The catalytic residues previously identified for all studied coronavirus and arterivirus PLPs are a cysteine followed by a histidine. In all coronavirus PLPs the catalytic Cys is followed by an aromatic res- idue (Trp or Tyr) (49), while in all known active arterivirus PLPs a Trp is adjacent to the catalytic Cys (30). Alignment of the amino acid sequences of the three SHFV PLP catalytic domains revealed a single Cys-Trp dyad upstream of a histidine in each: Cys115/Trp116/His130 in PLPα, Cys246/Trp247/His309 in PLPβ and Cys378/Trp379/His443 in PLPγ (Fig. 5.1A). However, the predicted PLPα Cys-Trp dyad was located only 14 aa upstream from the catalytic His residue while the PLPβ and PLPγ dyads were located 62 and 64 aa residues from their respective histidines. A conserved GV[Q/T]G motif was identified ap- proximately halfway between the catalytic Cys and His residues in the putative PLPβ and PLPγ. The posi- tion of this motif in PLPα (aa position 93 to 96) was used as an anchor to identify Cys63 in PLPα as the more likely catalytic Cys for PLPα. Cys63 aligned with the predicted catalytic cysteines in PLPβ and PLPγ and was located 67 aa residues from the PLPα catalytic histidine (Fig. 5.1A). However, Cys63 is followed by alanine instead of by an aromatic residue and a catalytic Cys/Ala dyad had not previously been identified in any known PLP. A recently resolved crystal structure of the PRRSV PLPα region (41) was next used to generate a homology model of each of the SHFV PLP domains (Fig. 5.1B). In the mod- eled structure of SHFV PLPα, Cys63 was located near the catalytic His130 in a position that was spatially
similar to those predicted for the putative catalytic Cys of both SHFV PLPβ and PLPγ (Fig. 5.1B) as well as for PRRSV PLPα (data not shown). These data supported the hypothesis that the SHFV PLPα catalytic residues are Cys63 and His130.
Both coronavirus and arterivirus PLPs cleave at Tyr or Gly residues that are adjacent to a Gly res- idue (142). Analysis of the SHFV nsp1 region sequence revealed seven Gly/Gly dyads and seven Tyr/Gly dyads. Sequence alignment and structure homology modeling of the three SHFV PLP regions suggested that PLPβ and PLPγ each cleave at a downstream Gly/Gly dyad and that PLPα cleaves at a unique Gly/Thr dyad (Fig. 5.2A). However, two Gly/Gly dyads (positions 150/151 and 164/165) detected 5 and 19 aa upstream from the predicted PLPα cleavage site are also possible PLPα cleavage sites. Also, three adjacent Gly residues were present at the predicted PLPγ cleavage site. The molecular masses of the pep- tides that would be produced by cleavage at the various sites were predicted (Fig. 5.2B).
A cDNA construct that expressed a polyprotein consisting of the N-terminal region of SHFV ORF1a (nts 210 through 1933) with an N-terminal Flag tag and a C-terminal c-Myc tag was made as de- scribed in Materials and Methods. The polyprotein encoded by this construct included the complete nsp1α, nsp1β and nsp1γ sequences and 93 aa of nsp2. Wild type and mutant plasmid DNAs were used as templates in coupled in vitro transcription/translation reactions done in the presence of 35S-methionine.
The products of PLP processing of polyproteins produced were immunoprecipitated (IP) using either an anti-Flag or an anti-c-Myc antibody and separated by SDS-PAGE. The masses of the precipitated proteins were compared to the those of the predicted protein products (Fig. 5.2B). The wild type construct pro- duced 9 bands with molecular masses of approximately ~63, ~54, ~39, ~30, ~28, ~27, ~23, ~18, and ~15 kDa. The 63, 54, 39, 30, 28, 27, 23, and 18 kDa bands were pulled down by anti-FLAG antibody, while the 63, 39, 30, and 23 kDa bands were pulled down by the anti-c-Myc antibody (Fig. 5.3A). The 63 kDa band pulled down by both antibodies is the full-length polyprotein, while the 54 kDa band pulled down by only anti-FLAG antibody contains nsp1α, nsp1β and nsp1γ but not nsp2 sequence. The FLAG-tagged 39 kDa band most likely contains both nsp1α and nsp1β, while the FLAG-tagged 18 kDa band most like- ly contains only nsp1α. These data indicate that the nsp1α protein is efficiently cleaved from the polypro-
tein. The c-Myc-tagged 23 kDa band most likely contains nsp1γ and nsp2. The 15 kDa band is most likely nsp1γ because neither the anti-FLAG nor anti-c-Myc antibody pulled down this peptide (Fig. 5.2B). These data indicate that nsp1γ is also efficiently cleaved from the polyprotein and nsp2.
The predicted molecular mass for nsp1β is 22.1 kDa. Because this is an internal peptide it would not be tagged. The nsp1γ-nsp2 band was predicted to be 24.5 kDa. Comparison of the intensity of the ~23 kDa band detected in the lysate and c-Myc pulldown lanes indicated that the band intensity was greater in the lysate than in the c-Myc IP lane suggesting that the lysate band contained both nsp1β and the nsp1γ- nsp2 precursor. In an attempt to verify that the ~23 kDa band contained nsp1β, two different antibodies specific for nsp1β were used for IP of the lysate. Unfortunately, neither of these pulldowns produced de- tectable bands (data not shown).
A 30 kDa band produced by the wild type construct was present in the lysate as well as in the FLAG and the c-Myc IP lanes, but not in the IgG IP lane, suggesting that this band contained both nsp1α
and nsp2 peptides. The 30 kDa bands in each of the IP lanes had decreased intensity compared to same band in the lysate lane. It is likely that the 30 kDa lysate band contained two precursor peptides, one con- sisting of nsp1α plus part of nsp1β and a second that consisted of nsp2 and nsp1γ plus part of nsp1β. These bands were thus designated as nsp1α+tβ and tβ+nsp1γ+nsp2, respectively. The detection of these bands suggested that the catalytic region of nsp1β contains a functional cleavage site but no Gly/Gly or Tyr/Gly dyads were present in this region. However, a Gly271/Val272 dyad was present within the PLPβ
catalytic domain.
Three additional unexpected bands of 23, 27 and 28 kDa were present in both the lysate and the FLAG IP lane. Based on the presence of the FLAG tag and the molecular masses of these bands, these peptides were likely produced by cleavage at non-canonical sites located downstream of the predicted nsp1α cleavage sites but before the predicted cleavage site Gly271/Val272 in the nsp1β catalytic region. However, the intensity of each of these protein bands was low suggesting that cleavage at these sites was not efficient. These bands were designated nsp1α+ttβ due to the further truncation of nsp1β. The presence of a 39 kDa band in the c-Myc IP lane supports cleavage of at least one of these non-canonical sites be-
cause a peptide with the C-terminal region of ttβ attached to nsp1γ and nsp2 would have an approximate molecular mass of 37 kDa. These bands were thus designated as ttβ+nsp1γ+nsp2. The intensity of each of these protein bands was also low suggesting that cleavage at these sites was not efficient.