SARS-CoV-2 NSP5 antagonizes MHC II expression by subverting histone deacetylase 2

ABSTRACT SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.

Fig. S1.Detection of MHC II, CIITA, and NSP5 Expression Levels.A-C) Human moDCs were transduced with lentiviral vectors bearing a zsGreen selectable marker and identified by excluding cell debris using the FSC-A/SSC-A scattergrams (A), selecting selected via FSC-A/FSC-H (B), and live moDCs identified by gating on SSC-A and the cell viability dye eFluor670-FVD (C).Up to 10,000 cells were recorded per condition in each experiment.D) CIITA expression in macrophages treated with ivermectin (Ivm) and/or INFγ.E) Regions cloned for the FISH-FRET and dual-luciferase vectors.For CIITA, two FISH promoters were cloned covering the PI promoter and its 5' region (PI FISH), as well as the region between the end of the PI promoter and 80 bp downstream of the PIII/PIV promoters (PIII/ IV FISH).Dual-luciferase vectors containing the PI (blue), PIII/IV (green), and IRF-binding site (orange) deleted PIII/IV promoters were generated.For MHC II, a FISH probe consisting of a 3500 bp region covering from 3200 bp 5' to the HLA-DRA promoter to 100 bp after the promoter was cloned, as was a dual-luciferase vector containing the promoter itself (blue).P-GCATC ACCGG TAGAC TACAA GGACC P-TGTGT CCGTA CCAGC TGCTT G Note: "P-" indicates that the 5′ end of the primer is phosphorylated.
F-G) RT-PCR quantification of NSP5 (F) and MHC II and CIITA expression (G) in A549 cells that were uninfected, infected with the USA-WA1/2020 strain of SARS-CoV-2, transduced with empty or NSP5-expressing lentiviral vectors, or treated with INFγ.Data is expressed as ΔΔCt relative to GAPDH.Data quantifies (D,F,G) or is representative of (A-C) 3 independent experiments.* = p < 0.05 compared to uninfected, n.s.= p > 0.05 between compared bars, Mann-Whitney U Test.

Fig. S2 .
Fig. S2.Quantification of NSP5 Proteolytic Activity.A) Structure (top) and FRET signal (bottom) of the positive FRET control, comprised of mVenus and mTurquoise2 separated by a 12-amino acid linker.B) Structure (top) and FRET signal (bottom) of the negative FRET control, comprised of mVenus and mTurquoise2 expressed as separate proteins.C) top: structure of the NSP5 proteolysis intramolecular FRET probe in which mVenus and mTurquoise2 are linked by the final 10 amino acids of SARS-CoV-2 NSP4 and the first 10 amino acids of SARS-CoV-2 NSP5.The known NSP5 cleavage site is indicated by the arrow.Bottom: localization and FRET efficiency of the NSP5 proteolysis probe when expressed in the absence of NSP5 (No NSP5), in the presence of wild-type NSP5 (NSP5), or when expressed with the NSP5 H41A , or NSP5 C145S point mutants.D) Quantification of NSP5 proteolysis reporter cleavage by immunoblotting for mVenus showing the expected mass of the uncleaved reporter (55 kDa) and the N-terminal fragment produced by cleavage (27 kDa).E) Measurement of NSP5 proteolysis reporter cleavage via quantification of whole-cell FRET.Cells are transfected with the positive or negative control vectors, or with the NSP5 cleavage reporter plus either empty vector (--), wild-type NSP5 (WT), or the NSP5 H41A or NSP5 C145S point mutants.n = 5, * = p < 0.05 compared to the positive control (+) and is presented as interquartile range (box) ± 95% (whiskers), Kruskal-Wallis test with Dunn correction.

Fig. S3 .
Fig. S3.HDAC2 Cleavage and NSP5 Half-Life.A-B) Structure (A) and FRET signal (B) of a HDAC2 intramolecular cleavage FRET probe in which mVenus is fused to the N-terminus, and mTurquoise2 to the C-terminus, of human HDAC2.The putative NSP5 proteolytic site is indicated in purple.C) Quantification of HDAC2 cleavage by NSP5 as quantified by whole-cell FRET.Cells are transfected with either the FRET negative control [(-) Control], FRET positive control [(+) Control], or with the HDAC2-FRET reporter plus one of empty NSP5 vector (-), wild-type NSP5 (WT) or with the H41A or C145S NSP5 mutants.D) Confirmation of HDAC2 knockdown in cells that are untreated (UT), transfected with a cell-permeant non-targeting siRNA (siScrm), or transfected with a cell-permeant HDAC2-targeting siRNA (siHDAC2).E) NSP5 lifespan immunoblots from cells transfected with wildtype NSP5 (NSP5), the NSP5 H41A or NSP5 C145S point mutants, or the NSP5 Δ1-192 and NSP5 Δ199-306 deletion mutants.Protein synthesis was inhibited at t= 0 min with cycloheximide and equal volumes of cell lysate loaded from each indicated timepoint.F) Determination of NSP5 half-life by densitometry, with non-linear regression used to calculate the half-life.* = p < 0.05 compared to the positive control (+), Kruskal-Wallis test with Dunn correction.Data is presented interquartile range (box) ± 95% (whiskers).

Table S1 .
PCR Primers Used in This Study