ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication

Abstract

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.

Keywords: COVID-19; ORF3a; SARS-CoV-2; UVRAG; autophagy.

FIGURE 1

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) ORF3a triggers incomplete autophagy. (A–B) SARS-CoV-2 infection triggers incomplete autophagy. HeLa-hACE2 (A) or Vero-E6 (B) cells were infected with SARS-CoV-2 (MOI = 1) and cell lysates were collected at indicated time point post-infection for immunoblotting (IB) with indicated antibodies. (C) Screening of SARS-CoV-2-encoded proteins for LC3-I to LC3-II conversion in HeLa cells. (D,E) SARS-CoV-2 ORF3a induces incomplete autophagy. HeLa-vector and HeLa-ORF3a stable cells were treated with or without BafA1 (100 nM) for 4 h and the cell lysates were collected for IB with indicated antibodies (D). HeLa cells were transfected with increasing amount ORF3a expressing plasmid and the cell lysates were collected for IB with indicated antibodies at 48 post-transfection (E). (F) ORF3a expression modulates autophagy in Vero-E6 cells. (G) ORF3a blocks SQSTM1/p62 degradation upon starvation. HeLa-vector or HeLa-ORF3a stable cells were treated by serum starvation for 4 h and the cell lysates were collected for IB with indicated antibodies. (H,I) SARS-CoV-2 ORF3a induces LC3 puncta formation. HeLa-vector or HeLa-ORF3a cell line were treated with BafA1 (100 nM) and endogenous LC3 puncta were immunostained (H) and quantified (I). Scale bar, 15 μm. Arrow: representative autophagosomes. (J,K) HeLa-mRFP-GFP-LC3 stable cells were transfected with ORF3a or vector control and transfected cells were fixed and the LC3 puncta were captured (J,K) quantified as indicated. Yellow puncta, autophagosomes; Red puncta, autolysosomes; Arrow, representative autophagosomes. p62/GAPDH or LC3-II/GAPDH levels were quantified by the band intensity in (A–G). Similar results were obtained by three independent experiments. Mean ± SEM; n = 50; ***p < 0.001 and ****p < 0.0001 by Student’s t test in panels (I,K).

FIGURE 2

Severe Acute Respiratory Syndrome Coronavirus-2 ORF3a targets UVRAG to modulate PI3KC3 complex formation. (A) SARS-CoV-2 ORF3a interacts with UVRAG in yeast two-hybrid system. (B) SARS-CoV-2 ORF3a binds to UVRAG in HEK293T cells. HEK293T cells were co-transfected with indicated plasmids and cell lysates were collected and subjected to immunoprecipitation (IP) and IB with indicated antibodies at 48 h post-transfection. (C) Schematic diagram of SARS-CoV-2 ORF3a-UVRAG interaction. (D) The C2 and CCD of UVRAG bind to SARS-CoV-2 ORF3a. (E) SARS-CoV-2 ORF3a co-localizes with endogenous UVRAG. HeLa-ORF3a stable cells were subjected to immunostaining with antibodies against Flag or UVRAG. Scale bar, 15 μm. (F) SARS-CoV-2 ORF3a binds to endogenous UVRAG in HEK293T cells. (G–I) SARS-CoV-2 ORF3a blocks PI3KC3-C2 but facilitates PI3KC3-C1. HEK293T cells were co-transfected with indicated plasmids and cell lysates were collected and subjected to IP and IB with indicated antibodies at 48h post-transfection.

FIGURE 3

Cellular autophagy is required for efficient SARS-CoV-2 replication. (A) Genetic abrogation of Atg3 or Atg5 blocks autophagy response in MEF-hACE2 cell lines. (B–E) Atg3 or Atg5 KO reduces SARS-CoV-2 replication efficiency in MEF-hACE2 stable cells. MEF-hACE2^WT, MEF-hACE2^{Atg3 KO}, or MEF-hACE2^{Atg5 KO} were infected with SARS-CoV-2 (MOI = 1) and virus replication was examined by IB of viral N protein (B,C) or quantitative RT-PCR of viral transcripts (D,E). (F,G) ORF3a expression facilitates SARS-CoV-2 replication. Calu3-vector or Calu3-ORF3a stable cells were infected with SARS-CoV-2 (MOI = 1) and virus replication was examined by IB of viral N protein (F) or quantitative RT-PCR of viral transcripts (G). Mean ± SEM; n = 3 (D,E) or n = 4 (G); ns, not significant, *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test.

FIGURE 4

SARS-CoV ORF3a cannot induce autophagy. (A) Sequence alignment between SARS-CoV-2 ORF3a and SARS-CoV ORF3a. (B–D) SARS-CoV ORF3a does not affect autophagy. HeLa-vector, HeLa-ORF3a^{SARS–CoV–2}, or HeLa-ORF3a^SARS–CoV cell lines were treated with BafA1 (100 nM) and endogenous LC3 puncta were immunostained (B) and quantified (C). Scale bar, 15 μm. Arrow: representative autophagosomes. Mean ± SEM; n = 50; ns and ****p < 0.0001 by one-way ANOVA and Bonferroni’s post hoc test. HeLa-vector or HeLa-ORF3a^SARS–CoV cell lines were treated with BafA1 (100 nM) for 4 h and the cell lysates were collected for IB with indicated antibodies (D). (E) ORF3a^{SARS–CoV–2} but not ORF3a^SARS–CoV interacts with endogenous UVRAG. HEK293T cells were transfected with Flag-ORF3a^SARS–CoV or Flag-ORF3a^{SARS–CoV–2} and cell lysates were collected and subjected to IP and IB with indicated antibodies at 48h post-transfection. (F–H) SARS-CoV ORF3a does not affect the formation of UVRAG complex (F), Beclin 1 complex (G), or Atg14 complex (H). HEK293T cells were co-transfected with indicated plasmids and cell lysates were collected and subjected to IP and IB with indicated antibodies at 48 h post-transfection.

FIGURE 5

Schematic diagram of SARS-CoV-2 ORF3a-mediated incomplete autophagy. SARS-CoV-2 ORF3a interacts with UVRAG to positively regulate PI3KC3-C1 but suppress the PI3KC3-C2, leading to elevated autophagosome formation but blockage of autophagosome maturation.