key: cord-1021005-7gjk20td
authors: Waisner, Hope; Grieshaber, Brandon; Saud, Rabina; Henke, Wyatt; Stephens, Edward B; Kalamvoki, Maria
title: SARS-CoV-2 harnesses host translational shutoff and autophagy to optimize virus yields: The role of the envelope (E) protein
date: 2022-03-25
journal: bioRxiv
DOI: 10.1101/2022.03.24.485734
sha: 4d6f1ad26a6ea0f7343cf85739f3fe6707d2ec1a
doc_id: 1021005
cord_uid: 7gjk20td

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 phosphorylation of the translation initiation factor eIF2α and LC3 lipidation 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. In support of this, we found that the γ1 34.5 protein of herpes simplex virus 1 (HSV-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 prevented E accumulation. While γ1 34.5 protein prevented deleterious effects of E on the host cells, it had a negative effect on SARS-CoV-2 replication. This 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 pathways. 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 production. Importance In 2020, a new β-coronavirus, SARS-CoV-2, entered the human population that has caused a pandemic resulting in 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. Co-expression 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.

Compared with other highly pathogenic coronaviruses (CoVs), the mortality rate of SARS-CoV-2 is approximately 2% among unvaccinated individuals (1) . This mortality rate, along with the lack of pre-existing immunity, the fact that about 20% of infected individuals without preexisting immunity require medical attention, and the highly transmissible nature of the virus, has led to the disruption of normal activities worldwide. Several highly effective vaccines have received use authorization, but the slow global vaccination rate and accumulation of adaptive mutations in different proteins of the virus, particularly the Spike protein, yield novel variants with different immunoevasion properties (2) (3) (4) (5) . Coronaviruses are enveloped viruses with a single-stranded, positive sense RNA genome. The coronavirus particle is composed of four structural proteins: nucleocapsid (N), membrane (M), envelope (E), and spike (S) (6) . E is a small integral membrane protein that ranges from 75-106 aa (7) (8) (9) . E protein localizes to the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the Golgi complex (10) (11) (12) (13) (14) (15) . The protein exists in different forms, including a monomeric form that potentially interacts with cellular proteins to alter the secretory machinery and to communicate signals, and a high-molecular weight homo-oligomer with a function in virion assembly (15) (16) (17) (18) .

In addition, a pentameric form of E protein is an ion channel (viroporin) with mild selectivity for cations that has been linked to virus pathogenesis (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) .

The importance of E during SARS-CoV and SARS-CoV-2 infections is highlighted by the fact that viruses lacking the gene for E protein display significantly reduced virus yields, due to aborted viral assembly that gives rise to immature virions with a strikingly aberrant morphology (12, 25, (30) (31) (32) . For example, during infection with a mouse hepatitis virus (MHV) deleted of E, the virions display pinched, elongated rather than spherical shapes and smaller plaques, with irregular-shaped and jagged edges (33). How E protein facilitates virion morphogenesis remains unclear considering that only a small fraction of E is incorporated into the virions (34). A role of E in inducing membrane curvature has been proposed for MHV that is perhaps associated with E homo-polymerization and its interactors, but a mechanism is currently unknown (13) .

The role for the cation channel activity of E during SARS-CoV-2 infection is also unclear although mutations within the transmembrane domain that inhibit the ion channel activity in SARS-CoV E are reversed by this virus (35). However, as most known mutations that impair the ion channel activity of E also impair E oligomerization, it is currently unknown if one or both properties of the protein are rescued (21, 25, (36) (37) (38) . The transport of Ca 2+ by SARS-CoV E has been correlated with inflammatory-mediated lung damage in vivo, highlighting the importance of E in viral pathogenesis (39) (40) (41) (42) (43) (44) (45) . The channel activity of E could also alter the secretory pathway or the luminal environment, leading to efficient trafficking of virions (18, 27, 46, 47) .

Consistently, some of the proposed interactors of E are associated with ion transport and others with vacuoles and mitochondria, suggesting that E may participate in re-organizing membranes and the recruitment of lipid processing machineries at sites of virion assembly (11, (48) (49) (50) (51) (52) (53) (54) .

Considering that E protein localizes in the ER-ERGIC-Golgi compartments and forms an ion channel we sought to determine the type of responses activated in cells ectopically expressing E.

We found that E protein triggered ER-signaling pathways that led to phosphorylation of the translation initiation factor eIF-2α with a concomitant translational shutoff and LC3 lipidation.

Both effects indicate that major metabolic alterations occur in cells expressing E that impact protein synthesis and potentially mobilize energy resources. We also found that E protein accumulation was restricted in cells ectopically expressing E protein. To further understand the functions of E we determined whether proteins from heterologous viruses known to prevent eIF-2α phosphorylation and LC3 lipidation could reverse the adverse effects of E on the host. The γ 1 34.5 protein of HSV-1 is known to prevent host translational shutoff during HSV-1 infection by recruiting the protein phosphatase 1α (PP1α) to dephosphorylate eIF-2α, which is phosphorylated by activated protein kinase R (PKR) following foreign RNA sensing (55-59). In addition, γ 1 34.5 protein inhibits autophagy by binding to the autophagy-inducing protein Beclin-1 that is downstream of activated PKR (59-62). Mutant HSV-1 viruses lacking the Beclin-1interacting domain of γ 1 34.5 display reduced viral replication in vitro and in vivo, due to robust activation of autophagy (59-66). We found that γ 1 34.5 could reverse eIF-2α phosphorylation, but not LC3 lipidation induced by E, and enabled E protein accumulation.

An interesting observation was that HSV-1 γ 1 34.5 inhibited SARS-CoV-2 infection. One mechanism was through inhibition of the host translational shutoff by γ 1 34.5 that is imposed by the virus to gain translational advantage over the host. Additionally, disruption of autophagy pathways by γ 1 34.5 during SARS-CoV-2 infection led to formation of aberrant vacuolar structures, most likely containing engulfed organelles, instead of viral membrane factories.

Taken together, our data suggest that SARS-CoV-2 harnesses stress response pathways of the host for optimal progeny virus production.

The E protein of SARS-CoV-2 initiates autophagy and interferes with translation initiation. The E protein accumulates in the ER and ERGIC where it can form a channel with weak cation specificity which may exhibit Ca 2+ transport activity in the ERGIC (10, 11, 14, 15) ( 19-22, 24-26, 28) . While only a small amount of E protein expressed during SARS-CoV-2 infection is incorporated into the virions, the protein appears to also induce membrane curvature, and participate in membrane scission (13, 34, (67) (68) (69) . Thus, we sought to determine ER signaling responses that may be activated by E expression. We found that ectopic expression of E in HEK-293 cells caused LC3 lipidation that was apparent by 48 h post-transfection ( Figure 1A ).

Moreover, we observed reduced p62/SQSTM1 accumulation ( Figure 1B ). The p62/SQSTM1 is an adaptor protein that sorts ubiquitinated cargo to autophagosomes for degradation and subsequently is degraded itself (70, 71) . This reduction of p62/SQSTM1 accumulation is indicative of autophagy activation. We did not observe changes in the levels of optineurin (OPTN) suggesting that mitophagy was not induced by E expression ( Figure 1B) . Also, we did not observe changes in the levels of ATG5 protein (autophagy related 5), which along with ATG12 protein acts as an E1-activating enzyme during autophagy ( Figure 1B) (72) . In addition, we tested whether E expression could trigger phosphorylation of the translation initiation factor eIF-2α, a modification that is usually observed when unfolded protein response (UPR) pathways are activated (73, 74) . We observed that ectopic expression of E triggered accumulation of p-eIF-2α ( Figure 1C ). As a control, HEK-293 cells were infected with an HSV-1 γ 1 34.5-null mutant, which cannot reverse phosphorylation of eIF-2α. Finally, following infection of Caco-2 cells with SARS-CoV-2 we observed some accumulation of p-eIF-2α ( Figure 1D ). We conclude that ectopic expression of E protein activates stress responses that lead to phosphorylation of eIF-2α and LC3 lipidation. Figure 2D , LC3 lipidation due to E expression was not reversed by γ 1 34.5. This is perhaps because γ 1 34.5 interferes with phagophore elongation through Beclin-1 binding, which does not necessarily interfere with LC3 lipidation. We conclude that γ 1 34.5 protein inhibits the phosphorylation of eIF-2α triggered by E expression, but does not inhibit LC3 lipidation. were lower when co-expressed with GFP compared with E alone, most likely because of competition of the two plasmids for transport into the nucleus, gene transcription and protein translation. However, when E was co-expressed with the γ 1 34.5 protein, accumulation of E protein was strongly enhanced (Figures 3A). In contrast, we did not observe N protein accumulation when it was co-expressed with the γ 1 34.5 protein ( Figure 3B ). These data suggest that stress responses activated following E expression negatively impact E accumulation, however, HSV-1 γ 1 34.5 protein can reverse these effects.

Considering that signaling responses activated following E expression have deleterious effects on the host, SARS-CoV-2 must control E functions to ensure optimal virus replication (39) (40) (41) (42) (43) (44) (45) .

Thus, we sought to determine if any SARS-CoV-2 proteins can reverse E effects allowing for E protein accumulation. We chose to analyze the effects of other virion proteins that may impact E functions through interactions. HEK-293 cells were co-transfected with plasmids expressing the E protein and either the M, N, or S proteins of SARS-CoV-2. HEK-293 cells were also cotransfected with plasmids expressing the E protein and either the EGFP or the HSV-1 γ 1 34.5 protein to serve as negative and positive controls, respectively. As shown in Figure 3C , both N and M proteins prevented p-eIF-2α accumulation due to E expression and enhanced E protein accumulation. In contrast, the S protein did not prevent p-eIF-2α accumulation and did not support E protein accumulation. Nevertheless, the γ 1 34.5 protein was more effective than the M and N proteins of SARS-CoV-2 in enabling E protein accumulation. We also assessed the impact of different proteolytic machineries on E protein accumulation and found no significant effect (supplemental data and Figure S1 ). Thus, E protein expression negatively impacts its own accumulation but this effect is reversed by proteins from SARS-CoV-2 or by heterologous viruses that appear to counterbalance E effects.

2α. In the next series of experiments, we determined whether specific E mutants could decrease the propensity of E protein to induce ER stress responses. Two point mutations were inserted in the transmembrane domain of the E protein, asparagine (N) at position 15 was converted to alanine (A) and the valine (V) at position 25 was converted to phenylalanine (F) (N15A/V25F).

These mutations are known to reduce E oligomerization to some extent (21, 22, 36, 75) . The N15A mutation reduces pentamerization of E, while V25F reduces higher order oligomers (75) . Figure 4A , the E N15A/V25F-expressing cells accrued similar levels of p-eIF-2α as cells expressing wild-type E (compare lane 4 to lane 3), suggesting that reduced E oligomerization does not reduce ER stress responses triggered by E. This level of p-eIF-2α was again reversed by γ 1 34.5 protein (compare lane 8 to lane 4). We also tested a mutant of E in which the conserved proline at position 54 was changed to a glycine (E-P54G). P54 is located within the cytoplasmic domain at the turn of a β-coil-β motif and likely affects E topology. E-P54G also triggered p-eIF-2α that was partially reversed by γ 1 34.5 protein (compare lane 5 to lane 3, and lane 9 to lane 5). LC3 lipidation was triggered by the unmodified E protein and all mutants tested, albeit to a greater extent by E-P54G. We conclude that disruption of E pentamerization or oligomerization does not reduce ER stress responses triggered by E expression.

We then sought to determine whether the E protein of SARS-CoV, MERS-CoV, and HCoV-OC43 trigger similar responses as SARS-CoV-2 E (31). Cells were co-transfected with Eexpressing and γ 1 34.5 -expressing plasmids as described above. Like SARS-CoV-2 E protein, SARS-CoV, MERS-CoV, and HCoV-OC43 E homologs induced phosphorylation of eIF-2α ( Figure 4B -C). In each case the HSV-1 γ 1 34.5 protein blocked phosphorylation of eIF-2α ( Figure 4B ) and enabled E protein accumulation ( Figure 4B and D). We conclude that E homologs and E oligomerization mutants display the same propensity as SARS-CoV-2 E to induce ER stress responses that result in eIF-2α phosphorylation. Overall, γ 1 34.5 was able to inhibit the translational shutoff imposed during SARS-CoV-2 infection and which resulted in a decrease in virus replication and progeny virus production.

During SARS-CoV-2 infection autophagy appears to supplement the viral membrane factories with membranes and metabolites required for virus replication (76) (77) (78) . While γ 1 34.5 is known to interfere with autophagy by binding to Beclin-1, we observed that co-expression of γ 1 34.5

protein with E did not reduce, but rather enhanced LC3 lipidation ( Figure 2D ). LC3 lipidation was also induced during SARS-CoV-2 infection, however in the presence of γ 1 34.5, the levels of We also noticed a decrease in the amounts of S protein, Beclin-1 and ATG5 in infected γ 1 34.5expressing cells, although the levels of the ATG5/ATG12 complex remained unaltered. These data indicated that γ 1 34.5 disrupted autophagic responses during SARS-CoV-2 infection, causing a reduction in viral infection.

The γ 1 34.5 protein is known to combat autophagy during HSV-1 infection through both a direct mechanism, by interacting with Beclin-1, and an indirect mechanism, by inhibiting PKR-induced phosphorylation of eIF-2α. To assess the impact of Beclin-1 on SARS-CoV-2 infection we depleted cells of Beclin-1 using a specific siRNA followed by infection with the reporter virus.

Depletion of Beclin-1 caused a delay in SARS-CoV-2 infection ( Figure 6C ). Additionally, depletion of ATG16L, a critical factor for synthesis of the autophagosome precursor, reduced SARS-CoV-2 infection ( Figure 6D ). These data suggest that early autophagy events are essential during SARS-CoV-2 infection.

While early autophagy events are critical during SARS-CoV-2 infection, treatment with the autophagy inducer rapamycin was inhibitory, most likely due to degradation of factors critical for virus infection or virions per se ( Figure 6E ). The rapamycin effect could not be reversed by γ 1 34.5 ( Figure 6E) . A striking observation was that γ 1 34.5 sensitized the cells and the levels of both lipidated and non-lipidated LC3 and the levels of the autophagy adaptor p62/SQSTM1 were reduced following exposure to an autophagy inducer, including SARS-CoV-2 or rapamycin ( Figure 6F ). Notably, depletion of Beclin-1 during SARS-CoV-2 infection increased LC3 lipidation (data not shown).

Finally, we determined if γ 1 34.5 expression could cause overt changes in the viral membrane factories through its effects on autophagy. For this, doxycycline-treated Vero E6 + γ 1 34.5 cells and parental cells that were either exposed to SARS-CoV-2 or not, were processed for TEM A potential engulfment or fusion event with an organelle resembling a lysosome has been marked with a red arrow (Figure 7 , panel h). We conclude that γ 1 34.5 expression during SARS-CoV-2 infection altered autophagic responses and caused formation of abnormal vacuoles with different organelles entrapped undergoing degradation.

Our studies emanated from the observation that SARS-CoV-2 causes major rearrangements in the ER-Golgi membranes that form the viral membrane factories where the virus replicates and virions assemble. Since E is a small transmembrane protein that oligomerizes in the ER and the ERGIC, we hypothesized that it could disrupt the functions of these organelles (10) (11) (12) 14) .

Indeed, we observed that E protein caused LC3 lipidation, a hallmark of autophagy initiation, and phosphorylation of the translation initiation factor eIF-2α resulting in host translational shutoff. It is likely that E expression activates ER-stress responses, including the unfolded protein response that results in PERK activation, which phosphorylates eIF-2α (73, 74, 79) .

Disruption of ER homeostasis could subsequently lead to autophagy activation.

Coronaviruses are known to impose host translational shutoff during the early stages of infection to prevent the infected host from synthesizing new proteins, while translation of viral mRNAs is not affected (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) (91) (92) (93) . Phosphorylation of eIF-2α has been reported during infection by SARS-CoV, SARS-CoV-2, the transmissible gastroenteritis virus (TGEV) and other CoVs and both kinases PERK and PKR appear to participate in this process (94) (95) (96) (97) (98) (99) (100) . PERK could be activated following disruption of ER homeostasis by viral proteins accumulating in the ER (101, 102) . For example, E could disrupt ER Ca 2+ homeostasis through its viroporin function. PKR could be activated following viral RNA sensing. Ectopic expression of several SARS-CoV proteins including S and ORF3a triggers p-eIF-2α due to disruption of ER homeostasis (103) (104) (105) (106) .

We observed that ectopic expression of E protein had deleterious effects on the host cell that impacted E protein accumulation. It is not uncommon for viruses to develop mechanisms to regulate and harness the activity of proteins that trigger deleterious responses to ensure optimal replication. To test this, we co-expressed E with either other SARS-CoV-2 proteins that are known to interact with E, or with proteins from heterologous viruses that can evade host translational shutoff and autophagy. We discovered that the γ 1 34.5 protein of HSV-1 inhibited phosphorylation of eIF-2α triggered by E expression and allowed for E protein accumulation.

The mechanism by which γ 1 34.5 protein prevents accumulation of p-eIF-2α has been previously Besides γ 1 34.5, we found that the M and N proteins of SARS-CoV-2 prevent accumulation of p-eIF-2α in E-expressing cells, while S exacerbated accumulation of p-eIF-2α. Both M and N interact with E during virion assembly (6) . It is likely that this binding alters the localization of E, its oligomerization status, or its potential binding with host factors, which alters its propensity to cause ER stress responses. Alternatively, the immunoevasion properties of M and N could reverse the translation inhibition imposed by E (110) (111) (112) . On the other hand, ectopic expression of S is known to trigger phosphorylation of eIF-2α, and in the presence of E such an effect was exacerbated (103) . Consistent with this, we have observed increased LC3 lipidation when E and S were co-expressed ectopically (data not shown).

The properties and functions of the E proteins are generally conserved among beta coronaviruses, as the E proteins of SARS-CoV, MERS-CoV, and HCoV-OC43 were found to trigger similar responses as SARS-CoV-2 E (31). Mutations that obstructed the ion channel function of E or decreased E oligomerization did not suppress p-eIF-2α accumulation. Perhaps the intracellular localization of E and its interactors are sufficient to trigger ER stress responses leading to eIF-2α phosphorylation.

An interesting observation was that SARS-CoV-2 displayed decreased replication and progeny virus production in γ 1 34.5-expressing cells ( Figure 7B ). One explanation is that by preventing host translational shutoff, γ 1 34.5 decreased the efficiency of viral gene expression, and enabled expression of host defense genes that are known to combat SARS-CoV-2 infection (80, 113 ). An additional possibility was that γ 1 34.5 disrupted autophagy pathways utilized by SARS-CoV-2.

Coronaviruses are known to exploit autophagosome formation to support DMV biogenesis, while stalling lysosome fusion to evade autophagy-mediated degradation. Transient depletion of Beclin-1, which functions as a scaffold in forming a multiprotein assembly during autophagy initiation and nucleation, and is a known target of γ 1 34.5, obstructed the infection. In addition, depletion of ATG16L, an integral part of the complex involved in LC3 lipidation that is essential for autophagosome formation and expansion, had a negative effect on SARS-CoV-2 infection.

Thus, it appears that LC3 lipidation is essential for SARS-CoV-2 DMV formation. Another striking observation was that infection of γ 1 34.5-expressing cells with SARS-CoV-2 led to formation of enormous size vacuoles, almost half the size of the nucleus, containing what appeared to be various organelles undergoing degradation. This abnormal phenotype of vacuoles may be the result of either defective autophagy or defective proteolysis. While SARS-CoV-2 through ORF3a can inhibit fusion of autophagosomes with lysosomes and decrease lysosomal activity by increasing lysosomal pH, this was not sufficient to yield the abnormal vacuole phenotype, and γ 1 34.5 was required to sensitize the cells by altering early autophagy events (114) .

Overall, we provide novel evidence that ectopic expression of E causes adverse effects on the host cell. These effects were found to be antagonized by γ 1 34.5, a protein from a heterologous virus. This led us to discover that the activity of E is likely regulated during SARS-CoV-2 infection by other viral proteins to ensure optimal virus production. Finally, we demonstrated that pathways inhibited by γ 1 34.5 are required for optimal SARS-CoV-2 growth, therefore these pathways could be considered novel antiviral targets.

The A549 cells (human lung adenocarcinoma), Caco-2 (human colorectal adenocarcinoma), HEK-293 (human embryonic kidney epithelial cells), and Vero E6 (normal monkey kidney epithelial cells) were obtained through ATCC. The SARS-related coronavirus 2 isolate USA-WA1/2020 was obtained through BEI resources (NR-52281). The icSARS-CoV-2-mNG was obtained through the World Reference Center for Emerging Viruses and Arboviruses ("WRCEVA") at the University of Texas Medical Branch at Galveston ("UTMB").

A plasmid expressing the γ 1 34.5 ORF with a FLAG-tag was digested with Hind III and Xba I to extract only the FLAG-tagged γ 1 34.5. This fragment was then inserted into the pLenti-mCherry-Mango II x 24 plasmid (Addgene #127587) digested with Nhe I and BamH I. HEK-293 cells were seeded in a 60 mm dish at 60% confluence and were co-transfected with the pLenti-mCherry-FLAG-γ 1 34.5 plasmid described above, the Gag-Pol-expressing plasmid, and the vesicular stomatitis virus G (VSV-G)-expressing plasmid at a ratio of 7:7:1 (5 μg total amount of DNA) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. At 48 h after transfection, the supernatant from the cultures was collected, filtered through a 0.45-μm-pore-size filter, and used to infect Vero-E6 cells, as described before (115, 116) . Puromycin selection initiated at 24 h after exposure of cells to lentiviruses and continued until only resistant clones survived. Resistant cultures were then plated in a 6-well plate and exposed to doxycycline 

The procedures for immunoblotting were described elsewhere (see also supplemental materials and methods) (115, 117) .

Cells were uninfected or infected with SARS-CoV-2 for indicated times. Starvation of cells was 

Cell lysates were collected in TRIzol reagent (Ambion) at indicated times post-infection. Total RNA was extracted via phenol-chloroform extraction method. Reverse-transcription PCR was then performed using LunaScript RT Master Mix Kit (NEB) using a gene specific primer. This primer was used to specifically detect the negative strand RNA from SARS-CoV-2, as described previously (118) . The reverse transcription primer, 5'-ACAGCACCCTAGCTTGGTAGCCGAACAACTGGACTTTATTGA -3', contains IAC (internal amplification control) tag-2 and a part targeting the ORF1ab gene of SARS-CoV-2.

Real-time PCR was then performed using SYBR Green reagent (Invitrogen) according to the manufacturer's recommendations in a 7500 fast real-time PCR system (Applied Biosystems).

The forward primer, 5'-AGGTGTCTGCAATTCATAGC-3' (743-762bp), and the reverse primer, 5'-ACAGCACCCTAGCTTGGTAG -3' (IAC tag-2), were used for amplification.

Cell monolayers on Thermanox plastic coverslips (13 mm 

The p values were calculated using a standard unpaired Student's t-test with a p ≤0.05 considered significant. All statistical analyses were performed using at least three biological replicates. 

COVID-19 Vaccination and Non-COVID-19 Mortality Risk -Seven Integrated Health Care Organizations

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

Effectiveness of the Single-Dose Ad26.COV2.S COVID Vaccine

Phase 3 Safety and Efficacy of AZD1222 (ChAdOx1 nCoV-19) Covid-19 Vaccine

Molecular Architecture of the SARS-CoV-2 Virus

The coronavirus E protein: assembly and beyond

Coronavirus envelope protein: current knowledge

Coronavirus envelope protein: A small membrane protein with multiple functions

Identification of a Golgi complex-targeting signal in the cytoplasmic tail of the severe acute respiratory syndrome coronavirus envelope protein

Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein

The missing link in coronavirus assembly. Retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and physical interaction between the envelope and membrane proteins

Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E

Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells

Structure of a conserved Golgi complextargeting signal in coronavirus envelope proteins

The transmembrane oligomers of coronavirus protein E

A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway

Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein

Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel

Structure and inhibition of the SARS coronavirus envelope protein ion channel

Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids

Model of a putative pore: the pentameric alpha-helical bundle of SARS coronavirus E protein in lipid bilayers

MERS coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels

Coronavirus virulence genes with main focus on SARS-CoV envelope gene

Characterization of the SARS-CoV-2 E Protein: Sequence, Structure, Viroporin, and Inhibitors

Coronavirus Proteins as Ion Channels: Current and Potential Research

On the Alert for Cytokine Storm: Immunopathology in COVID-19

COVID-19 cytokine storm: the interplay between inflammation and coagulation

Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis

Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damage and constitutes an antiviral target

Is There a Link Between the Pathogenic Human Coronavirus Envelope Protein and Immunopathology? A Review of the Literature

Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease

Virus glycosylation: role in virulence and immune interactions

Post-translational modifications of coronavirus proteins: roles and function

Bcl-xL inhibits T-cell apoptosis induced by expression of SARS coronavirus E protein in the absence of growth factors

The SARS coronavirus E protein interacts with PALS1 and alters tight junction formation and epithelial morphogenesis

Interaction of ICP34.5 with Beclin 1 modulates herpes simplex virus type 1 pathogenesis through control of CD4+ Tcell responses

Release of coronavirus E protein in membrane vesicles from virus-infected cells and E protein-expressing cells

Nucleocapsid-independent assembly of coronavirus-like particles by coexpression of viral envelope protein genes

Infectious bronchitis virus E protein is targeted to the Golgi complex and directs release of virus-like particles

p62/SQSTM1 Binds Directly to Atg8/LC3 to Facilitate Degradation of Ubiquitinated Protein Aggregates by Autophagy

Homeostatic Levels of p62 Control Cytoplasmic Inclusion Body Formation in Autophagy-Deficient Mice

The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy

Translational control and the unfolded protein response

Regulation of translation initiation in eukaryotes: mechanisms and biological targets

Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals? Viruses

Manipulation of autophagy by SARS-CoV-2 proteins

Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues

Human Coronavirus: Host-Pathogen Interaction

Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase

Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells

Translational control in murine hepatitis virus infection

Coronavirus nonstructural protein 1: Common and distinct functions in the regulation of host and viral gene expression

A two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1 protein

Severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

Nonstructural Protein 1 of SARS-CoV-2 Is a Potent Pathogenicity Factor Redirecting Host Protein Synthesis Machinery toward Viral RNA

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

7a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen-activated protein kinase

Coronavirus spike protein inhibits host cell translation by interaction with eIF3f

The nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha

Perk is essential for translational regulation and cell survival during the unfolded protein response

Porcine Reproductive and Respiratory Syndrome Virus Infection Induces Stress Granule Formation Depending on Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) in MARC-145 Cells

Downregulation of protein kinase PKR activation by porcine reproductive and respiratory syndrome virus at its early stage infection

Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

Porcine Reproductive and Respiratory Syndrome Virus Infection Induces both eIF2α Phosphorylation-Dependent and -Independent Host Translation Shutoff

Severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase R but is resistant to its antiviral activity

SARS-CoV-2 nucleocapsid protein impairs stress granule formation to promote viral replication

The PERK Arm of the Unfolded Protein Response Negatively Regulates Transmissible Gastroenteritis Virus Replication by Suppressing Protein Translation and Promoting Type I Interferon Production

Coronavirus infection, ER stress, apoptosis and innate immunity

Coronavirus-induced ER stress response and its involvement in regulation of coronavirus-host interactions

Modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein

The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligandindependent downregulation of the type 1 interferon receptor

A SARS-CoV protein, ORF-6, induces caspase-3 mediated, ER stress and JNK-dependent apoptosis

The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6

Role of Herpes Simplex Virus 1 γ34.5 in the Regulation of IRF3 Signaling

Control of TANK-binding Kinase 1-mediated Signaling by the γ134.5 Protein of Herpes Simplex Virus 1

Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

SARS-CoV-2 targets MAVS for immune evasion

Targeting liquid-liquid phase separation of SARS-CoV-2 nucleocapsid protein promotes innate antiviral immunity by elevating MAVS activity

SARS-CoV-2 Membrane Protein Inhibits Type I Interferon Production Through Ubiquitin-Mediated Degradation of TBK1

The role of host eIF2α in viral infection

The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes

The ICP0 Protein of Herpes Simplex Virus

Downregulates Major Autophagy Adaptor Proteins Sequestosome 1 and Optineurin during the Early Stages of HSV-1 Infection

Diverse Populations of Extracellular Vesicles with Opposite Functions during Herpes Simplex Virus 1 Infection

Circadian CLOCK histone acetyl transferase localizes at ND10 nuclear bodies and enables herpes simplex virus gene expression

Rapid detection of SARS-CoV-2, replicating or non-replicating, using RT-PCR

The cells were harvested at 24 and 48 h post-transfection and equal amounts of proteins were analyzed for LC3 lipidation 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 Δγ 1 34.5 virus (5 PFU/cell). The cells were harvested at 48 h posttransfection or at 14 h post-infection. Equal amounts of proteins were analyzed for p-eIF-2α. D: Caco-2 cells were

The plasmid expressing γ 1 34.5 -Flag was a gift from Dr. He Bin (University of Illinois, Chicago).