Fig 1: SSHHP sequences in SARS-CoV-2. (A) Three PLpro cleavage sites in the viral polyprotein are recognized by the SARS-CoV-2 PLpro. (B) The CoV PLpro enzymes also have deubquitinase and de-ISGylating activity. The SARS-CoV PLpro (white ribbon, PDB entry 5E6J(111)) bound to a diubiquitin activity-based probe (dark blue) is overlaid with the SARS-CoV-2 PLpro C111S variant (white ribbon, PDB entry 6YVA) bound to murine ISG-15 (cyan). In the inset is the structure of two molecules of the PLpro, showing the overlay of the Ub and ISG-15 proteins along the protease. The active-site residues of SARS-CoV-2 are labeled in black. (C) Sequences similar to the viral protease cleavage site sequences can be found in other host proteins, including ISG-15, Ub, MYH6, MYH7, FOXP3, PROS1, and ErbB4(HER4). (D) The host proteins containing the cleavage site sequences have loss-of-function phenotypes that are similar to the observed symptoms and causes of death of COVID-19, i.e., the virus-induced phenotype.
Fig 2: Analysis of the COVID-19 SARS-CoV-2 SSHHP sequences. (A) Hits from three PHI-BLAST searches. The shades of blue correspond to the distribution in (E). (B) Partial list of predicted hits sorted by their bit scores, alignment lengths, and % positives. The cleavage sequences tested in vitro are highlighted in green. (C) Three-dimensional graph showing the relationships between alignment length, % positives, and bit score. The histogram shows the counts of the PHI-BLAST hit results compiled from the cleavage sites. The most populated point was 80% positives over five amino acids. The hits at this point have low bit scores. (D) Summary of the in vitro cleavage assays. From left to right: CFP–PROS1–YFP immunoblot, CFP–PROS1–YFP, CFP–MYH6/7–YFP, CFP–FOXP3–YFP, and CFP–ErbB4–YFP SDS-PAGE gels. All of the substrates showed cleavage, and the preferred substrate was MYH6/7. (E) Distribution of the proteins in the graph in (A), colored in shades of blue to show where they can be found in the graph in (A).
Fig 3: Multispecies sequence alignments of predicted and in vitro-validated cleavage site sequences. Residues that match a predicted cleavage site sequence in consecutive order are shown in red, and residues that are tolerated at the subsite but are present in other cleavage sequences are shown in green. The human PROS1, MYH6/MYH7, FOXP3 and ErbB4(HER4), sequences were inserted between CFP and YFP and tested in vitro. Deubiquitinase and deISGylase activity was shown by Freitas et al.(22)
Fig 4: In vitro cleavage of myosin-6/7 (MYH6, MYH7) sequences by the SARS-CoV-2 PLpro and MERS PLpro. (A) Two CFP–MYH6/7–YFP substrates were tested; one containing 16-amino acids and a second containing 20-amino acids. The cleavage products are boxed in red. (B) Bovine heart lysates made from tissue taken from the bottom of the left ventricle were treated with the purified SARS-CoV-2 PLpro, MERS PLpro, or MERS C112A PLpro in vitro. The arrow points to the new band that appeared in immunoblots using an anti-MYH6 antibody (ab207926). MYH7 immunoblots can be found in the Supporting Information. (C) The LKGG cleavage site sequence in MYH6 and MYH7 corresponds to a helix-breaking sequence called a skip. Regions of myosin containing Skip 3 (PDB entry 4XA4) and Skip 4 (PDB entry 4XA6) are shown. The skip residues are shown in blue. (D) Mass spectra confirming the cleavage site in the CFP–MYH6/7–YFP substrate. (E) The LKGG or LRGG sequence can be found in the three cardiac myosins as well as the six skeletal myosins.
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