Study shows the SARS-CoV-2 accessory protein ORF7a inhibits HLA-I surface expression levels in human cells

By Pooja Toshniwal PahariaReviewed by Danielle Ellis, B.Sc.Jun 1 2022

In a recent study posted to the bioRxiv* preprint server, researchers identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), facilitating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune evasion by downregulating the surface expression of major histocompatibility complex (MHC) class I molecules.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

Cytotoxic T lymphocytes (CTLs) play a vital role in eliminating SARS-CoV-2-infected cells, mediated by the recognition of virus-derived peptides presented to MHC class I molecules. To counteract these antiviral effects, several viruses have developed mechanisms to interfere with antigen presentation by inhibiting the synthesis of MHC class I molecules and degrading them via viral proteasomes. The viruses decrease the cell surface antigen presentation of MHC class I, thereby interfering with viral recognition by CTLs.

About the study

In the present study, researchers assessed whether SARS-CoV-2 has developed mechanisms to counteract CTL activity by interfering with the human leukocyte antigen (HLA) class I antigen presentation pathway.

Vero E6 cells were infected with SARS-CoV-2 NL/2020 strain, and the surface MHC class I expression was assessed by performing flow cytometry analysis at one- and two days post-infection (dpi). The impact of SARS-CoV-2 infections was evaluated in human adenocarcinoma human alveolar basal epithelial cells (A549 cells) transduced with transmembrane serine protease 2 (TMPRSS2) and angiotensin-converting enzyme 2 (ACE2). The cells were subsequently challenged with the SARS-CoV-2 NL/2020 strain.

To identify the SARS-CoV-2 molecule downregulating surface HLA-I expression, the StrepII-tagged viral complementary deoxyribonucleic acid (cDNA) library was transduced in the 293T cells, after which the HLA-I surface expression was measured by flow cytometry analysis. In addition, ORF7a cDNA was subcloned in another lentiviral vector to validate the study findings.

Further, to assess if ORF7a downregulates cell surface HLA class I expression or affects the expression of secretory pathway proteins, the expression of several antigens on the surface of ORF7a-transduced 293T cells was monitored. Next, the team assessed if ORF7a regulated HLA-I among different cell lines, for which ORF7a was transduced in cell lines such as MelJuSo (cutaneous melanoma), HK-1 (nasopharyngeal carcinoma), and Huh7 (adult hepatocellular carcinoma).

To identify the ORF7a domains vital for HLA-I downregulation, three SARS-CoV-2 mutant strains were generated by replacement of the ORF7a peptide sequence with that of a human cluster of differentiation 8A (CD8A), mutating the C-terminal retrieval ER motifs (ORF7a tail-mut mutant), and completely deleting the cytoplasmic and transmembrane tail.

To determine the amino acid (aa) changes responsible for the gain-of-function phenotype of SARS-CoV-2 ORF7a, two chimeric mutants were generated by introducing three aa changes in the SARS-CoV-1 ORF7a sequence. To assess whether the phenylalanine at position 59 in the SARS-CoV-2 ORF7a was present in other sarbecoviruses, protein-sequence alignments were performed.

To assess if ORF7a induces surface HLA class I downregulation by degradation of proteins, the team assessed if the total HLA-I pool (surface and intracellular expression) is depleted on ORF7a expression in 293T cells. To assess if p97 or proteasome were involved in ORF7a-mediated HLA-I surface downregulation, the ORF7a-transduced 293T cells were treated with MG132 (proteasome inhibitor) or CB 5083 (p97 inhibitor) before assessment of the surface and intracellular HLA class I expression. Immunoprecipitation analysis was performed to assess if ORF7a was directly associated with HLA-I expression. 

Results

MHC class I surface expression was downregulated in SARS-CoV-2 spike (S)-expressing cells and transduced cells at two dpi. SARS-CoV-2 infection also reduced MHC class I surface expression in the cell lines tested. In the flow cytometry analysis, of all viral cDNAs, only ORF7a and its constructs significantly downregulated surface HLA-I expression. Both untagged and StrepII-tagged ORF7a induced potent cell surface HLA-I downregulation, an effect not seen by ORF8 due to low protein levels in ORF8. However, HLA-I downregulation was transient, and the phenotype was lost after ~14 days. ORF7a did not impact the expression of CD9, CD49b, CD46, and CD58 from 293T cells, indicative of the specific action of ORF7a.

The transmembrane domain and the ORF7a peptide sequence were essential, whereas the ER-retrieval motif was expendable for HLA-I downregulation. The transfer of the luminal region II from SARS-CoV-2 ORF7a to SARS-CoV-1 ORF7a and the T59F/H62Q/A68P chimeric ORF7a molecules led to substantial downregulation of cell surface HLA-I expression.

The T59F mutation caused the gain-of-function phenotype of SARS-CoV-2 ORF7a and cell surface HLA class I downregulation. The mutation was detected in all sarbecovirus isolates. Position 59 of ORF7a showed considerate variability among the viral isolates. Surprisingly, the intracellular HLA class I expression was potently upregulated in ORF7a-expressing 293T cells at seven and 21 dpi, whereas HLA class I surface expression was downregulated.

Immunofluorescent imaging of ORF7a-transduced MelJuSo cells indicated that HLA-I accumulated in perinuclear ER and probably the ER-Golgi intermediate compartment (ERGIC). Treatment with MG132 or p97 inhibitors abrogated the ORF7a-mediated HLA-I cell surface downregulation without affecting the intracellular HLA-I expression, indicating that ORF7a-mediated downregulation of cell surface HLA-I was p97- and proteasome-dependent. Strong and specific co-precipitation of ORF7a and HLA-I was observed in the immunoprecipitation analysis.

Conclusion

Overall, the study findings showed that SARS-CoV-2 can interfere with antigen presentation, evading immune surveillance. By downregulating the expression of HLA-I and antigen presentation, ORF7a facilitates the evasion of cytotoxic T lymphocyte responses by SARS-CoV-2.

*Important notice: 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.