Table 1.
List of the different HA subtypes and subtype isolates with the corresponding peptide sequence used for the cleavage assays.
P1, P2, P3 and P4 designate the amino acid residues next to the the cleaved bond towards the N-terminus with P1 being the residue proximal to the cleavage site [40].
Fig 1.
Comparison of HA subtypes that caused pandemics.
(A) Cleavage site mimetic peptide representative of H1N1 strains was monitored for cleavage by the indicated proteases. Red letter in the peptide sequence emphasizes a different amino acid compared to the cleavage sequence of the H1 isolate A/WSN/33 (B). (C) and (D) display cleavage assays of peptide mimics representative for H2N2/H2N6 and H3N2 strains, respectively. RFU—relative fluorescence units.
Fig 2.
Cleavage profile of the H10 subtype.
(A) The peptide mimicking the cleavage motif of the H10N8 strain was incubated with the indicated proteases and cleavage was monitored by the increase of fluorescence at 390 nm. Red indicates the amino acid change that was created in the H1 peptide mimic to resemble the H10 sequence (B).
Fig 3.
Differences in cleavage pattern of two H6N1 isolates.
(A) and (B) show the cleavage site sequence of two H6N1 strains circulating in the early 21st century and more recent ones, respectively. Red letter indicates the changed amino acid at the P3 position in the cleavage site.
Fig 4.
Comparison of H5N1 LPAI and HPAI subtypes.
Peptides mimicking the LPAI sequence (A) and the HPAI sequence (B) of H5N1 were treated with the indicated proteases. (C) Furin was incubated with the H5N1 HPAI peptide mimic on the left and a designed control peptide which carries an optimal furin cleavage site on the right.
Fig 5.
Cleavage profile of H7 isolates.
Cleavage rates of three different peptides representing the HA subtypes of the H7N9 (A), H7N3 (B) and H7N2 (C) isolates were analyzed. Cleavage by the indicated proteases was monitored by the increase of fluorescence at 390 nm. Red letters in the peptide sequence highlight the distinct amino acid at the P2 position in the different isolates.
Fig 6.
A peptide mimetic representative of the H9N2 strain was incubated with the indicated proteases and cleavage was measured as an increase of fluorescence at 390 nm.
Fig 7.
Cleavage analysis of distinct HA subtypes by the Factor Xa protease.
The different peptides mimicking the cleavage motifs of the indicated HA subtypes were incubated with Factor Xa and the cleavage was monitored by the increase of fluorescence at 390 nm. H6N1 P3 E and H6N1 P3 A designate the H6N1 peptides that carry Glu and Ala at the P3 position, respectively. H7 P2 G, H7 P2 P and H7 P2 T indicate peptides that have Gly, Pro or Thr in the P2 position, respectively.