The Envelope Protein of SARS-CoV-2 Inhibits Viral Protein Synthesis and Infectivity of Human Immunodeficiency Virus type 1 (HIV-1)

  1. Wyatt Henke
  2. Hope Waisner
  3. Sachith Polpitiya Arachchige
  4. Maria Kalamvoki
  5. Edward Stephens

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Abstract

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.

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  1. ScreenIT

    SciScore for 10.1101/2022.03.23.485576: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Expression of different coronavirus E proteins, the SARS-CoV-2 S, and SARS-CoV S protein were confirmed by transfection with the Turbofect transfection reagent (ThermoFisher) followed by radiolabeling and immunoprecipitation analysis using a mouse monoclonal antibody directed against the HA-tag (Thermo-Fisher, #26183) or an antibody against SARS-CoV Spike protein (Sino Biologicals, #40590-T62).
    antibody against SARS-CoV Spike protein
    suggested: None
    The cultures were then incubated at 4C overnight with an and a rabbit monoclonal antibody against either Golgin-97 (Abcam, #ab84340) or Calnexin (Cell Signaling Technology, #2679).
    Calnexin (Cell Signaling Technology, #2679
    suggested: (Cell Signaling Technology Cat# 2679, RRID:AB_2228381)
    The cells were washed in PBS, incubated with a secondary goat anti-rabbit antibody conjugated to AlexaFluor™-488 (Invitrogen, A11008) for 1 h at room temperature, washed, and reacted with rabbit anti-mouse conjugated to AlexaFluor™- 594 (Invitrogen, A11062) for 1 h.
    anti-rabbit
    suggested: (Thermo Fisher Scientific Cat# A-11008, RRID:AB_143165)
    anti-mouse
    suggested: (Innovative Research Cat# A11062, RRID:AB_1500656)
    Expression of E was detected using an HA-tag antibody (Invitrogen).
    HA-tag
    suggested: None
    Aliquots of cells from the same co-transfections were also analyzed for protein expression by immunoblots using antibodies directed against the HA-tag (E proteins, SARS-CoV-2 S protein, and Vpu), the SARS-Cov-1 S protein, or against GFP to monitor SARS-CoV-2 N-GFP expression.
    SARS-CoV-2 S protein,
    suggested: None
    GFP
    suggested: None
    The lysates were transferred to new tubes and BST-2 (using an anti-BST-2 antibody), the E proteins (all having a C-terminal HA-tag), and SARS-CoV and SARS-CoV-2 S proteins, and N-GFP proteins, or GAPDH (to equalize loading) immunoprecipitated using appropriate antibodies.
    anti-BST-2
    suggested: None
    GAPDH
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Immunofluorescence studies: To examine the intracellular localization of the SARS-CoV-2 E protein, COS-7 cells grown on 13 mm cover slips were transfected with either the empty pcDNA3.1(+) vector or one expressing the SARS-CoV-2 E protein using Turbofect transfection reagent (ThermoFisher).
    COS-7
    suggested: None
    At 48 h post-transfection (pt), the culture medium was collected, clarified by low-speed centrifugation, and the supernatants analyzed for infectious virus by titration on TZM-bl cells (29, 30, 91–95).
    TZM-bl
    suggested: RRID:CVCL_B478)
    Levels of infectious virus were determined by preparing a series of 10-fold dilutions of the culture supernatant followed by inoculation of Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    At 24 h, equal levels of HIV-1ΔEnv-/VSV-G pseudotyped virus (M.O.I. of 0.1) were used to infect HEK293 cells for 48h.
    HEK293
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids with the entire HIV-1 NL4-3 genome (pNL4-3) and pNL4-3Δvpu were obtained from the NIH AIDS Reagent Branch.
    pNL4-3Δvpu
    suggested: None
    HEK293 cells were co-transfected with empty pcDNA3.1(+) or the vector expressing E proteins and pNL4-3.
    pNL4-3
    suggested: None
    HEK293 cells were transfected with either the empty pcDNA3.1(+) vector or one expressing SARS-CoV-2 E protein.
    pcDNA3.1(+)
    suggested: RRID:Addgene_129020)
    Analysis of the phosphorylation of eIF-2α: To determine if the expression of the E protein of SARS-CoV-2 resulted in phosphorylation of eIF2α, HEK293 cells seeded in 6-well plates were either left untransfected or transfected with 1 μg of pUC19 or of a SARS-CoV-2 E-expressing plasmid.
    pUC19
    suggested: RRID:Addgene_50005)
    SARS-CoV-2 E-expressing
    suggested: None
    HEK293 cells were co-transfected with either empty pcDNA3.1(+) vector and pcDNA3.1(+) expressing the human BST-2 protein, or pcDNA3.1(+) expressing each of the proteins described above and BST-2.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    Software and Algorithms
    SentencesResources
    Plasmids expressing the SARS-CoV and SARS-CoV-2 S proteins were purchased from Sino Biologicals (catalog # VG40150-G-N; VG40589-CY).
    Sino Biologicals
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  2. Version published to 10.1101/2022.03.23.485576 on bioRxiv