In a recent study posted to the bioRxiv* preprint server, researchers observed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) downregulates the expression of major histocompatibility complex (MHC) class I molecules.
MHC-I is a heterodimer of beta-2-microglobulin (β2m) and a glycosylated heavy chain. The MHC-I molecules are loaded with peptides in the endoplasmic reticulum (ER), including viral peptides. MHC-I-peptide complexes are trafficked to the cell surface where self, non-self, or viral peptides are displayed and presented to the immune cells. The cluster of differentiation 8 (CD8+) T cells identify and kill cells with non-self-peptides.
Viruses downregulate MHC-I expression to prevent elimination by the adaptive arm of the immune system. This could be achieved by downregulating transcription of MHC-I genes or inhibition of MHC-I function. It has been reported that SARS-CoV-2 infection downregulates a specific MHC-I allele, HLA-A*02:01 (HLA-A2), with open reading frame-6 (ORF6) and ORF8 implicated in the process.
The study and findings
In the current study, researchers investigated the roles of ORF3a and ORF7a proteins of SARS-CoV-2 because these have been predicted to localize to the secretory pathway. First, Vero E6 or HEK293T-hACE2 cells expressing angiotensin-converting enzyme 2 (ACE2) were infected with SARS-CoV-2 WA1 at 10 multiplicity of infection (MOI) for 24 hours. When MHC-I expression was estimated using flow cytometry, a 20% – 30% decline in surface expression was noted in the infected cells relative to controls (non-infected cells).
ORF3a, ORF7a, and ORF8 were separately expressed in HEK293T or HeLaM cells, and after 24 hours of transfection, the authors noted a 25% – 30% reduction in surface expression of MHC-I. Further, the epidermal growth factor receptor (EGFR) and CD47 levels were unaffected by ORF8 or ORF7a, whereas ORF3a downregulated CD47 and EGFR in both cell lines. This implied that ORF7a or ORF8 expression affects MHC-I levels, while ORF3a expression impairs protein trafficking via the secretory pathway.
Moreover, the ORF3a protein from SARS-CoV-1 and SARS-CoV-2 were individually expressed in HeLaM cells, and Golgi morphology was examined by confocal microscopy. The team detected Golgi fragmentation in both cases, while non-infected cells had intact Golgi system. A transient ORF3a expression for 24 hours reduced MHC-I levels by approximately 30%. The researchers posit that ORF3a downregulates MHC-I on the surface by disrupting the Golgi apparatus and blocking protein trafficking to the cell surface.
In subsequent experiments involving inducible expression of ORF7a on doxycycline (Dox) treatment in HeLaM cells (henceforth HeLaM-iORF7a cells), the team observed that MHC-I expression on the surface was 20% lower, albeit the messenger ribonucleic acid levels (mRNA) levels remained unchanged relative to controls. This confirmed that the downregulation of MHC-I was not transcriptional.
Overall, the MHC-I levels in ORF7a-expressing HeLaM cells were similar to controls in lysates. Therefore, the team investigated whether ORF7a affected intracellular MHC-I distribution and found that the MHC-I export rate from the ER was slowed by ORF7a expression. Next, the team observed co-immunoprecipitation of ORF7a with MHC-I heavy chain but not β2m, or MHC-I-peptide complex, indicating that ORF7a interferes with β2m association with the MHC-I heavy chain.
Further, a β2m-encoding plasmid or a control plasmid (no β2m) was transiently expressed in HeLaM-iORF7a cells. Expectedly, cells with a control plasmid showed reduced MHC-I surface expression upon Dox treatment relative to untreated cells (no Dox). However, this was eliminated when β2m was expressed, implying that excess β2m levels could overcome ORF7a action. Confocal examination revealed localization of ORF7a to both the Golgi system and ER.
The researchers generated a mutant variant of ORF7a (ORF7a-ARA) wherein lysine residues from a dibasic sequence (an ER-retrieval sequence) at the C-terminus were substituted with alanine residues. They observed that this mutant was primarily localized to the Golgi system. In the presence of ORF7a, MHC-I was retained to ER, while this was not the case with ORF7a-ARA. The team generated Dox-inducible versions of ORF7a or ORF7a-ARA in HEK293T-hACE2 cells to investigate the effects on surface levels of MHC-I and antigen presentation by HLA-A2 (since HeLaM cells lack this allele).
Surface MHC-I levels were reduced by 40% when ORF7a expression was induced, but not with ORF7a-ARA induction. Last, the authors used a specific MHC-I-peptide complex, with latent membrane protein 2A (LMP2A) from Epstein-Barr virus as the peptide. LMP2A is presented by and binds to HLA-A2 with high affinity. To this end, HEK293T-hACE2 cells and their ORF7a or ORF7a-ARA inducible versions were transduced with human influenza hemagglutinin (HA)-tagged fusion construct of ubiquitin: LMP2A-derived peptide (pLMP2A).
The presentation of the LMP2A-HLA-A2 complex was assessed using a specific monoclonal antibody. pLMP2A and ORF7a were expressed upon treatment with Dox, but only in HA-positive cells with ORF7a but not ORF7a-ARA, the presentation of pLMP2A was reduced due to decreased HLA-A2 levels mediated by ORF7a.
The study demonstrated that two SARS-CoV-2 accessory proteins (ORF3a and ORF7a) downregulate MHC-I with distinct mechanisms. MHC-I downregulation by ORF3a was more general with the inhibition of protein trafficking, whereas, by ORF7a, it was specific in that it interacts with the MHC-I heavy chain, slowing the export of MHC-I-peptide complexes.
Further, the downregulation of MHC-I by ORF7a was attenuated when the ER retrieval sequence was mutated by replacing lysine with alanine. In conclusion, the results demonstrated that ORF3a and ORF7a reduce surface MHC-I levels, with the downregulation mediated by ORF7a being more specific and novel, allowing immune evasion by SARS-CoV-2.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.