Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig

The Molecular Chaperone Heat Shock Protein90Beta Regulates the Proliferation of PRRSV

Author WangShaoHua
Tutor FangLiuRong
School Huazhong Agricultural University
Course Preventive Veterinary Medicine
Keywords PRRSV Hsp90 17-AAG Nsp2
CLC S858.28
Type Master's thesis
Year 2012
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Porcine reproductive and respiratory syndrome (PRRS), induced by porcine reproductive and respiratory syndrome virus (PRRSV), is a serious infectious diseasesharmful to the pig industry, which major clinical manifestations are reproductive failure of pregnant sows and respiratory symptoms of weaned piglets. The United States first discovered PRRS in1987, then Guobao Qing and Chenzhang Shui confirmed that PRRSV infected Chinese herd in1996. Currently, PRRS has spread to the world’s major pig-raising countries and regions, caused huge economic losses for breeding industry.Heat shock proteins (Hsp-s) are proteins produced by cells to protective themselves when cells in stress conditions of heat shock, infection of pathogenic microorganisms and so on. Hsp-s is a highly conserved protein family widespread in eukaryotes and prokaryotes. As a chaperone, Hsp-s are mainly involved in correcting folding and transferring of proteins and promoting degradation of not properly folded proteins, consequently participate in cell signal transduction, immune response and biological evolution. According to molecular size, Hsp-s are divided into Hsp100, Hsp90, Hsp70, Hsp60and small Hsp-s. Hsp90plays an important role in the conformation maturation of intracellular proteins and it is a significant chaperone for viral proteins synthesis. To study the role of Hsp90in PRRSV replication process, do following researches:1. PRRSV promotes Hsp90β changedAfter PRRSV WUH3respectively infected Marc-145cells or PAM, Hsp90p were detected in mRNA and western blotting levels by means of relative fluorescence quantitative PCR and western blotting experiments, found that PRRSV can raise Hsp90P at the two levels.2.17-AAG, Hsp90-specific inhibitor, influences the proliferation of PRRSVWith experiment of PRRSV dose-dependent to17-AAG in Marc-145and toxicity test of17-AAG in Marc-145, the research chose5μM (17-AAG) as subsequent experiments standard. Then adding17-AAG at different time points in early viral replication respectively, confirmed that17-AAG inhibited the proliferation of PRRSV and Nsp2expression, in addition, adding17-AAG in early viral replication could significantly inhibit the proliferation of PRRSV. Moreover, after PRRSV WUH3infected Marc-145cells, added17-AAG to cells, found that17-AAG could inhibit the proliferation of PRRSV and significantly suppress Nsp2expression at12h,24h,36h and48h by way of plaque experiment and western blotting experiment.3. The influence of siRNA Hsp90β on PRRSV proliferationTo confirm the role of Hsp90β in the proliferation of PRRSV, the research designed interference molecules of monkey Hsp90β and porcine Hsp90, then verified the effect of interference molecules and selected appropriate concentration of them by means of relative fluorescence quantitative PCR and western blotting experiments. Next, the research transfect transiently interference molecules in Marc-145cells or PAM, found that interference molecules could inhibit the proliferation of PRRSV and Nsp2expression by way of plaque experiment and western blotting experiment.4. The influence of siRN A Hsp90β on non-structural proteins of PRRSVNsp2has a cysteine proteinse activity and mediates Nsp2/Nsp3cutting. In addition, Nsp2and Nsp3both are necessary proteins of PRRSV replication and transcription; Nsp9is an RNA-dependent RNA polymerase. Nsp9, Nsp10, Nsp11and Nsp12form replicase complex, which complete synchronously the genome replication and subgenomic transcription of Arteritis virus. The research transfect transiently Nsp2, Nsp3, Nsp9and Nspl2connected to PCAGGS-HA in Marc-145cells, then detected the influence of17-AAG on non-structural proteins. The results showed that17-AAG could inhibit Nsp2expression, but no significantly effect on Nsp3, Nsp9and Nsp12.5. The influence of17-AAG on NF-κB signal pathwayNF-κB signal pathway palys an important role in PRRSV replication process. The previous experiments confirmed that17-AAG could inhibit the proliferation of PRRSV. The research detected the effect of17-AAG on NF-κB signal pathway. The results indicated that PRRSV could activate NF-κB signal pathway, while17-AAG suppressed the activity of NF-κB.

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