An epidemic Zika virus isolate suppresses antiviral immunity by disrupting antigen presentation pathways

Zika virus (ZIKV) has emerged as an important global health threat, with the recently acquired capacity to cause severe neurological symptoms and to persist within host tissues. We previously demonstrated that an early Asian lineage ZIKV isolate induces a highly activated CD8 T cell response specific for an immunodominant epitope in the ZIKV envelope protein in wild-type mice. Here we show that a contemporary ZIKV isolate from the Brazilian outbreak severely limits CD8 T cell immunity in mice and blocks generation of the immunodominant CD8 T cell response. This is associated with a more sustained infection that is cleared between 7- and 14-days post-infection. Mechanistically, we demonstrate that infection with the Brazilian ZIKV isolate reduces the cross-presentation capacity of dendritic cells and fails to fully activate the immunoproteasome. Thus, our study provides an isolate-specific mechanism of host immune evasion by one Brazilian ZIKV isolate, which differs from the early Asian lineage isolate and provides potential insight into viral persistence associated with recent ZIKV outbreaks.


Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, seeAuthors & Referees and theEditorial Policy Checklist .

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
Martin J. Richer
Flow cytometry data were analyzed using Flowjo software (version 9.9.5, BD Biosciences). RT-qPCR data were first analzyed using CFX Maestro software (version 4.1.2434.0124, BioRad) and fold changes or PFU equivalents were calculated using Excel (version 16.43,Microsoft). Type I IFN quantification data were analyzed using Excel. All graphs were made and statistics calculated using Prism 9 (version 9.1.0, GraphPad).
The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files.

nature research | reporting summary
October 2018

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Validation Experiments were conducted with at least 3-5 mice per group, chosen to ensure that data were reproducible and held biological significance. Further, these sample sizes are standard for the field and have been previously used to demonstrate differences following ZIKV infection (PMID 28081442;PMID 28835502;PMID 30677097;PMID 28231312).
No data were excluded from the analyses.
The findings herein were reliably reproduced and number of times experiments were repeated are indicated in figure legends (minimum twice for in vivo experiments, range 2-4, minimum 3 times for in vitro experiments).
Mice were randomly assigned to indicated groups. For in vitro experiments, different groups were derived from the same cell suspensions/ stock materials so randomization was not required but controlled for.
No blinding was performed. All data analysis was undertaken impartially and in a strict unbiased manner and is reported as it was collected. Flow cytometry, RT-qPCR, and B16-Blue assay were collected using automated software that did not require intervention by the experimenters.
The following antibodies were used in an appropriate combination of fluorochromes: All antibodies are commercially available and were validated by the manufacturers from which they were purchased. For BioLegend antibodies, validation includes (from https://www.biolegend.com/en-us/quality-control): 1) Staining of 1-3 target cell types with either single-or multi-color analysis detailed in the QC specification (including positive and negative controls). The tested cells can be primary cells and/or cell lines known to be positive or negative for the target antigen. 2) Each batch product is validated by QC testing with a series of dilutions to make sure the product is working within expected antibody titer range. 3) Each batch is compared to an internally established "gold standard" to maintain batch-to-batch consistency. 4) When applicable, our products are side-by-side tested with our competitors' products to make sure that BioLegend's products exceed or are at least the same quality. 5) For most tandem dye-conjugated products, color compensation is examined in order to verify tandem integrity.
BD Biosciences antibodies are QC tested to ensure species and antigen reactivity, and are routinely tested for the intended purpose (flow cytometry -see https://www.bdbiosciences.com/us/applications/research/t-cell-immunology/th-1-cells/surfacemarkers/mouse/buv395-rat-anti-mouse-cd4-gk15/p/563790). Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Instrument Vero cells: Dr. Steven Varga, University of Iowa (originally purchased from ATCC); B16-Blue cells: Dr. Maziar Divangahi, McGill University (originally purchased from Invivogen) All cell lines used were authenticated by the manufacturers and checked morphologically under the microscope.
All cell lines used tested negative for mycoplasma contamination.
No commonly misidentified cell lines were used.
6-12 week old wild-type, IL-10 knock-out, and OT-I mice on the C57BL/6 background of both sexes were used for this study. Mice were maintained on a 12-hour light/dark cycle (light 7am-7pm; dark 7pm-7am), with an ambient temperature of between 20°C and 23°C and between 40% and 60% humidity.
The study did not involve wild animals.
The study did not involve samples collective from the field.
All animal procedures were carried out in accordance with the Canadian Council on Animal Care and were approved by the McGill University Animal Care Committee.
For flow cytometry analysis of cells in the blood, blood was collected, and erythrocytes lysed using Vitalyse (Cedarlane), cells were incubated with TruStain fcX (anti-mouse CD16/CD32, clone 93, BioLegend) and stained using indicated antibodies, followed by fixation with IC Fixation Buffer (eBioscience). For spleen analysis, spleens were isolated and mechanically disrupted to generate single-cell suspensions. Erythrocytes were lysed with Ammonium-Chloride-Potassium buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1mM Na2EDTA in distilled H2O, pH 7.2), cells were incubated with TruStain fcX (anti-mouse CD16/CD32, clone 93, BioLegend) and stained with the indicated antibodies, followed by fixation using IC Fixation Buffer (eBioscience). For tetramer staining, following erythrocyte lysis cells were incubated with TruStain fcX (anti-mouse CD16/CD32, clone 93, BioLegend) prior to staining for 45 minutes with H-2Db Env294-302 tetramer, with gentle vortexing every 20 minutes, followed by surface antibody staining and fixation with IC Fixation Buffer (eBioscience). For dendritic cell detection, spleens were cut into pieces and incubated with 10 ng/mL DNase and 1 mg/mL collagenase for 30 minutes at 37°C and 5% CO2 prior to generation of a single-cell suspension by mechanical disruption through a 70 micron basket filter (Fisher Scientific), erythrocyte lysis and antibody staining. Intracellular staining for granzyme B was performed using Perm/Wash Buffer (eBioscience) following fixation with IC Fixation Buffer (eBioscience). Intracellular staining for Ki67 was performed using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience), as per the manufacturer's instructions. Detection of BrdU accumulation was performed using the Phase-Flow FITC BrdU Kit (BioLegend), as per the manufacturer's instructions. Staining for TCR V" diversity was performed using the Anti-Mouse TCR V" Screening Panel (BD Biosciences), as per the manufacturer's instructions. Samples were analyzed with a BD LSRFortessa flow cytometer (BD Biosciences) and FlowJo software (BD Biosciences). For ex vivo restimulation, spleens were harvested and processed as above. Erythrocytes were lysed with ACK buffer and samples were simulated for 5.5 hours at 37°C with 5% CO2 in the presence of 200nM Env294-302 peptide or media alone and brefeldin A (BioLegend). Cells were then stained with surface antibodies and fixed with IC Fixation Buffer (eBioscience), followed by intracellular staining for IFN-# in Perm/Wash Buffer (eBioscience). Samples were analyzed with a BD LSRFortessa flow cytometer (BD Biosciences) and FlowJo software (BD Biosciences).