SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits immune suppression

Abstract

Over 950,000 whole-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been determined for viruses isolated from around the world. These sequences are critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We isolate one of these mutant viruses from a patient sample and use viral challenge experiments to link this isolate (ORF7a^Δ115) to a growth defect. ORF7a is implicated in immune modulation, and we show that the C-terminal truncation negates anti-immune activities of the protein, which results in elevated type I interferon response to the viral infection. Collectively, this work indicates that ORF7a mutations occur frequently, and that these changes affect viral mechanisms responsible for suppressing the immune response.

Keywords: IFN response; ORF7a; SARS-CoV-2.

Graphical abstract

Figure 1

SARS-CoV-2 genomic surveillance identifies global reoccurrence of ORF7a truncations (A) Symptom onset (purple) and PCR-based SARS-CoV-2 test results (coral) for patients in Bozeman, Montana, are shown with vertical bars. Seven-day moving averages, shown with lines, were used to indicate epidemiological trends. (B) Phylogenetic analysis of SARS-CoV-2 genomes sampled in Bozeman and globally. The tree was constructed from an alignment of 55 Bozeman samples and 4,871 genomes subsampled from GISAID. Subsampling was performed using Augur utility (https://nextstrain.org) by selecting 10 genomes per country per month since the start of the pandemic. Outer ring shows SARS-CoV-2 lineages assigned to genome sequences (Rambaut et al., 2020a). Major lineages include A (pink) that is associated with initial outbreak in China and B (blue) that emerged later in Europe. Minor lineages (i.e., C-N) are offshoots of lineage B. Red branches identify truncated ORF7a variants (n = 205) detected in the global data and merged into the alignment. The red dot highlights 7 of the 55 ORF7a variants that were isolated in Bozeman between April and July (2020). White dots highlight 48 viral genomes isolated in Bozeman that have wild-type ORF7a sequences. (C) Distribution of different mutations that occur along the ORF7a coding sequence.

Figure 2

ORF7a truncation results in loss of function (A) Amino acid (aa) sequence alignment of SARS-CoV-2 ORF7a^WT, ORF7a^Δ115, and SARS-CoV-1 ORF7a. Gaps show non-matching positions; red shows 17-aa sequence resulting from a frameshift in the ORF7a mutant. Beta strands (arrows) and alpha helices (coil) are shown above the alignment. (B) Diagram of SARS-CoV-2 ORF7a Ig-like fold. Disulfide bonds that stabilize the β sandwich structure are shown with red lines. The portion of the protein eliminated by the deletion is shown in gray. (C) C-terminally FLAG-tagged ORF7a^WT and ORF7a^Δ115 were cloned and overexpressed in HEK293T-hACE2. Protein expression was confirmed with western blot using anti-FLAG antibody. β-Actin (ACTB) was used as a loading control. (D and E) FLAG-tagged ORF7a^WT (D) or ORF7a^Δ115 (E) expressed in HEK293T-hACE2 cells. Immunostaining was performed using an anti-FLAG antibody (green). Cell nuclei were stained with Hoechst 33342 (blue). White scale bar is 10 μm. (F) HEK293T cells with integrated ISRE-luciferase reporter were transfected with pLV-mCherry (“-”), ORF7a^WT, or ORF7a^Δ115 plasmids. Transfected cells were treated with 5 ng/mL human recombinant IFN-α2b for 24 h, and induction of type I IFN signaling was measured with luciferase assay. Fold induction versus non-treated control was determined and normalized to mCherry control. Means (n = 6) were compared with one-way ANOVA (p = 0.00162). Pairwise comparisons were performed using post hoc Tukey’s test. Data are shown as mean ± SD. ^∗∗p < 0.01; ^nsp > 0.05. (G) ORF7a and ORF7b sgRNAs were identified by RT-PCR. Lower molecular weight band corresponding to ORF7b sgRNA is indicated with asterisk (^∗). Diagram on the right shows primer (arrows) positions. Specificity of PCR products was confirmed with Sanger sequencing. GAPDH was used as a control for cDNA synthesis. (H) Lysates from SARS-CoV-2-infected cells were probed with antibodies raised against ORF7b protein. ACTB was used as a loading control.

Figure 3

The ORF7a^Δ115 mutation results in replication and immune suppression defects (A and B) VeroE6 (A) or HEK293T-hACE2 (B) cells were infected with ORF7a^WT or ORF7a^Δ115 SARS-CoV-2 strain at MOI = 0.05. Viral RNA was measured in supernatant using qRT-PCR at different time points after infection. Measured RNA levels were normalized (using ΔCt method) to RNA levels at 0.5 hpi. (C) Total RNA was extracted from infected HEK293T-hACE2 cells, and viral RNA was measured. The ΔΔCt method was used to normalize viral RNA levels to host RNA (ACTB) and 0.5 hpi time point. Data in (A)–(C) are presented as mean ± SD of three biological replicates. Significance levels: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, or ^nsp > 0.05 (no significant difference). (D) Volcano plot showing IFN-I response in 293T-hACE2 cells infected with ORF7a^WT SARS-CoV-2 strain at MOI = 0.05. Expression of IFN-I response genes was studied 24 hpi using qRT-PCR array targeting 91 human transcripts (88 targets and 3 references). Experiment was performed in three biological replicates. Dashed lines show regulation (≥2-fold) and statistical significance (p < 0.05) thresholds. Each dot represents mean (n = 3) normalized expression of a single gene relative to non-infected host. Genes that passed the threshold are labeled. (E) Volcano plot showing IFN-I response in ORF7a^Δ115 versus ORF7a^WT infection. (F) Data shown in (D) and Figure S3A were plotted as a heatmap. Genes were classified into three groups. Group #1 genes are oppositely regulated between the two viral strains. Group #2 genes are upregulated by both ORF7a^WT and ORF7a^Δ115. Group #3 genes are downregulated in both. Genes that have statistically significant difference in expression between two viral strains are marked with asterisk. Gray shows genes with no detectable expression over 40 cycles of qRT-PCR.