Expression And Fuction Analysis Of Non-structural Protein 4 Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is characterized by late term abortions and still birth in sows, and respiratory tract distress in sucking pigs. PRRS was first discovered in North America in 1987 and almost simultaneously in Europe (in 1990). Porcine reproductive and respiratory syndrome virus (PRRSV) responsible for the syndrome was first isolated on alveolar macrophage cultures by Wensvoort in 1991. PRRSV genome is a positive-strand RNA of approximately 15,000 nucleotides, comprising at least 9 open reading frames (ORFs). ORF1 is comprised of two large, overlapping ORFs, ORF1a and ORF1b. ORF1a is translated directly, whereas ORF1b is translated by a ribosomal frame-shift, yielding a large ORF1ab poly-protein. After the autocatalytic release of non-structural protein 1α(Nsp1α), Nsp1βand Nsp2, the remainder of the poly-protein is cleaved into 10 non-structural proteins (Nsp3–12) related to the virus transcription and replication by Nsp4. The PRRSV Nsp4 belongs to the 3C-like serine proteinases (3CLSP). Besides their crucial roles in viral replication, recent studies suggested that Nsp4 also contributed to modulate host immunity and virulence.In order to study the immunogenicity, cellular location, and function of Nsp4, Nsp4 gene of highly pathogenic PRRSV was amplified and cloned into pET-30a vector, designated pET30a-Nsp4. The soluble fusion protein expressed from pET30a-Nsp4 was purified. Western-blot showed that the purified protein could be specifically recognized by PRRSV positive serum, suggesting the fusion protein has a good immunogenicity.The anti-serum against Nsp4 was obtained by immunizing BALB/c mice using the purified Nsp4. The value of polyclonal antibody was about 1:16000 detected by ELISA. Immunofluorescence assay and Western-blot analysis showed that polyclonal antibody can recognize the native protein in Marc-145 cells infected by PRRSV.In order to characterize properties of PRRSV 3CLpro, the Nsp4 gene was placed downstream of the T7 promoter. The resultant plasmid designated pT7Pro, encoded the 3CLpro with a MHHHHHHSS sequence in the N-terminal. The cDNA encoding the Nsp3′4 covered the last 8 residues of Nsp3 and the complete sequence of Nsp4, was cloned into the pET32a vector, and the resultant plasmid was designated as pET32a-Nsp3′4. Next, the Ser118 to Tyr mutation of the 3CLpro was introduced into pET32a-Nsp3′4. The Nsp4 expressed from pT7Hispro and the mutant protein expressed from pET32a-Nsp3′4 S118Y were used as the enzyme and the substrate, respectively. The optimal temperature and pH of the PRRSV 3CLpro for the proteolytic activity were 8℃and 7.5, respectively. Increased concentration of Na+ (~1000 mM) was not significant inhibitory to the cleavage activity. Cu2+, Zn2+, PMSF and EDTA, remarkably inhibited the proteinase activity.In order to confirm the active center of PRRSV 3CLpro consist of His39, Asp64 and Ser118, several single amino acid substitutions were introduced into the plasmid pT7Pro by a modified PCR-based site-directed mutagenesis method. All the replacements of the predicted active site residues His39, Asp64and Ser118 abolished the enzymatic activity of recombinant PRRSV 3CLpro, in contrast, the replacements of Ser45, Asp60 and Asp69 didn't affect the activity of PRRSV 3CLpro, supporting the proposed catalytic function of His39, Ser118and Asp64.Nsp9, Nsp10, Nsp11, and Nsp12 genes of highly pathogenic PRRSV strain HuN4 were amplified and cloned into pET-32a vector. The recombinant Nsp9, Nsp10, Nsp11, and Nsp12 were expressed and putrified.The anti-serum against Nsp9, Nsp10, Nsp11, and Nsp12 were obtained, by immunizing BALB/c mice with these purified recombinant proteins describled above.