MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

Abstract

Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems-based approach, we examined differential regulation of IFN-γ-dependent genes following infection with robust respiratory viruses including influenza viruses [A/influenza/Vietnam/1203/2004 (H5N1-VN1203) and A/influenza/California/04/2009 (H1N1-CA04)] and coronaviruses [severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV)]. Categorizing by function, we observed down-regulation of gene expression associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down-regulation of antigen-presentation gene expression, which was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation, rather than histone modification, plays a crucial role in MERS-CoV-mediated antagonism of antigen-presentation gene expression; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Together, the results indicate a common mechanism utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.

Keywords: DNA methylation; antigen presentation; coronavirus; epigenetics; influenza.

Fig. 1.

Type II IFN treatment and diverse respiratory virus infections result in robust expression differences. Global transcriptional response of consensus genes following IFN-γ treatment or infection with H5N1-VN1203, H1N1-09, SARS-CoV, and MERS-CoV is shown. Genes were ordered by functional groups. Values represent logtwofold change compared with time-matched mock-infected samples.

Fig. 2.

Both H5N1-VN1203 and MERS-CoV down-regulate genes within the MHC locus and associated with antigen presentation. Global transcriptional response of gene located within the MHC locus on chr. 6 (MHC class I, II, III, and adjacent genes) or distant genes (not located on chr. 6) associated with antigen presentation following infection with H5N1-VN1203, H1N1-09, SARS-CoV, and MERS-CoV is shown. Genes are ordered by MERS-CoV expression levels at 24 hpi. Values represent logtwofold change compared with time-matched mock-infected samples.

Fig. 3.

Proteomics analysis confirmed down-regulation of MHC class I-associated molecules following H5N1-VN1203 and MERS-CoV infection. (A) Differential abundance analysis comparing virus to time-matched mock samples with statistically significant increases represented in red and decreases in blue (P values based on logtwofold changes, flagged for statistical significance based on P ≤ 0.05). P values are derived from ANOVA (quantitative comparison) when enough data are present; otherwise, P values are from the g test (qualitative comparison). (B–D) Peptide abundance normalized to mock for HLA-A (B), HLA-B (C), and HLA-C (D) molecules following infection with H1N1-09 (red), H5N1-VN1203 (blue), SARS-CoV (green), and MERS-CoV (orange). Each point represents peptide associated with an HLA molecule class and line plotted for average of multiple peptides.

Fig. 4.

Altered histone modifications play a role in H5N1-VN1203 influenza, but not MERS-CoV antagonism of antigen presentation. (A and B) ChIP with antibodies against H3K4me3 (A) or H3K27me3 (B) followed by qPCR of the 5′ UTR of the identified genes 18 hpi with H1N1-09 (red), H5N1-VN1203 (blue), SARS-CoV (green), or MERS-CoV (orange). Values represent fold increase binding compared with mock on Log2 scale. P values are based on Student’s t test and compare individual viral infection to mock value: *P < 0.05; **P < 0.01; ***P < 0.001. (C) Chromosome plot of chr. 6 showing depletion of H3K4 methylation following H5N1-VN1203, but not MERS-CoV, infection. Loss of H3K4 methylation was called against the time-matched mock reads as reference reads with mock values equaling 0. H3K4 methylation depletion was then quantified and plotted as bars according the location on chr. 6; each bar indicates total number of histone modification marks within 1 Mb.

Fig. 5.

Examination of host DNA revealed complete methylation at MHC gene loci following MERS-CoV infection. (A) DNA methylation status during MERS-CoV, SARS-CoV, H5N1, and H1N1 infection. MeDIP was performed with total DNA from Calu3 cells infected with MERS-CoV, SARS-CoV, H5N1-VN1203, or H1N1-09 for 18 h to determine the methylation status of MHC gene promoters. Methylated CpG islands within the promoter regions for HLA-A, B2M, and PDIA3 were quantified by qRT-PCR. Results were reported as relative percentage of methylated (red) and unmethylated (gray) DNA in each target genomic DNA sequence for each virus. (B) Chromosome plot of chr. 6 showing DNA methylation after H5N1 and MERS-CoV infection. Differential methylation levels were called against the time-matched mock reads as reference reads with mock values equaling 0. Methylation levels were then quantified and plotted as bars according their location on chr. 6; each bar indicates the total number of differential methylation marks within 1 Mb.

Fig. 6.

Proteomics analysis of H5N1-VN1203 and MERS-CoV mutants suggest that both viral and host processes contribute to reduction of antigen-presentation molecules. (A and B) Peptide abundance normalized to mock for HLA-A (A) and HLA-C (B) following infection with influenza H5N1-VN12033 WT (blue), PB2-K627E mutant (gray), or NS1-truncation (red). (C and D) Peptide abundance normalized to mock for HLA-A (C) and HLA-C (D) following infection with MERS-CoV WT (orange) or accessory ORF mutant (ΔORF3-5; gray). Each point represents a peptide associated with an HLA molecule class and line plotted for average of multiple peptides.