hpi, harvested, fixed, permeabilized, and stained with anti-E2 CHIKV (CHK-11) at decreasing (2-fold) serial dilutions. Cells were analyzed for E2 and VENUS expression by flow cytometry. (A) Representative flow cytometry plots. (B) Quantification of cell frequency for E2 and VENUS positive cells (red = infected, black = mock).

https://doi.org/10.1371/journal.ppat.1011794.s005

(EPS)

S6 Fig. Frequency of VENUS+ cells after transfection of EVA cell cultures.

EVA cells were co-transfected with the designated WT or mutant pCMV-nsP2 plasmids or an identical control plasmid lacking the methionine start codon for nsP2 (vector) and pCMV-VENKL. At 24 h post-transfection, cells were evaluated by flow cytometry for expression of VENUS. (A) Representative flow cytometry plots for vector (black, circle), nsP2 (red, circle) and nsP2QMS (blue, circle) and frequency of VENUS+ cells among live, CD45- cells. (B) Representative flow cytometry plots for vector (black, circle), nsP2 (red, circle), nsP2K192A (red, triangle), nsP2C478S (red, square) and nsP2QMS (blue, circle) and frequency of VENUS+ cells among live, CD45- cells.

https://doi.org/10.1371/journal.ppat.1011794.s006

(EPS)

Acknowledgments

We thank the University of Colorado Anschutz Medical Campus Cancer Center Flow Cytometry Shared Resource Core Facility (RRID:SCR_022035). We thank Michael S. Diamond (Washington University) for critical evaluation of the manuscript. Finally, deep thanks to Olga Prikhodko for technical support and critical assistance.

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