Interactions of SARS-CoV-2 protein E with cell junctions and polarity PDZ-containing proteins
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Abstract
The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified ten human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, LNX2) and MPP5/Pals1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2 and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo , sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4 and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.
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SciScore for 10.1101/2021.12.04.471219: (What is this?)
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Table 1: Rigor
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Antibodies Sentences Resources Soluble detergent extracts were incubated with Glutathione resins for 2 hr at 4°C prior to washing three times with PBS supplemented with NaCl 200 mM and 0.1% Triton and processed for western blot analysis with GFP antibody (Novus NB600-313). GFPsuggested: (Novus Cat# NB600-313, RRID:AB_10002194)Experimental Models: Cell Lines Sentences Resources HEK 293 cells were transiently transfected with the GFP tagged constructs using the phosphate calcium method. HEK 293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)4.6 Immunofluorescence: HeLa cells were transfected using the Genejuice transfection reagent … SciScore for 10.1101/2021.12.04.471219: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Soluble detergent extracts were incubated with Glutathione resins for 2 hr at 4°C prior to washing three times with PBS supplemented with NaCl 200 mM and 0.1% Triton and processed for western blot analysis with GFP antibody (Novus NB600-313). GFPsuggested: (Novus Cat# NB600-313, RRID:AB_10002194)Experimental Models: Cell Lines Sentences Resources HEK 293 cells were transiently transfected with the GFP tagged constructs using the phosphate calcium method. HEK 293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)4.6 Immunofluorescence: HeLa cells were transfected using the Genejuice transfection reagent (Novagen) according to the manufacturer protocol. HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)Recombinant DNA Sentences Resources The PDZ domains were cloned into pETG-41A plasmid vectors as an N-terminal fusion to a histidine tag and a maltose-binding protein (His-MBP-tag). pETG-41Asuggested: NoneInvitrogen), PDZ domains of ZO-1, LNX2 and PARD3 were subcloned into pDEST™17 vector and MPP5-PDZ into pDEST™15 vector, allowing the production of recombinant proteins as a fusion to an N-terminal histidine and GST tag, respectively. pDEST™17suggested: NonepDEST™15suggested: NoneThe pCMV ALFA E vector was constructed as follows: DNA sequence encoding the ALFA tag (MSRLEEELRRRLTE) followed by a linker (GGGGS) fused to the sequence corresponding to the alpha-variant of SARS-CoV2 E cDNA (GenBank: BCM16077.1) synthetized by Eurofins. pCMV ALFAsuggested: NoneThe ALFA-linker-E sequence was subsequently cloned into a pCMV backbone vector using ClaI and XhoI. pCMVsuggested: RRID:Addgene_16459)The sequences corresponding to the PDZ domains were cloned into the pCMV GFP vector using EcoRI and XhoI sites. pCMV GFPsuggested: NoneSoftware and Algorithms Sentences Resources Models were rebuilt using COOT (Emsley et al., 2010), and refinement was done with phenix. COOTsuggested: (Coot, RRID:SCR_014222)refine of the PHENIX suite (Adams et al., 2010). PHENIXsuggested: (Phenix, RRID:SCR_014224)All structural figures were generated with the PyMOL Molecular Graphics System, Version (Schrödinger). PyMOLsuggested: (PyMOL, RRID:SCR_000305)Results from OddPub: Thank you for sharing your data.
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