SARS-CoV-2 Harnesses Host Translational Shutoff and Autophagy To Optimize Virus Yields: the Role of the Envelope (E) Protein

Abstract

The SARS-CoV-2 virion is composed of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope (E). E spans the membrane a single time and is the smallest, yet most enigmatic of the structural proteins. E is conserved among coronaviruses and has an essential role in virus-mediated pathogenesis. We found that ectopic expression of E had deleterious effects on the host cell as it activated stress responses, leading to LC3 lipidation and phosphorylation of the translation initiation factor eIF2α that resulted in host translational shutoff. During infection E is highly expressed, although only a small fraction is incorporated into virions, suggesting that E activity is regulated and harnessed by the virus to its benefit. Consistently, we found that proteins from heterologous viruses, such as the γ₁ 34.5 protein of herpes simplex virus 1, prevented deleterious effects of E on the host cell and allowed for E protein accumulation. This observation prompted us to investigate whether other SARS-CoV-2 structural proteins regulate E. We found that the N and M proteins enabled E protein accumulation, whereas S did not. While γ₁ 34.5 protein prevented deleterious effects of E on the host cells, it had a negative effect on SARS-CoV-2 replication. The negative effect of γ₁ 34.5 was most likely associated with failure of SARS-CoV-2 to divert the translational machinery and with deregulation of autophagy. Overall, our data suggest that SARS-CoV-2 causes stress responses and subjugates these pathways, including host protein synthesis (phosphorylated eIF2α) and autophagy, to support optimal virus replication. IMPORTANCE In late 2019, a new β-coronavirus, SARS-CoV-2, entered the human population causing a pandemic that has resulted in over 6 million deaths worldwide. Although closely related to SARS-CoV, the mechanisms of SARS-CoV-2 pathogenesis are not fully understood. We found that ectopic expression of the SARS-CoV-2 E protein had detrimental effects on the host cell, causing metabolic alterations, including shutoff of protein synthesis and mobilization of cellular resources through autophagy activation. Coexpression of E with viral proteins known to subvert host antiviral responses such as autophagy and translational inhibition, either from SARS-CoV-2 or from heterologous viruses, increased cell survival and E protein accumulation. However, such factors were found to negatively impact SARS-CoV-2 infection, as autophagy contributes to formation of viral membrane factories and translational control offers an advantage for viral gene expression. Overall, SARS-CoV-2 has evolved mechanisms to harness host functions that are essential for virus replication.

Keywords: ER stress; HSV-1; HSV-1 γ1 34.5 protein; PERK; SARS-CoV-2; SARS-CoV-2 E protein; autophagy; protein synthesis; translation control; virus replication.

FIG 1

SARS-CoV-2 E expression causes LC3 lipidation, reduction in p62/SQSTM1 levels, and increased eIF-2α phosphorylation. (A) HEK-293 cells were either left untransfected, transfected with a control pUC19 plasmid, or an E-HA expressing plasmid. The cells were harvested at 24 and 48 h posttransfection and equal amounts of proteins were analyzed for LC3lipidation and E expression. The ratio of LC3-II/LC3-I is shown below. (B) Transfections were as in panel A. Equal amounts of proteins were analyzed for p62/SQSTM1, Optineurin and ATG5. (C) HEK-293 cells were transfected with an E-HA expressing plasmid or infected with a HSV-1 Δγ₁ 34.5 virus (5 PFU/cell). The cells were harvested at 48 h posttransfection or at 14 h postinfection. Equal amounts of proteins were analyzed for p-eIF-2α. (D) Caco-2 cells were infected with SARS-CoV-2 (2 PFU/cell). The cells were harvested at 48 h and at 72 h postinfection and equal amounts of proteins were analyzed for p-eIF-2α and total eIF-2α. Spike served as a control for the infection. B-actin served as a loading control in panels A-D.

FIG 2

The γ₁ 34.5 protein of HSV-1 prevents p-eIF-2α accumulation induced by E expression but not LC3 lipidation. (A) HEK-293 cells were either left untransfected, or transfected with the control plasmid pUC19, an E-HA expressing plasmid, cotransfected with E-HA and pUC19, E-HA and γ₁ 34.5-expressing plasmids, or with a γ₁ 34.5-expressing plasmid (Flag-tagged). The cells were harvested at 48 h posttransfection and equal amounts of proteins were analyzed for p-eIF-2α, total eIF-2α, E-HA, or γ₁ 34.5 protein expression (Flag tagged). B-actin served as a loading control. (B) Experiment was as in panel A, but it was performed in A549-ACE2 cells. A549-ACE2 transfection efficiency was lower than in HEK-293 and that could account for some variability in the results. (C) Quantification of data from at least three independent experiments performed as in panel A. The fold change of p-eIF-2α/eIF-2α ratio of each sample compared with untransfected cells is depicted.(D) HEK-293 cells were either left untransfected or transfected with the control plasmid pLenti CMV GFP Puro expressing EGFP, an E-HA expressing plasmid, cotransfected with E-HA and pLenti CMV GFP Puro, E-HA and γ₁ 34.5-expressing plasmids, or with a γ₁ 34.5-expressing plasmid (Flag-tagged). At 46 h postinfection the cells were starved for 3 h in RPMI Medium 1640 without l-methionineand subsequently incubated with medium supplemented with Click-iT AHA reagent for 2 h. Click chemistry reaction to monitor nascent protein synthesis was performed as described in Materials and Methods. Both a Ponceau S staining of the membrane and the reaction of HRP with the ECL substrate (Pierce) are depicted. Quantification of total protein signal per sample relative to the signal of total proteins from untransfected cells, after normalization to the signal from Ponceau S staining is depicted. (E) Transfections in HEK-293 cells were as in panel A and samples were analyzed for LC3 lipidation with the ratio of LC3-II to LC3-I shown below. The pUC19 plasmid was replaced with pLenti CMV GFP Puro that expresses the control protein EGFP. B-actin served as a loading control.

FIG 3

The γ₁ 34.5 protein of HSV-1 and the SARS-CoV-2 M and N proteins resulted in E protein accumulation. (A) HEK-293 cells were transfected with an E-HA expressing plasmid, a γ₁ 34.5-expressing plasmid (Flag-tagged), cotransfected with E-HA and γ₁ 34.5-expressing plasmids, or E-HA and EGFP-expressing plasmids. The cells were harvested at 48 h posttransfection and equal amounts of proteins were analyzed for p-eIF-2α, E-HA, γ₁ 34.5 expression (Flag tagged), or EGFP. B-actin served as a loading control. (B) Transfections were as in panel A. Cell lysates prepared at 48 h posttransfection were analyzed using a GFP antibody with β-actin serving as a loading control. (C–D) HEK-293 cells were transfected with an E-HA, M-HA, N-EGFP expressing plasmid, an EGFP-expressing plasmid, or cotransfected with an E-HA expressing plasmid and plasmids expressing SARS-CoV-2 M, N, S, or the HSV-1 γ₁ 34.5 protein, respectively. Single transfections were done using 500 ng per well and cotransfections were performed using 1 μg per well (500 ng per plasmid). The cells were harvested at 24 h posttransfection and equal amounts of proteins were analyzed for expression of E-HA, S-HA, M-HA, γ₁ 34.5 (Flag-tagged), or EGFP (control protein EGFP and N fused to EGFP). B-actin served as a loading control. Arrows indicate the E and M proteins that are both tagged with an HA epitope.

FIG 4

Homologs of E or oligomerization mutants do not abrogate phosphorylation of eIF-2α. (A) HEK-293 cells were transfected with plasmids expressing either wild-type E or various E oligomerization mutants. In addition, HEK-293 cells were transfected with a γ₁ 34.5-expressing plasmid, or cotransfected with various E forms and a γ₁ 34.5-expressing plasmid. The cells were harvested at 48 h posttransfection and equal amounts of proteins were analyzed for p-eIF-2α, total eIF-2α, E-HA, LC3, or γ₁ 34.5 expression (Flag tagged). The ratio of LC3-II to LC3-I is also shown. B-actin served as a loading control. (B–D) Plasmids encoding E protein from different CoVs, including SARS-CoV, MERS-CoV, and HCoV-OC43 were transfected in HEK-293 cells or cotransfected with a γ₁ 34.5-expressing plasmid. The cells were harvested at 48 h posttransfection and equal amounts of proteins were analyzed for p-eIF-2α, total eIF-2a, E-HA, and γ₁ 34.5 expression (Flag-tagged). B-actin served as a loading control.

FIG 5

HSV-1 γ₁ 34.5 inhibits SARS-CoV-2 infection. (A) Vero E6 + γ₁ 34.5 cells were treated with doxycycline (5 μg/mL for 48 h) to induce γ₁ 34.5 expression. Induced cells along with parental Vero E6 cells were infected with icSARS-CoV-2-mNG (10⁻⁴ PFU/cell). Images were captured at 24 h postinfection using an Olympus microscope. A quantification of mNeonGreen-positive cells in control versus Vero E6 + γ₁ 34.5 cells is depicted. (B) Expression of γ₁ 34.5-Flag protein following doxycycline treatment (20 μg/mL) of Vero E6 + γ₁ 34.5 cells for 48 h. (C) HEK-293 ACE2-expressing cells were transfected with either γ₁34.5 (Flag-tagged) expressing plasmid or the control plasmid pUC19. At 48 h posttransfection the cells were infected with SARS-CoV-2 (10 PFU/cell). The cells were harvested at 18 h postinfection and equal amounts of cell lysates were analyzed for p-eIF-2α, total eIF-2α, γ₁ 34.5 (Flag), Spike (S), and β-actin. (D) Vero E6 and Vero E6 + γ₁ 34.5, either untreated or treated with doxycycline (20 μg/mL) to induce γ₁34.5 expression, were infected with SARS-CoV-2 (10⁻⁴ PFU/cell). At 34 h postinfection the cells were starved for 3 h in RPMI Medium 1640 without l-methionine (Thermo-Fisher) and subsequently incubated with medium supplemented with Click-iT AHA (l-azidohomoalanine) reagent (Invitrogen) for 2 h. Cells were lysed in a solution containing 1% SDS in 50 mM Tris-HCl, pH 8.0, and labeled proteins were reacted with biotin-alkyne (PEG4 carboxamide-propargyl biotin) in a Click-chemistry reaction according to manufacturer’s instructions using the Click-iT Protein Reaction Buffer kit (Invitrogen). Biotinylated proteins were analyzed in a denaturing polyacrylamide gel and detected with streptavidin-HRP. Both a Ponceau S staining of the membranes and the reaction of HRP with 4-chloro-1-naphthol supplemented with hydrogen peroxide are depicted. Quantification of total protein signal per sample relative to the signal of total proteins from uninfected cells, after normalization to the respective signal from Ponceau S staining is depicted. (E) Infections were performed with SARS-CoV-2 (10⁻⁴ PFU/cell) in replicate cultures of Vero E6 or doxycycline-treated (20 μg/mL) Vero E6 + γ₁ 34.5 cells. The cells were harvested at 24 h postinfection and intracellular progeny virus was quantified by plaque assays in Vero E6 cells. (F–G) Infections were performed with either the wild type (panel F) or the reporter virus (panel G) as in panel E, in replicate cultures. Cells were harvested at 24 h postinfection and the –ssRNA was quantified by real-time PCR analysis.

FIG 6

A PERK inhibitor inhibits SARS-CoV-2 infection. (A) Vero E6 cells were infected with icSARS-CoV-2-mNG (10⁻⁴ PFU/cell). Cells were then either left untreated or treated at 1 h postinfection with GSK2656517 (Sigma-Aldrich) at a dose of either 500 nM or 1 μM. Images were captured at 24 h postinfection using an Olympus microscope. (B) A quantification of mNG –expressing cells over total number of cells from panel A is depicted. (C) Vero-E6 cells treated as in panel A were harvested at 24 h postinfection and equal amounts of lysates were analyzed for expression of p-eIF-2α, total eIF-2α, S, or β-actin. (D) HEK293 cells were untransfected or transfected with plasmids expressing either E-HA or EGFP. At 6 h posttransfection cells were either left untreated or treated with GSK2656517 at a dose of either 500 nM or 1 μM. At 24 h posttransfection cells were harvested and equal amounts of lysates were analyzed for expression of p-eIF-2α, total eIF-2α, HA, EGFP, or B-actin.

FIG 7

Effect of γ₁ 34.5 protein on autophagy during SARS-CoV-2 infection. (A–B) Replicate cultures of Vero E6 and doxycycline-induced Vero E6 + γ₁ 34.5 cells were infected with either the SARS-CoV-2 USA-WA1/2020 (panel A) or the reporter virus icSARS-CoV-2-mNG (panel B) (10⁻⁴ PFU/cell). Cells were harvested at 36 h postinfection and equal amounts of proteins were analyzed for p-eIF-2α, total eIF-2α, LC3, γ₁ 34.5 (Flag-tagged), ATG5, Beclin-1, S, E, N protein expression, and β-actin. Numbers represent ratio of LC3-II/LC3-I (panel A) and a quantification of LC3-I and LC3-II (panel B). (C) Quantification of band intensity of p-eIF-2α versus total eIF-2α relative to uninfected, Vero E6 cells from at least three independent experiments is depicted. (D) Vero-E6 cells were transfected at 50% confluence with either a control (scrambled) siRNA (Santa Cruz; sc-37007) or Beclin 1 siRNA (Santa Cruz; sc-29797) using Lipofectamine 3000 according to manufacturer’s instructions (Invitrogen). Both siRNAs were used at a 300 nM concentration and the cells were transfected for 72 h before infection. Efficiency of Beclin-1 depletion is depicted. Vero E6 cells treated with either the scrambled siRNA, or the Beclin-1 siRNA as above were infected with icSARS-CoV-2-mNG (10⁻⁴ PFU/cell). Images were captured at 24 h postinfection using an Olympus microscope. (E) Vero E6 cells treated with Beclin-1 and scrambled siRNA as above were infected with wild type SARS-CoV-2 (10⁻⁴ PFU/cell). The cells were harvested at 24 h postinfection followed by total RNA extraction. Quantification of the negative-sense RNA was done by RT-qPCR analysis. (F) Vero E6, ATG16L KD and ATG5 KD derivatives were infected with icSARS-CoV-2-mNG, as above and images were captured at 24 h postinfection. (G) Infections of Vero E6 cells and ATG16L KD or ATG5 KD derivatives were performed with the wild-type SARS-CoV-2. Samples were harvested at 24 h postinfection and quantification of progeny virus production was performed by plaque assays. All values were derived after analyzing samples from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (H–I) Efficiency of ATG16L or ATG5 depletion using specific shRNAs (Sigma) expressed from integrated lentiviral vectors is depicted. To deplete ATG16L four different cell lines were established using four different shRNAs (1–4). The cell line expressing shRNA number 2 was selected as depletion of ATG16L was more efficient.

FIG 8

Aberrant vacuolar structures in the cytoplasm of SARS-CoV-2 infected γ₁ 34.5 – expressing cells. (A) Replicate cultures of Vero E6 and doxycycline-induced Vero E6 + γ₁ 34.5 cells seeded on coverslips were infected with SARS-CoV-2 USA-WA1/2020 (10⁻⁴ PFU/cell). The cells were fixed with 2% glutaraldehyde at 42 h postinfection and processed for TEM analysis, as detailed in Materials and Methods. At least 50 cells were analyzed per sample. Representative images are depicted. (B) Model summarizing the mechanism of interference of SARS-CoV-2 replication by the HSV-1 γ₁ 34.5 protein. SARS-CoV-2 infection causes extensive reorganization of the ER/ERGIC compartments that leads to formation of viral membrane factories where the virus replicates. It also imposes a translational shutoff that offers an advantage to viral over host genes for expression, while suppressing host defense gene expression. E protein could contribute to virus replication by facilitating membrane rearrangements through activation of autophagy that supports the growth of the viral factories. E along with other viral proteins could be responsible for the translational shutoff during SARS-CoV-2 infection. γ₁ 34.5 protein could disrupt autophagy activated during SARS-CoV-2 infection by binding to Beclin-1 and/or through other mechanisms. Also, γ₁ 34.5 protein suppresses host translational shutoff during SARS-CoV-2 infection. Both effects could result in inhibition of SARS-CoV-2 replication. Autophagic vacuoles with abnormal size and morphology observed in SARS-CoV-2 infected γ₁ 34.5 -expressing cells could represent defects in the autophagolysosome pathway.