Fluorescent labeling of SARS-CoV2 ORF products

From supplementary information of recent JACS paper

Figure 1 – Labeling of SARS-CoV-2 derived microproteins in fixed cells. 

COS-7PylRS-AF cells were transfected with the indicated constructs and incubated for 24 h with 50 µM TCO*K. Cells were subsequently fixed, permeabilized and labeled with met-tet-BDP-FL and analyzed by confocal microscopy. SARS-CoV-2 M (Membrane) protein was used as a control because of its known localization to the Golgi (Klumperman et al., 1994). ORF7b is a type I (luminal N-terminus) transmembrane protein implicated as virulence factor, generated by leaky ribosome scanning in an alternative frame within the main ORF7a (Pfefferle et al., 2009). The homologous microprotein in SARS1 is suggested to localize to the Golgi (Schaecher et al., 2008). Our observation is a wider distribution in Golgi, ER and plasma membrane. ORF9b is an alternative ORF within ORF9a and the SARS1 homolog is reported to localize to mitochondria (Shi et al., 2014). We also observe a distinct speckled pattern in the cytosol compatible with partial mitochondrial localization. ORF9c expression and function is currently unknown but we observe a distinct localization with cellular membranes. ORF3b of SARS-CoV-2 is truncated to a 22 amino acid fragment due to a premature stop codon as compared to the SARS1 homolog, but has been reported to potently suppress host cell antiviral interferon response (Konno et al., 2020). The truncation preserves a single predicted transmembrane domain and our imaging suggests an association with cellular membranes.

Movie 1: Widefield, live cell imaging of SARS-CoV-2-ORF6N-TCO*K. 24 h after transfection and TCO*K incubation, COS-7PylRS-AF cells were exposed for 30 min to 500 nM me-tet-BDP-FL. Right after removal of the dye, cells were imaged over a short period of time. In the movie, it is possible to see ORF6-containing vesicles travelling through the cytoplasm. Timestamps (min:sec) are displayed in the top-right corner of each frame. 

RESOURCES

IDNameReference/Repository
E407pAS1_4xh7SKPylT_EF1_PylRS AF_IRES_Puro Addgene 140023
K122.5pAS1_4x7SKPylT_EF1_Ub-*CoV2_MAddgene 162815
K124.1pAS1_4x7SKPylT_EF1_Ub-*CoV2_9bAddgene 162816
K125.2pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF6Addgene 162817
K126.1pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF7bAddgene 162818
K127.2pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF10Addgene 162819
K128.2pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF9c/14Addgene 162820
K132.2pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF3b_altAddgene 162821
K134.10pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF3b(CoV1h)Addgene 162822
Table 1 – Plasmids are available on request and via Addgene:

METHODS

COS-7 cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM), containing high glucose, GlutaMAXTM and pyruvate (Gibco). Medium was supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich). All cell lines were cultured in an ambient-controlled incubator at 37°C, 5% O2 and 5% CO2. Cells were forward transfected using Lipofectamine LTXTM with PLUSTM reagent (Invitrogen) according to manufacturer’s protocol. Axial trans-cyclooct-2-ene-l-lysine (TCO*K) was added at the time of transfection as indicated and cells were harvested after 24 h. TCO*K (SiChem, SC-8008) stock solution was prepared at 100 mM in 0.2 M NaOH/H2O, 15% DMSO. 6-Methyl-Tetrazine-BODIPY®-FL (me-tet-BDP-FL, Jena Bioscience) stocks were prepared in DMF and further diluted in either RIPA buffer (lysate labeling), TBS-T (fixed cells labeling) or the appropriate growth medium (live cell labeling). 

Immunofluorescence and live cell imaging

For immunofluorescence, cells were grown and transfected in 96-well µ-Plates (ibidi). After withdrawing the ncAA for 4 h, cells were fixed in 4% formaldehyde for 10 min at room temperature and permeabilized for 15 min with 0.1% (v/v) triton/PBS. Prior to incubation with the appropriate antibodies, cells were click-labeled with 500 nM 6-Methyl-tetrazine-BODIPY-FL (Jena Bioscience), washed 3 times with PBS and then blocked for 1 hour in 2% BSA in TBS supplemented with 0.1% Tween-20 (TBS-T). Cells were incubated with the HA-probe antibody (F-7, Santa Cruz Biotechnology) overnight at 4°C. After washing with TBS-T, cells were stained with Alexa555-conjugated secondary antibodies (Life Technologies) for 60 min at room temperature and counterstained with 1 mg/ml DAPI (Sigma-Aldrich). After washing, cells were imaged on a Zeiss LSM780 confocal laser scanning microscope using a 40x/1.3 oil objective.

For live cell imaging, cells were grown and transfected in 96-well imaging plates (BD Falcon). After withdrawing ncAA for 1 hour, cells were incubated 30 minutes at 37°C in presence of 500 nM me-tet-BDP-FL. Where stated, cells were co-stained with either 4 µM Hoechst, 10 mM ER-trackerTM Red or 250 nM MitoTrackerTM Orange CMTMRos (Invitrogen). After washing 2 times with PBS, cells were immediately imaged in Live Cell imaging Solution (Molecular Probes), on a Nikon eclipse Ti2 inverted widefield microscope equipped with a heated imaging chamber. Images were acquired using a 20×/0.75 air objective or a 40x/1.15 water objective. For the long-term imaging experiment shown in Supplementary Figure 3, cells were maintained in Leibovitz’s L-15 Medium (Gibco) and imaged using a 10x/0.45 air objective.

Bibliography

Klumperman, J., Locker, J.K., Meijer, A., Horzinek, M.C., Geuze, H.J., and Rottier, P.J. (1994). Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J. Virol. 68, 6523–6534.

Konno, Y., Kimura, I., Uriu, K., Fukushi, M., Irie, T., Koyanagi, Y., Sauter, D., Gifford, R.J., USFQ-COVID19 Consortium, Nakagawa, S., et al. (2020). SARS-CoV-2 ORF3b Is a Potent Interferon Antagonist Whose Activity Is Increased by a Naturally Occurring Elongation Variant. Cell Rep. 32, 108185.

Pfefferle, S., Krähling, V., Ditt, V., Grywna, K., Mühlberger, E., and Drosten, C. (2009). Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo. Virol. J. 6, 131.

Schaecher, S.R., Diamond, M.S., and Pekosz, A. (2008). The transmembrane domain of the severe acute respiratory syndrome coronavirus ORF7b protein is necessary and sufficient for its retention in the Golgi complex. J. Virol. 82, 9477–9491.

Shi, C.-S., Qi, H.-Y., Boularan, C., Huang, N.-N., Abu-Asab, M., Shelhamer, J.H., and Kehrl, J.H. (2014). SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome. J. Immunol. 193, 3080–3089.

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