Screening And Identifying Of Host Proteins Interacting With PRRSV Nucleocapsid Protein

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease that emerged in the late 1980s and severely threatened global pig industry, characterized by the reproductive deficiency in sows, respiratory symptoms in pigs of other ages, and high lethal rate of piglets. PRRSV, the pathogen of this disease, is a memember of arteritis viridae. Its genome is single-stranded positive sense RNA that encodes 7 structural protein, among which nucleocapsid protein (N) accounts for most in the virion and is the most conserved. The nuclear location seuquence and RNA-binding domains are closely associated with the process of viral replication. To screen the host proteins that interact with PRRSV N protein will help to further demonstrate the pathogenesis of the virus and the anti-viral mechanism of the host. Based on this, in this study, host proteins that interact with PRRSV N protein were screened from pig brain cDNA library through yeast two-hybrid technology and verified. The main contents include:1. The screening of host proteins interacting with N protein of PRRSV WuH3 strain. The ORF7 gene of PRRSV WuH3 strain was amplified and inserted into pGBKT7 vector to construct recombinant plasmid pGBKT7-N, which was then used as bait protein in yeast two-hybrid to screen host proteins interacting with PRRSV N protein from pig brain cDNA library.81 positive clones was obtained and 5 potential host proteins that interact with PRRSV N protein were identified by seuquence analysis. By the preliminary screening, we selected two proteins of interests for subsequent experiments: small nuclear ribonucleoprotein polypeptide G (SNRPG) and Homo sapiens lectine, galactoside-binding, soluble 1, LGALS1).2. The verification of interaction between PRRSV N protein and the interested host proteins identified by screening. The interaction between PRRSV N proteins and the two interested host proteins was verified. The yeast retest and GST-pull down assay was respectively conducted and it was proved that the two host proteins, SNRPG and LGALSA intereacted with PRRSV N protein. The two proteins of interest were reported to be located in both nuclear and plasma, and fluorescent co-localization test revealed that the protein interaction both occurred in cell plasma.3. The detection of inhibitive effect of SNRPG on PRRSV replication RNA interference technology was applied, and the cells were infected with PRRSV after intracellular SNRPG was interfered. Real-time PCR and TCID50 test were performed respectively to detect the effect of treated cells on PRRSV replication on mRNA and protein level. The results of the two test were in accordance, indicating the interference of SNRPG has no effect on PRRSV replication.