key: cord-0840206-7r9clf0u
authors: Henke, Wyatt; Waisner, Hope; Arachchige, Sachith Polpitiya; Kalamvoki, Maria; Stephens, Edward
title: The Envelope Protein of SARS-CoV-2 Inhibits Viral Protein Synthesis and Infectivity of Human Immunodeficiency Virus type 1 (HIV-1)
date: 2022-03-27
journal: bioRxiv
DOI: 10.1101/2022.03.23.485576
sha: 248f673ce300226534825904ebca66e49045ff2e
doc_id: 840206
cord_uid: 7r9clf0u

The human coronavirus SARS-CoV-2 encodes for a small 75 amino acid transmembrane protein known as the envelope (E) protein. The E protein forms an ion channel, like the viroporins from human immunodeficiency virus type 1 (HIV-1) (Vpu) and influenza A virus (M2). Here, we analyzed HIV-1 virus infectivity in the presence of four different β-coronavirus E proteins. We observed that the SARS-CoV-2 and SARS-CoV E proteins reduced HIV-1 yields by approximately 100-fold while MERS-CoV or HCoV-OC43 E proteins restricted HIV-1 infectivity to a lesser extent. This was also reflected in the levels of HIV-1 protein synthesis in cells. Mechanistically, we show that that the E protein neither affected reverse transcription nor genome integration. However, SARS-CoV-2 E protein activated the ER-stress pathway associated with the phosphorylation of eIF-2α, which is known to attenuate protein synthesis in cells. Finally, we show that these four E proteins and the SARS-CoV-2 N protein did not significantly down-regulate bone marrow stromal cell antigen 2 (BST-2) while the spike (S) proteins of SARS-CoV and SARS-CoV-2, and HIV-1 Vpu efficiently down-regulated cell surface BST-2 expression. The results of this study show for the first time that viroporins from a heterologous virus can suppress HIV-1 infection. IMPORTANCE The E protein of coronaviruses is a viroporin that is required for efficient release of infectious virus and for viral pathogenicity. We determined if the E protein from four β-coronaviruses could restrict virus particle infectivity of HIV-1 infection. Our results indicate that the E proteins from SARS-CoV-2 and SARS-CoV potently restricted HIV-1 while those from MERS-CoV and HCoV-OC43 were less restrictive. Substitution of the highly conserved proline in the cytoplasmic domain of SARS-CoV-2 E abrogated the restriction on HIV-1 infection. Mechanistically, the SARS-CoV-2 E protein did not interfere with viral integration or RNA synthesis but rather reduced viral protein synthesis. We show that the E protein-initiated ER stress causing phosphorylation of eIF-2α, which is known to attenuate protein synthesis. Companion studies suggest that the E protein also triggers autophagy. These results show for the first time that a viroporin from a coronavirus can restrict infection of another virus.

The E protein of coronaviruses is a viroporin that is required for efficient release of infectious virus and for viral pathogenicity. We determined if the E protein from four βcoronaviruses could restrict virus particle infectivity of HIV-1 infection. Our results indicate that the E proteins from SARS-CoV-2 and SARS-CoV potently restricted HIV-1 while those from MERS-CoV and HCoV-OC43 were less restrictive. Substitution of the highly conserved proline in the cytoplasmic domain of SARS-CoV-2 E abrogated the restriction on HIV-1 infection. Mechanistically, the SARS-CoV-2 E protein did not interfere with viral integration or RNA synthesis but rather reduced viral protein synthesis. We show that the E protein-initiated ER stress causing phosphorylation of eIF-2α, which is known to attenuate protein synthesis. Companion studies suggest that the E protein also triggers autophagy. These results show for the first time that a viroporin from a coronavirus can restrict infection of another virus.

The SARS-CoV-2 E protein did not inhibit HSV-1 infection. We next determined if the restriction of release of infectious HIV-1 was observed in other virus infections. HEK293 cells were transfected with the plasmid expressing SARS-CoV-2 E protein for 24 h followed by infection of cultures with HSV-1 (0.01 pfu/cell). Samples were collected at 3, 24, and 48 h post-infection. The results indicate that SARS-CoV-2 E protein did not interfere with infectious HSV-1 progeny virus production at 24 or 48 h post-infection (Fig. 3) .

As the E protein of SARS-CoV-2 appeared to decrease the levels HIV-1 virus released, we constructed pcDNA3.1(+) vectors that expressed the E proteins from three other βcoronaviruses that cause mild to severe pathogenicity in humans (HCoV-OC43, SARS-CoV, MERS-CoV). Like the SARS-CoV-2 E protein, examination of the intracellular expression revealed that these E proteins were primarily localized in the ER and Golgi regions of the cell with no expression at the cell surface (data not shown). We determined if these E proteins could also restrict HIV-1 particle infectivity by transfection of HEK293 cells with vectors expressing the SARS-CoV-2, SARS-CoV, MERS-CoV, or HCoV-OC43 E proteins, HSV-1 gD, or gD[ΔTMCT] and pNL4-3. At 48 h, the culture medium was collected, clarified, and the levels of infectious HIV-1 released determined using TZM.bl cell assays. The presence of gD restricted the release of infectious HIV-1 (0.04%) while the presence of gD [TMCT] did not affect levels of infectious virus released (~101%) (Fig. 4A) . The results indicate that the E proteins from SARS-CoV-2 and SARS-CoV potently restricted the release of infectious HIV-1 at 1.3% and 1.4%, respectively. However, MERS-CoV and HCoV-OC43 E proteins were less restrictive at 8 approximately 16% and 37%, respectively, of the pcDNA3.1(+) control (Fig. 4) .

Immunoprecipitation of gD and E proteins from cell lysates from the restriction assays confirmed that the gD and E proteins were expressed (Fig. 4B) . These results provide additional data on the specificity of the restriction of HIV-1. Immunoprecipitation of HIV-1 proteins from cell lysates of cells co-transfected with pcDNA3.1(+) and pNL4-3 revealed presence of the gp160 Env, gp120 cleavage product, Gag p55, and p24. Further, gp120 and p24 were immunoprecipitated from the clarified culture medium. In contrast, immunoprecipitation of HIV-1 proteins from lysates prepared from cells co-transfected with the vector expressing SARS-CoV-2 E and pNL4-3 revealed that the Env precursor gp160, the gp120, p55, and p24 were immunoprecipitated at much lower levels as were gp120 and p24 in the clarified culture medium. (Fig. 5A) . The E protein was detected in the cell lysates but not detected in culture medium (Fig. 5B) . We also determined if the E proteins from SARS-CoV, MERS-CoV and HCoV-OC43 also affected HIV-1 protein synthesis (Fig. 5C-H) . The presence of SARS-CoV E also reduced levels of viral proteins in the cell lysates and 9 culture medium compared with the pcDNA3.1(+)/pNL4-3 control (Fig. 5C-D Expression of SARS-CoV-2 E protein results in the phosphorylation of eIF-2α. As expression of the SARS-CoV-2 E protein significantly reduced HIV-1 protein expression, we determined if the E protein activated pathways associated with an unfolded protein response due to ER stress such as phosphorylation of eIF-2α, which 10 can inhibit protein synthesis. HEK293 cells were transfected with the vector expressing SARS-CoV-2 E-HA. At 48 h post-transfection (pt) cells were lysed and phosphorylated eIF-2α was analyzed by immunoblots using an antibody against phospho-eIF-2α. In cells transfected with the empty pUC19 vector, little phosphorylated eIF-2α was detected (Fig. 7) . In contrast, in cells transfected with the vector expressing E-HA phosphorylated eIF-2α was readily detected (Fig. 7) . These results indicate that expression of E-HA resulted in increased eIF-2α phosphorylation.

As the HIV-1 Vpu and the coronavirus E proteins have a similar overall structure and are both viroporins, we examined if the E proteins from different coronaviruses, like Vpu, could down-regulate bone marrow stromal antigen 2 (BST-2). Vectors expressing each of the four E proteins, SARS-CoV and SARS-CoV-2 S proteins, SARS-CoV-2 N protein, and the HIV-1 Vpu protein were co-transfected into HEK293 cells with a vector expressing human BST-2. At 48 h post-transfection, cells were immunostained with a monoclonal antibody against BST-2 tagged with Alexa 488 and BST-2 surface expression analyzed by flow cytometry. The mean and median fluorescent intensities were calculated using the FlowJo software program. Our results showed that neither the E proteins nor SARS-CoV-2 N down-regulated BST-2 cell surface expression (Fig. 8A-B) . However, the SARS-CoV and SARS-CoV-2 S proteins and the HIV-1 Vpu protein significantly down-modulated BST-2 cell surface expression (Fig. 8A-B) . Analysis of aliquots of cells from the same co-transfections revealed E proteins, S proteins, N and Vpu were all expressed well in co-transfected cells (Fig. 9C) .

We next examined the steady-state levels of BST-2 in the presence of the 11 proteins analyzed above. HEK293 cells were co-transfected with vectors as described above and radiolabeled as described in the Materials and Methods section. Cell lysates were prepared, and the BST-2 proteins and viral proteins were immunoprecipitated with appropriate antibodies as described in the Materials and Methods section. The results indicate that in the presence of SARS-CoV and SARS-CoV-2 S proteins and HIV-1 Vpu, levels of immunoprecipitated BST-2 were significantly less than from cells transfected with the vector expressing BST-2 alone or cells co-transfected with vectors expressing BST-2 and the E proteins or SARS-CoV-2 N protein ( Fig. 9) . Taken together, the E and SARS-CoV-2 N proteins had no effect on BST-2 expression, while SARS-CoV and SARS-CoV-2 S proteins, and Vpu caused the downregulation of BST-2 from the cell surface and a decrease in total amounts of BST-2.

The E protein of coronaviruses is a multi-functional protein that has several roles in the virus replication cycle. It is localized to the ER, ERGIC and Golgi compartments of the cell and associates with the M protein at the ERGIC during virus maturation (32-35).

The palmitoylation of the conserved cysteine residues of the MHV E protein is important as substitution of these cysteine residues results in an unstable protein, aggregation of the M protein, and leads to incompetent virus-like particle (VLP) formation (33, 36).

Other studies have shown that the E protein of infectious bronchitis virus (IBV) alters the secretory pathway by disrupting Golgi organization leading to the production of larger Mechanistically, we showed that neither viral integration nor RNA synthesis were impaired by the presence of SARS-CoV-2 E protein. We observed that in the presence of the SARS-CoV-2 E protein, the levels of viral protein synthesis were significantly reduced. Previously, the SARS-CoV E protein was shown to be a channel for calcium 14 ions via its viroporin activity (65) . The ER is the major store of Ca ++ within eukaryotic cells and is involved in lipid biosynthesis, detoxification of chemicals, and de novo protein synthesis and protein folding. Within the ER, molecular chaperones such as Bip/GRP78 are tasked to properly fold proteins such that they can transit to the Golgi complex. Thus, the ER provides a special environment for these chaperones for proper protein folding. Under conditions of Ca ++ depletion, the ability of calcium-dependent molecular chaperones to properly fold proteins is reduced, which leads to the accumulation of misfolded proteins, ER stress, and initiation of an unfolded protein response (UPR). The UPR is a cellular pathway that detects and attempts to alleviate ER stress (66, 67) . The UPR is initiated by the activation of three canonical ER stress sensors; the PKR-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1α (IRE1-α) (66-71). Here we show that expression of the SARS-CoV-2 E protein caused the phosphorylation of eIF-2α to attenuate general protein synthesis. Phosphorylation of eIF-2α allows the translation of ATF4 mRNA, which encodes a transcription factor controlling the transcription of genes involved in autophagy, apoptosis, amino acid metabolism and antioxidant responses. In a previous study that examined gene expression in VeroE6 and MA104 cells infected with SARS-CoV and SARS-CoVΔE, investigators showed that SARS-CoV E protein down-regulated the inositol requiring enzyme (IRE-1) signaling pathway of the unfolded protein response (UPR) pathway but not the PERK or ATF6 pathways (72) . They also found that expression of the E protein in trans reduced the stress response in cells infected with SARS-CoVΔE (72). There could be several reasons for the differences in the results of the two studies. These include the: a) cell types analyzed; b) transfection 15 of cells with vector expressing a single protein from SARS-CoV-2 versus the use of wild type SARS-CoV and SARS-CoV E-deleted viruses; c) differences in the genome structure of SARS-CoV-2 and SARS-CoV; and d) differences in the amino acid sequences of the two E proteins (4 amino acid substitutions). Thus, this is the first report of the SARS-CoV-2 E protein involved in ER stress and phosphorylation of eIF-2α (see accompanying paper). Our analysis suggested that the decreased HIV-1 protein synthesis in cells expressing the E protein was correlated with the phosphorylation of eIF-2α. We show that indeed a decrease in protein synthesis occurs in E protein transfected cells (see accompanying paper).

Another possible mechanism for the observed decrease in HIV-1 protein expression may be related to autophagy. Autophagy, first discovered in yeast, is a multistep degradative process that maintains cell homeostasis by eliminating misfolded/old elements (proteins and organelles) trapped in autophagosomes targeted to fuse with lysosomes to obtain nutrients (73, 74) . Autophagy is also relevant in the innate and adaptive immunity against viral infections (75) (76) (77) (78) . HIV-1 infection has been shown to induce (macrophages) or inhibit (CD4+ T cells) autophagy (78) (79) . During the initial entry step, the HIV-1 gp120/gp41 proteins at the surface of the virus bind to CD4 receptors and co-receptor CCR5 or CXCR4, initiating autophagic response in CD4+ T cells. This autophagic process represents an anti-HIV-1 response by the host cell leading to the selective degradation of HIV-1 Tat (80) (81) (82) . In later stages of HIV-1 infection there is an increased activation of mechanistic target of rapamycin complex 1 (mTORC1) (a regulator of autophagy) which leads to an inhibition of autophagy (83) .

Additionally, HIV-1 Nef protein interacts with Beclin 1 and its inhibitor BCL2 (84). In our accompanying paper, we show that the expression of the E protein leads to increased lipidation of microtubule-associated protein 1A/1B-light chain 3 (LC3) to yield LC3-II, which is required for autophagy. This could lead to the degradation of the viral proteins and explain our observations.

We also examined the E proteins from SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-OC43 for the ability to down-regulate bone marrow stromal antigen-2 (BST-2; also known as CD317 or tetherin). This type II transmembrane protein was first discovered as a cell surface marker for differentiated and neoplastic B cell types (85) and later was shown to be induced by type I and II interferons. It is now understood that BST-2 activates NF-κB resulting in the activation of IFN-I responses and proinflammatory responses against viruses (86) . The antiviral functions of BST-2 were first demonstrated with HIV-1 that lacked the vpu gene (20, 87). Since its initial identification as an antiviral restriction factor, BST-2 has been shown to tether several enveloped viruses including coronaviruses. BST-2 was shown to inhibit the release of HCoV-229E although the protein responsible for overcoming this restriction factor was not investigated (88). Later, it was shown that the SARS-CoV S protein caused the degradation of BST-2 (89) . We showed that unlike Vpu, the E proteins neither downregulated BST-2 expression at the cell surface nor caused BST-2 degradation while the S protein of SARS-CoV and SARS-CoV-2 clearly down-regulated surface expression of BST-2 and was accompanied by a decrease in total amounts of BST-2.

In conclusion, we showed that the E protein viroporin from SARS-CoV-2 and Perkin-Elmer) for 16 h. Cell lysates were prepared, and culture medium processed as previously described (29, 30, 90) . HIV-1 proteins were immunoprecipitated using a cocktail of antibodies previously described and is referred to in the figures as "anti-HIV" antibodies (29, 30, 90) . The E proteins were immunoprecipitated using a monoclonal antibody directed against the HA-tag. Immunoprecipitates were collected by incubation with protein A-Sepharose beads overnight at 4C, the beads were washed with RIPA buffer, and the samples resuspended in sample reducing buffer. The samples were 21 boiled, proteins separated by SDS-PAGE (10% or 12.5 % gels), and proteins visualized using standard radiographic techniques.

if the E proteins interfered with viral genome integration, we assessed the level of integration by amplification Alu repeat/gag sequences using the previously described procedure (96) . HEK293 cells were transfected with either the empty pcDNA3. (both antibodies were obtained from Cell Signaling). Expression of E was detected using an HA-tag antibody (Invitrogen). β-actin was used as a loading control. At 24 h, cells were fixed, permeabilized and blocked. The cover slips were reacted with a mouse monoclonal antibody against HA-tag and with a rabbit antibody against the ER marker calnexin or trans Golgi network protein Golgin-97 overnight at 4C followed by an appropriate secondary antibody for 1 h. Cells were washed, and counter stained with DAPI (1 μg/ml) for 5 min as described in the Materials and Methods section. The cover slips were mounted and examined using a Leica TCS SPE confocal microscope using a 63x objective with a 2x digital zoom using the Leica Application Suite X LAS X, LASX) software package. A 405nm filter used to visualize the DAPI and a 594nm filter to visualize HA staining and 488 nm filter to visualize the ER or Golgi marker staining. Panels A-C. Cells transfected with the vector expressing E-HA and immunostained for the HA tag and calnexin. Panels D-F. Cells transfected with the vector expressing E-HA and immunostained for the HA tag and Golgin-97. Figure 9 . The levels of BST-2 in cells in the presence of coronavirus proteins. HEK293 cells were co-transfected with either the empty pcDNA3.1(+) vector or pcDNA3.1(+) vector expressing each of the four E proteins, SARS-CoV-2 N protein, SARS CoV-2 S protein, SARS CoV S protein, or HIV-1 Vpu and a vector expressing human BST-2 protein. At 30 h posttransfection, cells were starved for methionine/cysteine and radiolabeled with 500 μCi of 35 Smethionine/cysteine for 16 h. Cell lysates were prepared in RIPA buffer and one third of the lysate was used to immunoprecipitate BST-2 proteins using an anti-BST-2 antibody; one third of the lysate was used to immunoprecipitate E proteins and Vpu (Panel B); S proteins (Panel C) using an anti-Spike antibody; or the N protein using an anti-GFP antibody (Panel D); and one third of the lysate was used to immunoprecipitate GAPDH to normalize loading as described in the Materials and Methods section.

Pneumonia of unknown aetiology in Wuhan, China: potential for international spread via commercial air travel

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

Pillai SK; Washington State 2019-nCoV Case Investigation Team. 2020. First Case of 2019 Novel Coronavirus in the United States

China novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in China

Membrane binding proteins of coronaviruses

Supramolecular architecture of the coronavirus particle

Ion flux in the lung: virus-induced inflammasome activation

Viral proteins function as ion channels

The small hydrophobic protein of the human respiratory syncytial virus forms pentameric ion channels

Classical swine fever virus p7 protein is a viroporin involved in virulence in swine

Vpu protein: The viroporin encoded by HIV-1

Pore-forming activity of pestivirus p7 in a minimal model system supports genus-specific viroporin function

The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy

Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1

Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV-1-infected cells

High-risk human papillomavirus E5 oncoprotein displays channel-forming activity sensitive to small-molecule inhibitors

Role of severe acute respiratory syndrome coronavirus viroporins E, 3a, and 8a in replication and pathogenesis

Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis

Structure and inhibition of the SARS coronavirus envelope protein ion channel

Structural model of the SARS coronavirus E channel in LMPG micelles

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

The infectious bronchitis coronavirus envelope protein alters Golgi pH to protect the spike protein and promote the release of infectious virus

Viroporins: structure, function and potential as antiviral targets

Viroporins from RNA viruses induce caspase-dependent apoptosis

The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles

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

Targeting the unfolded protein response in disease

Mechanisms, regulation and functions of the unfolded protein response

Beyond the cell factory: Homeostatic regulation of and by the UPR(ER)

Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress

Calcium and apoptosis: ER-mitochondria Ca 2+ transfer in the control of apoptosis

The regulation of autophagy by calcium signals: Do we have a consensus?

Severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway

Viruses and the autophagy pathway

Autophagy, EVs, and infections: A perfect question for a perfect time

Studies progression on the function of autophagy in viral infection

Human immunodeficiency virus type-1 infection inhibits autophagy

Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages

Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4 + T lymphocytes

Differential role of autophagy in CD4 T cells and macrophages during X4 and R5 HIV-1 infection

PKM2-mediated inhibition of autophagy facilitates Tat's inducing HIV-1 transactivation

Hyperactivation of mammalian target of rapamycin complex 1 by HIV-1 is necessary for virion production and latent viral reactivation

HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

Molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

Stimulation of NF-κB activity by the HIV restriction factor BST2

The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein

BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface

Severe acute respiratory syndrome coronavirus spike protein counteracts BST2-mediated restriction of virus-like particle release

Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy

Quantitative analysis of human immunodeficiency virus type 1 DNA dynamics by real-time PCR: integration efficiency in stimulated and unstimulated peripheral blood mononuclear cells

Immunoprecipitation of E proteins from cell lysates (C) and culture medium (D) from HEK293 cells co

Immunoprecipitation of HIV-1 proteins from cell lysates and culture medium from HEK293 cells co-transfected pcDNA3.1(+) alone, pcDNA3.1(+)/pNL4-3, or pcDNA3.1(+) expressing the MERS-CoV E-HA/pNL4-3. Panel G-H. Immunoprecipitation of HIV-1 proteins from cell lysates and culture medium from HEK2933 cells co

Figure 6. The SARS-CoV-2 E protein did not alter integration or tat transcription

Cells were harvested at 48h postinoculation, total DNA extracted, and Alu-gag products amplified using real time DNA PCR. An additional negative control included treatment of transfected/infected cells with 20μM raltegravir for 2 h prior to inoculation of cultures with pseudotyped virus. Panel B. Cells were transfected and infected as in panel A. At 48 h post-transfection, cells were washed, pelleted, and the RNA was extracted