Acta Veterinaria et Zootechnica Sinica ›› 2023, Vol. 54 ›› Issue (2): 683-693.doi: 10.11843/j.issn.0366-6964.2023.02.025

• PREVENTIVE VETERINARY MEDICINE • Previous Articles     Next Articles

Study on the Effect of Heat Shock Protein HSP90B1 on the Replication of Bovine Viral Diarrhea Virus

CHEN Junzhen, QUAN Ran, FU Qiang, GE Lijuan, YUAN Yuanyuan, ZHANG Chengyuan, LI Jianlin, SHI Huijun*   

  1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
  • Received:2022-02-14 Online:2023-02-23 Published:2023-02-21

Abstract: The present study was aimed to investigate the effect of heat shock protein 90 beta family member 1 (HSP90B1) on the replication of bovine viral diarrhea virus (BVDV). The expression of HSP90B1 mRNA and protein in MDBK cells after BVDV infection were detected by using quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. HSP90B1 KO cells were constructed using CRISPR/Cas9 technology. The effect of knockdown of HSP90B1 on cell growth was detected by counting. After infecting with BVDV TC strain, BVDV replication in HSP90B1 KO and control Scramble cells were detected using qRT-PCR, immunofluorescence, viral titer and cytopathic effects (CPE). The qRT-PCR results showed that the transcription level of HSP90B1 mRNA was significantly increased at 24 h after BVDV infection compared with the blank group (P<0.05), and was significantly increased after 36 h (P<0.01). Meanwhile, Western blot results showed that the expression level of HSP90B1 protein of BVDV infection group was significantly increased compared with the uninfected group; Western blot results showed that the expression of HSP90B1 protein in HSP90B1 KO cells was significantly lower than that in MDBK cells; There was no difference in the number of cells; qRT-PCR results showed that BVDV 5'UTR RNA levels in HSP90B1 KO cells were significantly reduced after 12 h of BVDV infection (P<0.05) and highly significantly reduced after 36 h compared with the control group (P< 0.01); the results of immunofluorescence detection showed that the green fluorescence in HSP90B1 KO cells was significantly reduced compared with the control group; viral titer after BVDV infection was significantly lower in HSP90B1 KO compared to control (P<0.05) after 12 h and highly significantly lower after 36 h (P<0.01), CPE showed that control cells showed The CPE showed that control cells showed significant CPE after 12 h of infection, while HSP90B1 KO cells showed only a small amount of CPE, and HSP90B1 KO cells showed significant CPE after 36 h of infection, at which time control cells showed a large amount of CPE with cell shedding. The above results show that BVDV induces the expression of HSP90B1 in MDBK cells; the HSP90B1 gene in MDBK cells was successfully knocked out by CRISPR/Cas9 technology, and HSP90B1 KO cells were constructed, experiments show that knockout of HSP90B1 can inhibit BVDV replication.

Key words: heat shock protein, bovine viral diarrhea virus, CRISPR/Cas9, replication

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