pathogens Article Chicken Heat Shock Protein 70 Is an Essential Host Protein for Infectious Bursal Disease Virus Infection In Vitro Yufang Meng, Xiaoxue Yu *, Chunxue You , Wenjuan Zhang , Yingfeng Sun, Liuan Li, Tianming Jin, Pengyu Pan and Ailing Xie Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China; [email protected] (Y.M.); [email protected] (C.Y.); [email protected] (W.Z.); [email protected] (Y.S.); [email protected] (L.L.); [email protected] (T.J.); [email protected] (P.P.); [email protected] (A.X.) * Correspondence: [email protected]; Tel.: +86-022-23781297 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 Citation: Meng, Y.; Yu, X.; You, C.; production and knockdown of cHsp70 using small interfering RNAs (siRNA) and reducedviral Zhang, W.; Sun, Y.; Li, L.; Jin, T.; Pan, P.; Xie, A. Chicken Heat Shock Protein production, implying the necessity of cHsp70 in IBDV infection. These results reveal that cHsp70 70 Is an Essential Host Protein for is essential for IBDV infection in DF-1 cells, suggesting that targeting cHsp70 may be applied as an Infectious Bursal Disease Virus antiviral strategy. Infection In Vitro. Pathogens 2021, 10, 664. https://doi.org/10.3390/ Keywords: IBDV; Hsp70; dsRNA; replication; host-virus interaction pathogens10060664 Academic Editor: Anna Honko 1. Introduction Received: 12 April 2021 Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is Accepted: 26 May 2021 a very acute and highly contagious disease which leads to great loss in poultry. IBDV is Published: 28 May 2021 a double-stranded RNA virus which belongs to family Birnaviridae [1]. There are two serotypes of IBDV. Serotype 1 strains can cause pathogenicity and mortality in chickens Publisher’s Note: MDPI stays neutral and serotype 2 strains are avirulent to chickens [2–4]. IBDV targets chicken B lymphocytes with regard to jurisdictional claims in primarily, disrupts the function of the bursa of Fabricius, leads to immunosuppression published maps and institutional affil- and makes chickens susceptible to other pathogens. Serotype 1 strains of IBDV are widely iations. propagated in chicken embryo fibroblast cells, such as DF-1 cells and chicken embryo fibroblasts (CEF) [5]. The IBDV genome consists of two segments, segment A encoding vp2, 3, 4, 5 and segment B encoding vp1 [6]. vp2 and vp3 are structural proteins of IBDV particles, and the binding between vp2 or vp3 with dsRNA is very important to maintain Copyright: © 2021 by the authors. the stability of virions [7]. As parasitic organisms, many processes in the viral propagation Licensee MDPI, Basel, Switzerland. rely on host factors to facilitate, such as adsorption, stripping, genome replication and This article is an open access article transcription, viral protein synthesis, virus particle packaging and so on [8–12]. Meanwhile, distributed under the terms and the defense mechanisms of the host cells are activated to combat the viral infection, such conditions of the Creative Commons Attribution (CC BY) license (https:// as the innate immune response [13]. Hence, host factors may play positive or negative creativecommons.org/licenses/by/ roles during viral infection. Thus, study of virus–host protein interactions is critical for 4.0/). understanding viral pathogenesis and development of antiviral drugs. Pathogens 2021, 10, 664. https://doi.org/10.3390/pathogens10060664 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 664 2 of 11 such as the innate immune response [13]. Hence, host factors may play positive or nega- Pathogens 2021, 10, 664 tive roles during viral infection. Thus, study of virus–host protein interactions is critical2 of 11 for understanding viral pathogenesis and development of antiviral drugs. Heat shock proteins (Hsps) are evolutionarily conserved proteins. The expression of Hsps,Heat including shock proteinsHsp70, increase (Hsps)s are following evolutionarily stress exposure, conserved such proteins. as heat Theshock expression and path- ofogenic Hsps, infection. including Hsp70 Hsp70, is found increases highly following expressed stress in many exposure, virus infections such as heat and shockis involved and pathogenicin virus entry, infection. replication Hsp70 of the is found virus genome, highly expressed modulating in manyvirus polymerase virus infections activity and [14] is , involvedpackaging in of virus virions, entry, and replication innate immune of the virus response genome, of host modulating [15]. Inhibition virus polymeraseof Hsp70 re- activityduces the [14 ],replication packaging and of virions, production and innate of reproduct immuneive response and respiratory of host [15 syndrome]. Inhibition virus of Hsp70(PPRSV) reduces [16], inhibit the replications the EB andvirus production infection [17] of reproductive, decreases the and virus respiratory titer of syndromeZika virus virus[18,19] (PPRSV) and affect [16s], the inhibits assembly the EB of virushepatitis infection C virions [17], [20] decreases. Previous the studies virus titershowed of Zika that viruschicken [18 ,Hsp9019] and which affects belongs the assembly to the H ofsp hepatitis family of C virionsproteins [20 is]. involved Previous in studies IBDV infection. showed thatcHsp90 chicken participates Hsp90 whichin IBDV belongs entry into to theDF- Hsp1 cells family through of proteinsinteraction is with involved subviral in IBDV parti- infection.cles which cHsp90 are formed participates from vp2 in [ IBDV10]. Appl entryication into DF-1of anti cells-cHsp90 through micro interactionRNAs could with in- subviralhibit IBDV particles infection which and are furthermo formed fromre cHsp90 vp2 [10α,]. not Application cHsp90β, offunctions anti-cHsp90 in IBDV microRNAs infection could[21]. Heat inhibit conditioning IBDV infection cause ands increased furthermore Hsp70 cHsp90 expressionα, not and cHsp90 a decreasedβ, functions bursal in IBDV histo- infectionlogical score [21]. Heatduring conditioning IBDV infection causes [22 increased]. A DNA Hsp70 vaccine expression developed and ausing decreased a full bursallength histologicalvp2 gene of score IBDV during fused IBDVwith truncated infection [Hsp7022]. A DNAof Mycobacterium vaccine developed tuberculosis using could a full provide length vp2very gene good of protection IBDV fused against with truncatedIBDV [23] Hsp70. 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We Inobserved this research, that the we expression aimed to reveal of cHsp70 whether increased cHsp70 isin involved IBDV-infected in IBDV DF infection.-1 cells, Weand observedsiRNA interference that the expression targeting of cHsp70 cHsp70 lead increased to lower in production IBDV-infected of IBDV. DF-1 cells,Furthermore, and siRNA we interferenceobserved the targeting direct interaction cHsp70 lead of toHsp70 lower and production dsRNA of IBDV.IBDV. Furthermore,Overexpression we observedof cHsp70 thepromoted direct interaction viral production of Hsp70 and and siRNA dsRNA towards of IBDV. cHsp70 Overexpression weakened viral of cHsp70 production promoted. These viralresults production indicate that and chicken siRNA towardsHsp70 play cHsp70s a positive weakened role viralin DF production.-1 cells during These IBDV results infec- indicatetion. In thatthis chickenstudy, for Hsp70 the first plays time a positive the interaction role in DF-1 between cells duringhost protein IBDV- infection.Hsp70 and In IBDV this study,is revealed, for the which first time would the provide interaction a new between sight into host IBDV protein-Hsp70 pathogenesis. and IBDV is revealed, which would provide a new sight into IBDV pathogenesis. 2. Results 2. Results 2.1.2.1. ViralViral Load Load Changes Changes in in IBDV-Infected IBDV-Infected DF-1 DF-1 Cells Cells DF-1DF-1 cells cells are are susceptible