Fig. 8: ORF7a transmembrane domain is responsible for counteracting SERINC5 restriction of SARS-CoV-2 entry.
From: SARS-CoV-2 ORF7a potently inhibits the antiviral effect of the host factor SERINC5
Naturally occurring SARS-CoV-2 ORF7a deletions and the β-sheets of the SARS-CoV-2 ORF7a ectodomain do not affect its anti-SERINC5 function. 293T-hACE2 cells were infected with SARS-CoV-2 S pseudoviruses produced in the presence or absence of SERINC5 along with a naturally occurring deletion variants of SARS-CoV-2 ORF7a or b β-sheet deletion mutants of SARS-CoV-2 ORF7a. c The SARS-CoV-2 ORF7a transmembrane domain is critical for its anti-SERINC5 function. 293T-hACE2 cells were infected with SARS-CoV-2 S pseudoviruses produced in the presence or absence of SERINC5 along with either wild type ORF7a or ORF7-CD4 transmembrane domain chimera (ORF7aTMCD4). For a, b, and c, luciferase levels were normalized to p24CA levels. The percentage (%) of relative infectivity with respect to pseudovirus produced in the presence of empty vector is shown. All results are presented as mean ± SD from 3 independent experiments. Statistical analysis among E.V. and SERINC5 + E.V. conditions were performed by one-sample t test (two-tailed) while unpaired t-test (two-tailed) was used for comparisons among SERINC5 + E.V. and SERINC5 + ORF7a groups. Representative immunoblotting results of the expression levels of SARS-CoV-2 ORF7a variants or mutants in the producer cell lysates is shown at the bottom. d Transmembrane domain of SARS-CoV-2 ORF7a is required for its interaction with SARS-CoV-2 S and SERINC5. 293T cells were cotransfected with SARS-CoV-2 S, SERINC5, SARS-CoV-2 ORF7a, SARS-CoV-2 ORF7aTM CD4or empty vector as indicated. Cells were harvested and lysates were immunoprecipitated with anti-V5 antibody followed by immunoblot analyses probing for the indicated proteins. Representative immunoblotting results from n = 3 independent experiments are shown. Uncropped blots are in Source Data. (empty vector, E.V.; wild type SARS-CoV-2 ORF7a, WT; SARS-CoV-2 ORF7aΔ9nt, Δ9nt; SARS-CoV-2 ORF7aΔ18nt, Δ18nt; SARS-CoV-2 ORF7aΔ57nt, Δ57nt; SARS-CoV-2 ORF7aΔ96nt, Δ96nt; SARS-CoV-2 ORF7aΔA, ΔA; SARS-CoV-2 ORF7aΔB, ΔB; SARS-CoV-2 ORF7aΔC, ΔC; SARS-CoV-2 ORF7aΔD, ΔD; SARS-CoV-2 ORF7aΔE, ΔE, SARS-CoV-2 ORF7aΔF, ΔF; SARS-CoV-2 ORF7aΔG, ΔG; SARS-CoV-2 S, S; SARS-CoV-2 Spike S2 subunit, S2; not significant, ns).
