Abstract
Viral proteases are essential for the proteolytic processing that liberates individual proteins crucial for infection and replication. This work focused on two of the three proteases that contain a ZF motif: the PCP from HEV and Nsp1a from PRRSV, as no current treatments exist for these two viruses. Both of these proteases have been established in the Pandelia lab to contain an Fe-S cluster, serving as a potential novel therapeutic target. If these are bonafide Fe-S cluster-containing proteins, they must obtain this cofactor from the host machinery as viruses lack the biosynthetic machineries for Fe-S cluster assembly. A dedicated chaperone, Hsc20, accredits proteins as recipients of Fe-S clusters by recognizing an LYR tripeptide. Interestingly, all of the viral proteases examined contain this sequence.</p><p class="ql-align-justify"> Among the over 1,000 retrieved PCP sequences, no Cys-His dyad is structurally identified, suggesting that it is not a protease. Furthermore, the hexa-cysteine motifs at the N-terminus are strictly conserved, indicating that these residues play critical roles in the metal site. Evolutionary and occurrence inspections demonstrate that the LYR motif is conserved, suggestive of PCP binding an Fe-S cluster and interacting with Hsc20.</p><p class="ql-align-justify"> Among the inspections of 2,223 retrieved Nsp1a sequences, Nsp1a does not have similarity to known proteins. The metal binding (zinc-finger motifs) and protease sites (catalytic dyad) are conserved. The LYR motif is also conserved, suggestive of potential Fe-S cluster binding. Nsp1 evolution matches that of Arteriviruses, in which Oliver Shrews are considered the most ancient. It appears that Nsp1a diversified and specialized towards infecting pigs. Experimental analysis with respect to metal sites (Fe-S cluster or Zinc), indicate that cleavage of Nsp1 WT is not metal dependent in cellulo, and proteolysis comes from Nsp1a only. The metal binding Nsp1 mutant (C8/C10/C25/C28A) cleaved similar to WT indicating that the ZF is not critical for proteolytic.</p><p class="ql-align-justify"> Alpha Fold does not predict an interaction of the PCP or the Nsp1a protease with Hsc20 in two dimensional plots, albeit predicts an interaction interface on the structural models. Experimental validation using Size Exclusion Chromatography and Ni-NTA pull down column confirmed successful interactions between IscU and Hsc20, however interaction with viral proteases is not detected by size exclusion chromatography.