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.

Increased mTOR signaling, impaired autophagic flux and cell-to-cell viral transmission are hallmarks of SARS-CoV-2 infection

“…The results of this multi-omics study are in agreement with our findings, showing that SARS-CoV-2 regulates mTOR activity and autophagy at different levels. Nonetheless, another study showed that SQSTM1/p62 levels did not change during SARS-CoV-2 infection [21]. We demonstrated that the ratio of the autophagosome markers LC3-II to LC3-I and the content of p62/SQSTM1 increase in infected cells (Figure 1A), which is associate with the accumulation of autophagosomes by blockage of the autophagy flux [22].…”

“…The cell-to-cell transfer of viral components demonstrated here might represent an additional mechanism that SARS-CoV-2 uses to infect new cells while escaping the immune system and shed light on the virulence mechanisms and transmissibility SARS-CoV-2. For example, viral particle accumulation inside cells could explain cases of persistent systemic infection by SARS-CoV-2, the late onset of the symptoms, and the fact that many asymptomatic individuals are still able to produce infectious viral particles [21,85,86].…”

“…For the bafilomycin experiment, cells were cultured in 24-well plates as described above, followed by washing with phosphate-buffered saline (PBS) and treated with 10 nM of bafilomycin in DMEM 10% FBS for 3 hours. Treatment was withdrawn two hours prior to infection and 24 hpi [21,22] cells and supernatant samples were titrated by PFU as previously described.…”

Betacoronavirus Assembly: Clues and Perspectives for Elucidating SARS-CoV-2 Particle Formation and Egress

“…This observation raises the question of using this membrane reservoir for viral replication and as a way to hide from the immune system. This might be a partial answer in determining DMV origins in the case of SARS-CoV-2 ( 9 ). For SARS-CoV-2, the DMVs were very well characterized thanks to recent high-resolution microscopy techniques: DVMs are composed of two membranes separated by 18-nm luminal spacing, with an average diameter of 340 nm for the inner membrane, similar to SARS-CoV-1 DMVs ( 7 ).…”

Autophagy in Viral Infection and Pathogenesis

“…Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), an enveloped, single-stranded and positive-sense RNA β-coronavirus, is the cause of the COVID-19 pandemic (Wang et al, 2020;Wu et al, 2020;Zhou et al, 2020). ORF3a, an accessory protein of SARS-CoV-2, could strongly inhibit autophagy activity by blocking the fusion of autophagosomes with lysosomes (Hayn et al, 2021;Miao et al, 2021;Qu et al, 2021;Zhang et al, 2021). Specifically, ORF3a is localized in the late endosome and directly interacts with and sequestrates VPS39, an essential component of the homotypic fusion and protein sorting (HOPS) complex, thereby preventing the FIGURE 4 | Viruses hijack and subvert host autophagy to aid their own infections and pathogenesis.…”