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, which is identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified 10 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, and 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 the 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.
Highlights
There have been more than 263 million individuals infected by SARS-CoV-2, including more than 5.2 million deaths.1 While significant constant advances are made in understanding this virus, the knowledge of the SARS-CoV-2 protein interactions with host cell proteins is still limited.Targeting multiple cellular PDZ (PSD-95/Dlg/ZO-1)containing proteins through short linear PDZ-binding motifs (PBMs) is a common strategy used by viruses to facilitate their viral replication and dissemination to new hosts (Javier and Rice, 2011)
To investigate whether the five PDZ domains can interact with the full-length E protein and in a PBM-dependent manner, glutathione S-transferase (GST) pull-down assays were performed with the purified GST-tagged PDZ domains of ZO-1, LNX2, PARD3, MLLT4, and MPP5 and the lysates of HEK293 cells overexpressing constructs of GFP alone, GFPtagged wild-type full-length E protein (GFP-E-WT), GFP-tagged full-length E protein with the PBM mutated with glycines (GFP-E-GGGG), and GFP-tagged cytoplasmic tail of protein E designated 1, 2, 3, and 4 in Figure 2 (Figure 2A)
From this previous study and others (Teoh et al, 2010; Toto et al, 2020), we selected five human PDZ-containing proteins expressed in SARS-CoV-2 target cells showing significant interactions with the SARS-CoV2 protein E PBM and involved in cellular junctions and cell polarity: ZO-1/TJP1, PARD3, MLLT4, LNX2, and MPP5/PALS1
Summary
There have been more than 263 million individuals infected by SARS-CoV-2, including more than 5.2 million deaths. While significant constant advances are made in understanding this virus, the knowledge of the SARS-CoV-2 protein interactions with host cell proteins is still limited.Targeting multiple cellular PDZ (PSD-95/Dlg/ZO-1)containing proteins through short linear PDZ-binding motifs (PBMs) is a common strategy used by viruses to facilitate their viral replication and dissemination to new hosts (Javier and Rice, 2011). While significant constant advances are made in understanding this virus, the knowledge of the SARS-CoV-2 protein interactions with host cell proteins is still limited. PDZ proteins are involved in processes of particular interest in viral infection: cell junction formation, cell polarity establishment, and immune system signaling (Javier and Rice, 2011; James and Roberts, 2016; Gutiérrez-González and Santos-Mendoza, 2019). The two SARS-CoV-2 viroporins, proteins E and 3a, contain a C-terminal PBM targeting specific PDZ domain-containing proteins (Castaño-Rodriguez et al, 2018; Caillet-Saguy et al, 2021). PBMs are mainly located at the C-terminus of target proteins and interact directly with PDZ domains. They are classified into three types: type I PBM (-X-S/T-X-φCOOH), type II PBM (-X-φ-XφCOOH), and type III PBM (-X-D/E-X-φCOOH), with φ signifying a hydrophobic residue. The E protein is highly conserved with 94.7% identity between SARS-CoV and SARS-CoV-2, and their PBMs are strictly conserved
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