Structure And Function Of Nonstructural Protein 4 Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is characterized by the reproductive failure in pregnant gilts and sows and the respiratory tract illness in pigs of all ages, especially in suckling and nursery-age pigs. PRRSV is a small enveloped virus with a positive-sense, single-stranded RNA genome of approximately 15 kb. The PRRSV genome contains nine open reading frames (ORFs) coding for the viral replicase (ORFla and 1b), four membrane-associated glycoproteins (ORFs 2a to 5), two unglycosylated membrane proteins (ORF2b and ORF6) and the nucleocapsid protein N (ORF7). The replicase of PRRSV, an arterivirus, is processed by at least three viral proteases. Comparative sequence analysis suggested that nonstructural protein 4 (NSP4) is a 3C-like serine protease(3CLSP) that shares properties with chymotrypsin-like enzymes belonging to two different groups. The PRRSV NSP4 was predicted to utilize the His39-Asp64-Ser118 catalytic triad found in classical chymotrypsin-like proteases. On the other hand, its putative substrate-binding region contains Thr and His residues, which are conserved in viral 3C-like cysteine proteases and determine their specificity for (Gln/Glu) ↓ (Gly/Ala/Ser) cleavage sites.In order to understand the biochemical and structural characterization of PRRSV NSP4, E.coli expression system was used to expressed the wild-type NSP4 alone with 4 NSP4 mutants (H39R, D64V, S118Y, and H133L), a N-truncated NSP4, and a C-truncated NSP4. These recombinant proteins were further purified using Ni-NTA affinity chromatography. Western-blot was employed to confirm the authenticity of the purified proteins. Reversed-phase high-pressure liquid chromatographic (RP-HPLC) study was carried out to detect the 3CLSP activity using synthetic substrate peptide S (LGSLLEGAFRT) . The cleavage mixture of NSP4 and substrate peptide S was analyzed by RP-HPLC. The relative molecular weights of the products were then measured by MALD1-TOF MS. The result showed that substrate peptide S was cleaved at the peptide bond between Glu and Gly by the wild-type NSP4, which confirmed that PRRSV NSP4 has 3CLSP activity. However, 4 NSP4 mutants, N-truncated NSP4 and C-truncated NSP4 did not demonstrate the 3CLSP activity. The result suggests that His39, Asp64, Ser118 and His133 are indispensable for the 3CLSP activity of NSP4. It verifies the previous hypothesis that His39-Asp64-Ser118 constitutes a catalytic triad in PRRSV NSP4, and the substrate-binding region contains of Hisl33. Additionally, both N-terminus and C-terminus are indispensable for the 3CLSP activity of NSP4.Furthermore, the in vitro study of PRRSV NSP4 was conducted by using size-exclusion chromatography and SDS-PAGE. The result revealed that NSP4 could form many dimmers and a few trimmers in vitro even at low-concentration (0.2mg/ml). The dimmers were increased significantly at high-concentration (2mg/ml). The dimeric form was speculated to be the major form for biological activity of PRRSV NSP4. The N-truncated NSP4 mostly developed into trimers in vitro even at low-concentration (0.2mg/ml), whilst the C-truncated NSP4 mostly present as monomer in vitro even the concentration increased to 2mg/ml.The result suggests that C-terminus is indispensable for the dimerization of NSP4. N-terminus could stabilize the dimeric construction and prevent the formation oftrimer or polymer.The secondary structures of wild-type NSP4, mutants and truncated NSP4 were probed using CD. The results revealed that the wild-type PRRSV NSP4 folded into (J-pleated sheet. All point mutations have various effects on the secondary structure of NSP4 to certain extents. Remarkably, both terminal truncations significantly altered the structure of PRRSV NSP4, whereas the structure of truncated NSP4 became a mixture of various secondary structure types.PRRSV NSP4 3D model was created by MODELLER based on EAV NSP4 structure. PROCHECK and Verify-3D were used to evaluate the 3D model. The result showed that PRRSV NSP4 contains three domains. The first two domains form a two-p-barrel fold, which is responsible for the catalytic reaction. The structural analysis further confirms that His39-Asp64-Serll8 constitutes the catalytic triad, and Hisl33 is at the substrate-binding region. Lastly, the substrate peptide was docked in the catalytic center of the wild-type PRRSV NSP4 by using molecular docking method. The Glu (E) at the position of PI interact with Thrll3> Hisl33-. Serl36. The substrate peptide was docked into the negative charge region of the catalytic center.The work described in this paper provides biological and structural understandings of the PRRSV 3C-like Serine protease. It is also the first experimental analysis of PRRSV 3C-like Serine protease.