Host PDZ-containing proteins targeted by SARS-CoV-2
- PMID: 33864728
- PMCID: PMC8250131
- DOI: 10.1111/febs.15881
Abstract
Small linear motifs targeting protein interacting domains called PSD-95/Dlg/ZO-1 (PDZ) have been identified at the C terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A, and N showing significant interactions with dissociation constants values ranging from 3 to 82 μm. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Among the binders of the SARS-CoV-2 proteins E, 3a, or N, seven significantly affect viral replication under knock down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.
Keywords: 3A and N; PDZ-binding motif; PDZ-containing protein; SARS-CoV-2; human PDZ library; viral proteins E; viral replication.
© 2021 Federation of European Biochemical Societies.
PSD-95/Dlg/ZO-1 (PDZ)-binding motifs (PBMs) are SLiMs that interact with
a large family of protein interaction domains called PDZ. PBMs are
usually located at the extreme carboxyl terminus of proteins [[8]].
The PDZ-PBM interactome is one of the most prominent instances of a
SLiM-mediated protein interaction network serving key cell signaling
purposes [[9]]. PBMs were identified in diverse proteins of many viruses responsible for acute to chronic infections [[10, 11]].
Cellular PDZ-viral PBM interactions were shown to be directly involved
in viral pathogenicity of SARS-CoV, as well as of rabies virus and in
the oncogenicity of human papillomavirus (HPV) [[10]].
During infection, the viral proteins compete with the endogenous
ligands through the binding to the PDZ domain of the host protein
targets allowing a cellular relocalization, sequestration, or
degradation of protein [[12]] but can also affect the catalytic activity of signaling proteins [[13]].
Functional perturbations of cellular processes due to interactions
between viral PBMs and cellular PDZ-containing proteins can facilitate
the virus life cycle in the host and the dissemination to new hosts.
Thus, targeting the PBM-PDZ interface could potentially lead to novel
antiviral therapies.
ttps://pubmed.ncbi.nlm.nih.gov/33864728/
Sitaatti:
The four major structural proteins encoded by the coronavirus genome, the spike (S) protein, the nucleocapsid (N) protein, the membrane (M) protein, and the envelope (E) protein, participate in different aspects of the replication cycle and ultimately form a structurally complete viral particle. While a minor fraction is incorporated into the virion envelope, the integral membrane E protein (~ 8–12 kDa) of CoVs including SARS-CoVs is a multifunctional protein that forms ion channels with its transmembrane (TM) domain. This channel activity is mediated by formation of pentameric oligomers and is only mildly selective for cations [[14]]. The channel would be active in the secretory pathway, altering luminal environments, rearranging secretory organelles, and leading to efficient trafficking of virions [[15]]. In addition, E protein interacts with host proteins, related to its extramembrane domain particularly the C-terminal domain.
Sitaatti:
The C-terminal sequence in extra membrane position is predicted to be partially folded (helices and β-coil-β motif) which could affect the global conformation of the pentameric E channel suggesting that the channel activity of E and its protein interactions most likely interplay [[14, 16]]. In addition, the E protein encodes a putative PBM sequence at its extreme C terminus (Fig. 1A) that could also interfere through protein–protein interactions with the oligomeric organization of E protein and consequently its channel activity. The PBM sequence of E protein has been identified as a virulence factor for SARS-CoV virus, influencing replication level, virus dissemination, and pathogenicity of SARS-CoV in animal models [[17]]. To date, E protein of SARS-CoV has only been reported to interact with five host proteins: Bcl-xL, PALS1/MPP5, syntenin, sodium/potassium ATPase a1 subunit, and stomatin [[16]]. The relevance of these interactions is not yet fully understood. Among these five partners of SARS-CoV, PDZ domains of the PALS1/MPP5 tight junction protein and of Syntenin interact with the C-terminal PBM of E protein. PBM-PDZ interactions strongly impact the structure of mammalian epithelial cells and could contribute to the abrupt deterioration of the lung epithelium observed in patients infected by SARS-CoV [[18]]. Therefore, to better understand the influence of host PDZs and viral PBM motifs in the pathogenicity of SARS-CoV-2, we performed a targeted screen to identify cellular PDZ domains capable of interacting with PBM motifs CoV proteins.
PSD-95/Dlg/ZO-1 (PDZ)-binding motifs (PBMs)
Sitate
We established the list of PDZ-containing partners potentially targeted by SARS-CoV-2 E protein through its PBM. This work identified 10 PDZ-containing proteins and determined the affinities of their PDZ to SARS-CoV-2 E protein PBM. Most of them are involved in cellular junction dynamics (PARD3, TJP1/ZO-1, LNX2, MLLT4, NHERF1). To further understand the potential implication of the PDZ-PBM in pathogenicity, we have also determined the PDZ-containing proteins recognized by the E protein of SARS-CoV and MERS-CoV viruses, as well as the PDZ binders of the SARS-CoV-2 3a and N proteins that also encode for a C-terminal PBM sequence. From this PDZ list, we have selected 20 PDZ-containing partners of SARS-CoV-2 to assess their influence on SARS-CoV-2 replication in infected cells and found that the expression of seven of them is relevant for the efficiency of viral replication.
| A | B | C | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| SARS-CoV E | BI | St | K d | SARS-CoV-2 E | BI | St | K d | MERS E | BI | St | K d |
| TJP1_2 | 0.72 | 0.01 | 7.0 | TJP1_2 | 0.87 | 0.00 | 2.6 | HTRA3 | 0.81 | 0.15 | 4.2 |
| PTPN13_4 | 0.44 | 0.08 | 24 | NHERF1_2 | 0.51 | 0.07 | 18 | LAP2 | 0.72 | 0.07 | 6.9 |
| HTRA1 | 0.40 | 0.06 | 28 | PTPN13_4 | 0.44 | 0.06 | 24 | PDZD2_2 | 0.67 | 0.08 | 8.9 |
| MLLT4 | 0.28 | 0.03 | 51 | HTRA1 | 0.32 | 0.10 | 41 | MAGI2_6 | 0.52 | 0.01 | 17 |
| PARD3_3 | 0.26 | 0.04 | 55 | MAST2 | 0.27 | 0.10 | 54 | GORASP2 | 0.51 | 0.04 | 18 |
| PDZD2_6 | 0.26 | 0.06 | 57 | LNX2_2 | 0.25 | 0.04 | 61 | HTRA1 | 0.51 | 0.03 | 18 |
| LNX2_2 | 0.25 | 0.02 | 59 | PARD3_3 | 0.24 | 0.09 | 63 | RGS3 | 0.46 | 0.12 | 22 |
| SNX27 | 0.23 | 0.10 | 67 | MAGI1_6 | 0.45 | 0.03 | 23 | ||||
| RADIL | 0.21 | 0.03 | 75 | TJP3_3 | 0.44 | 0.10 | 24 | ||||
| MLLT4 | 0.20 | 0.02 | 82 | SNX27 | 0.44 | 0.03 | 25 | ||||
| APBA1_2 | 0.43 | 0.01 | 26 | ||||||||
| GORASP1 | 0.33 | 0.00 | 40 | ||||||||
| RIMS2 | 0.31 | 0.06 | 43 | ||||||||
| RIMS1 | 0.31 | 0.06 | 43 | ||||||||
| FRMPD4 | 0.31 | 0.00 | 43 | ||||||||
| PTPN13_2 | 0.30 | 0.04 | 46 | ||||||||
| TJP1_2 | 0.29 | 0.07 | 48 | ||||||||
| PARD3_3 | 0.28 | 0.08 | 50 | ||||||||
| InaDl_9 | 0.25 | 0.02 | 61 | ||||||||
| ARHGAP21 | 0.24 | 0.06 | 63 | ||||||||
| LNX2_1 | 0.24 | 0.06 | 64 | ||||||||
| GIPC2 | 0.24 | 0.01 | 65 | ||||||||
| TJP3_1 | 0.23 | 0.00 | 68 | ||||||||
| SIPA1L2 | 0.22 | 0.22 | 69 | ||||||||
| LRRC7 | 0.22 | 0.03 | 70 | ||||||||
| SYNPO2L | 0.22 | 0.01 | 72 | ||||||||
| LNX2_2 | 0.21 | 0.01 | 77 | ||||||||
| SDCBP_1 | 0.20 | 0.06 | 80 | ||||||||
| PTPN13_4 | 0.20 | 0,01 | 82 | ||||||||
The C-terminal motifs of SARS-CoV-2 N and 3a proteins also bind to cellular PDZ-containing proteins
Apart from the E protein, following a systematic search of the sequences of the 29 proteins found in SARS-CoV-2, the 3a protein and the nucleocapsid protein N were also found to contain PBMs. The PBM sequences of E and 3a proteins (Fig. 3A), two viroporins, belong to the same class of PBM, the class II (Fig. 1C). While the PBM of the E protein is involved in SARS-CoV virulence and replication [[17]], the motif of the protein 3a has no significant impact on viral growth [[24]]. However, it has been shown that this accessory 3a protein might exhibit compensatory function in the absence of E, on viral replication in vitro and in vivo. This homology suggests that the E and 3a proteins could interact with common host PDZ-containing proteins.
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