Table 2.
Comparative cleavage of peptides corresponding to polyprotein junctions in PRRSV JXA1
| Peptide | Polyprotein junction | Sequence(P–P′) |
Cleavage | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| P10 | P9 | P8 | P7 | P6 | P5 | P4 | P3 | P2 | P1 | P1′ | P2′ | P3′ | P4′ | P5′ | P6′ | P7′ | P8′ | |||
| SP1 | nsp3/nsp4 | S | Q | L | G | S | L | L | E | G | A | F | R | T | Q | K | P | + | ||
| SP2 | nsp4/nsp5 | L | A | A | K | P | E | L | E | G | G | L | S | T | V | Q | L | − | ||
| SP3 | nsp5/nsp6 | F | F | L | R | Y | F | A | E | G | K | L | R | E | G | V | S | − | ||
| SP4 | nsp6/nsp7 | Q | S | C | G | M | S | H | E | S | L | T | G | A | L | A | M | − | ||
| SP5 | nsp7/nsp8 | E | N | R | R | A | Q | W | E | A | A | K | L | S | V | E | Q | − | ||
| SP6 | nsp9/nsp10 | E | K | L | R | S | N | H | E | G | K | K | S | R | M | C | G | − | ||
| SP7 | nsp10/nsp11 | R | A | I | C | A | D | L | E | G | S | S | S | P | L | P | K | − | ||
| SP8 | nsp11/nsp12 | K | T | A | Y | F | Q | L | E | G | R | H | F | T | W | Y | Q | +++ | ||
| SP8-1 | nsp11/nsp12 | K | D | K | T | A | Y | F | Q | L | E | G | R | H | F | T | W | +++ | ||
| SP8-M | K | D | K | T | A | Y | F | Q | L | Q | G | R | H | F | T | W | +/− | |||
Synthetic 16-mer peptides that represent the eight predicted junction sequences in the replicative polyprotein were incubated with 25 μM recombinant PRRSV nsp4 as described in Materials and Methods and analyzed by HPLC for the extent of cleavage. After incubation at 37 °C for 15 h, if more than 50% of the peptide was found to be cleaved as is determined by the decrease in area of the peak representing the parental peptide, “+++” is given; if 10–20% of the parental peptide is cleaved, we perceive it as “+”; if less than 10% is cleaved but still detectable amounts of products are observed as marked by the appearance of new peaks in the HPLC profile, we mark it “+/−”. “−” means no visible product peaks in the HPLC profile could be observed at all. All the cleaved products were verified by MS and/or comparison with HPLC traces of peptide standards. SP8-1 and SP8 correspond to the same junction site in pp1b. SP8-1 was synthesized and analyzed for the purpose of separating the two product peaks from each other.