Interaction Of PRRSV Nonstructural Proteins With RNA And Preparation Of Polyclonal Antibodies Against NSP12

Porcine reproductive and respiratory syndrome(PRRS), which is caused by the infection of PRRS virus(PRRSV), has become an economically devastating disaster for the swine industy. Our knowledge about the function of structural proteins encoded by the virus has promoted in recent years. However, little is known about the functions of nsps(nonstructural proteins) of PRRSV. FL12 plasmid containing the full length cDNA of PRRSV strain NVSL#97-7895 were used as a template for construction of the expression vector pTriEx-nsp12 and subsequently used for abundant expression of nsp12. After purification by Ni2+ affinity chromatography, highly purified protein was obtained and used as immunogen for the preparation of polyclonal antibodies. A rabbit was immunized following by an appropriate immune program, and the antiserum aginst nsp12 was harvested 40 days after the primary immunization. Dot blot and Western blot assays showed that the antiserum reacted to nsp12 expressed from prokaryotic system with good sensitivity and specificity. Moreover, it showed the specific binding activity to nsp12 expressed from the virus infected cells.The localization of nsp12-GFP in 293T(subclones of human embryonic kidney) and MARC145(subclones of MA104 monkey kidney cell line) indicated that nsp12 distributed at some regions of the cytoplasm, probably locating at the organelles.The construction of the infectious cDNA clone was followed by two strategies. Unfortunately, no virus was rescued from both of the plasmid vectors. GFP expression analysis revealed that the process of protein expression was impeded by ribozymes which could be excised from the mRNA by self-cleavage. However, the infectious clone without ribozyme DNA sequence made no difference on the protein expression efficiency. We speculate that the transcription events could develop normally from the newly designed vectors. Nevertheless, because of the disfigurement on design, several redundant nucleotides attached at the 3’UTR(untranslated region) of PRRSV could not be completely cleaved though the HDV ribozyme functioned well. These additional nucleotides were likely to block the binding activity between RNA and RdRp(RNA-dependent RNA polymerase) which could efficiently perform replication or transcription processes. In addition, the low transfection efficiency of MARC145 could decrease the replication process of PRRSV and no CPE(cytopathic effect) could be detected.The 3’UTR and 5’UTR of arteriviruses are responsible for the genomic replication and transcription as their potential activity binding with RdRp(RNA-dependent RNA polymerase) or helicase. The infectious cDNA clone driven by T7 promotor could be transcribed into RNA in vitro and subsequently interacted with nsps. Gel shift studies showed that nsp9 could interact with both of the 3’UTR and 5’UTR of PRRSV as expected. However, the capsid protein N revealed no binding ability of RNA of PRRSV on either side. We speculate that the binding domain of N was locked by GST because of the steric hindrance effect. In addition, nsp8 showed good RNase activity to 3’UTR and 5’UTR of PRRSV which could digest RNA efficiently. No signs of binding activity were detected on the research of nsp7 and nsp12.