Chicken Heat Shock Protein 70 Is an Essential Host Protein for Infectious Bursal Disease Virus Infection In Vitro

Abstract

Infectious bursal disease virus (IBDV) infection causes pathogenicity and mortality in chickens, leading to huge economic losses in the poultry industry worldwide. Studies of host-virus interaction can help us to better understand the viral pathogenicity. As a highly conservative host factor, heat shock protein 70 (Hsp70) is observed to be involved in numerous viral infections. However, there is little information about the role of chicken Hsp70 (cHsp70) in IBDV infection. In the present study, the increased expression of cHsp70 was observed during IBDV-infected DF-1 cells. Further studies revealed that Hsp70 had similar locations with the viral double-stranded RNA (dsRNA), and the result of pull-down assay showed the direct interaction between cHsp70 with dsRNA, viral proteins (vp)2 and 3, indicating that maybe cHsp70 participates in the formation of the replication and transcription complex. Furthermore, overexpression of cHsp70 promoted IBDV production and knockdown of cHsp70 using small interfering RNAs (siRNA) and reducedviral production, implying the necessity of cHsp70 in IBDV infection. These results reveal that cHsp70 is essential for IBDV infection in DF-1 cells, suggesting that targeting cHsp70 may be applied as an antiviral strategy.

Keywords: Hsp70; IBDV; dsRNA; host-virus interaction; replication.

Figure 1

The viral load in IBDV-infected DF-1 cells at different times (MOI = 1): (a) TCID₅₀ was measured by IFA. (b) The expression of the vp2 gene by qRT-PCR.

Figure 2

The expression of cHsp70 at mRNA and protein levels increased during Ts infection. (a) Changes of cHsp70 expression at mRNA level detected by qRT-PCR. p ≤ 0.001 (***). (b) Changes of cHsp70 expression at protein levels detected by Western blot.

Figure 3

Direct interaction between Hsp70 and dsRNA. (a) Location of Hsp70 and dsRNA after IBDV infection; (b) Western blot results after pull-down assay. Left: anti-Hsp70 antibodies-pulling out proteins from mock-infected and IBDV-infected DF-1 cells. Middle: J2-pulling out proteins from mock-infected and IBDV-infected DF-1 cells. Right: total proteins from mock-infected and IBDV-infected DF-1 cells.

Figure 4

Overexpression of cHsp70 promotes the production of IBDV. (a) The transfection of pEGFP-N1 and pEGFP-N1-cHsp70 into DF-1 cells was observed under a fluorescence microscope. (b) Western blot results for detecting overexpression of cHsp70 proteins (from left to right, where three lanes of pEGFP-N1-cHsp70 transfected DF-1 cells means different time points post transfection): 24, 48, 72 h. (c) The changes of viral titers after cHsp70 were overexpressed at 24, 48, 72 h post IBDV infection. Differences were regarded as significant at p ≤ 0.05 (*) and very significant at p ≤ 0.01 (**).

Figure 5

Inhibition of cHsp70 by siRNA attenuates the production of IBDV. (a) Interference efficiencies of siRNAs targeting cHsp70 in DF-1 cells were calculated by qRT-PCR. (b) The changes of viral titers after siRNA was added. (c) The cell viability detected by CCK-8 assay. (d) The changes in vp2 gene expression when siRNA interference was carried out. (e) Comparison of the cytopathy effects by the addition of siRNA by visual study when CCK-8 assay was conducted. Differences were regarded as significant at p ≤ 0.05 (*) and very significant at p ≤ 0.01 (**).