Structure and Cleavage Specificity of the Chymotrypsin-Like Serine Protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

Abstract

Biogenesis and replication of the porcine reproductive and respiratory syndrome virus (PRRSV) include the crucial step of replicative polyprotein processing by self-encoded proteases. Whole genome bioinformatics analysis suggests that nonstructural protein 4 (nsp4) is a 3C-like serine protease (3CLSP), responsible for most of the nonstructural protein processing. The gene encoding this protease was cloned and expressed in Escherichia coli in order to confirm this prediction. The purified protein was crystallized, and the structure was solved at 1.9 A resolution. In addition, the crystal structure of the Ser118Ala mutant was determined at 2.0 A resolution. The monomeric enzyme folds into three domains, similar to that of the homologous protease of equine arteritis virus, which, like PRRSV, is a member of the family Arteriviridae in the order of Nidovirales. The active site of the PRRSV 3CLSP is located between domains I and II and harbors a canonical catalytic triad comprising Ser118, His39, and Asp64. The structure also shows an atypical oxyanion hole and a partially collapsed S1 specificity pocket. The proteolytic activity of the purified protein was assessed in vitro. Three sites joining nonstructural protein domains in the PRRSV replicative polyprotein are confirmed to be processed by the enzyme. Two of them, the nsp3/nsp4 and nsp11/nsp12 junctions, are shown to be cleaved in trans, while cis cleavage is demonstrated for the nsp4/nsp5 linker. Thus, we provide structural evidence as well as enzymatic proof of the nsp4 protein being a functional 3CLSP. We also show that the enzyme has a strong preference for glutamic acid at the P1 position of the substrate.

Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating β2-Microglobulin Transcription

“…The function of Nsp4 domain III is currently unknown. Analysis of the crystal structure of the Nsp4 crystal indicates the presence of two patches of solvent-exposed hydrophobic residues in domain III, implying possible interactions with other molecules (20), thus reinforcing our observation of the binding of Nsp4 domain III to the B2M promoter.…”

“…The active site of the 3C-like serine protease of Nsp4 is located between domains I and II and harbors a canonical catalytic triad comprising Ser118, His39, and Asp64 (20). In the current study, we observed that domain III of Nsp4 bound to the enhancer PAM element of the porcine B2M promoter and contributed to inhibition of B2M transcription, suggesting that its 3C-like serine protease activity was not involved in this inhibitory activity.…”

“…Nsp4 bound to the porcine B2M promoter to interfere with its transcription, and the interaction between Nsp4 domain III and the enhancer PAM element of the porcine B2M promoter was identified as essential for this inhibitory effect. Nsp4 is a 3C-like serine protease responsible for most nonstructural protein processing of PRRSV and plays an essential role in PRRSV infection (20). Nsp4 also impairs type I IFN expression by targeting the NF-B essential modulator (NEMO) and virus-induced signaling adaptor (VISA) (10,21), supporting Nsp4's critical role in modulating the host antiviral response.…”

“…4A and B), with Flag-Nsp4 having a greater effect than Flag-Nsp2. Furthermore, Nsp4 is a 3C-like serine protease responsible for most nonstructural protein processing and for antagonizing interferon (IFN) expression (20,21), playing an essential role in PRRSV infection, and we therefore selected Nsp4 for further studies.…”

Partial Sequence of a Novel Virus Isolated from Pelodiscus sinensis Hemorrhagic Disease

“…The sequence analysis implied TSHSV has a similar domain of replicase polyprotein of Arteriviridae, which is related to virus-coded proteinase [26,27] . RT-PCR and fluorescent quantitative RT-PCR based on the sequences we obtained are highly specific and can be used for molecular detection of this pathogen.…”