Mitochondrial Heat Shock Response Induced by Ectromelia Virus is Accompanied by Reduced Apoptotic Potential in Murine L929 Fibroblasts

Zbigniew Wyżewski,Marek G Niemiałtowski,Matylda B Mielcarska,Felix N Toka,Joanna Szczepanowska,Justyna Struzik,Karolina P Gregorczyk-Zboroch,Lidia Szulc-Dąbrowska,Magdalena Bossowska-Nowicka

doi:10.1007/s00005-019-00554-5

Abstract

Poxviruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxviruses on mtHsps has never been studied. We used highly infectious Moscow strain of ectromelia virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.

Highlights

Apoptosis of the infected cell is an event that leads to disruption of the viral replication cycle and, to elimination of the infectious agent from the host tissues
No differences in Hsp60 localization were detected compared to control cells, and its distribution was mostly perinuclear and probably reflected location viral factories were clearly visible within the cytoplasm of of mitochondria (Fig. 1b)
Scale bars: 20 μm and 10 μm. b Mean fluorescent intensity (MFI) of red, green (Hsp60), and blue fluorescence measured along the line marked by the white arrows in magnified images. c Correlation between Hsp60 level and mitochondrial potential in control and Ectromelia virus (ECTV)-infected cells at 24 hpi (Pearson correlation coefficient 0.94 and 0.96, respectively; *p < 0.05; n = 100). d Western blot analysis of Hsp60 level in control (C) and ECTV-infected L929 cells at 4, 8, 12, 18, and 24 hpi

Summary

Introduction

Apoptosis of the infected cell is an event that leads to disruption of the viral replication cycle and, to elimination of the infectious agent from the host tissues. Programmed cell death serves as a mechanism of unspecific antiviral immune response. For this reason, many viruses delay apoptosis to preserve infected cells as a replication platform and to facilitate further dissemination (Gregorczyk et al 2014a; Mehta et al 2015). Ectromelia virus (ECTV) is a member of the Orthopoxvirus genus of the Poxviridae family and is the causative agent of mousepox—a lethal disease of certain strains of mice (Esteban and Buller 2012). The Poxviridae family contains DNA viruses that are classified into two subfamilies:

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Conclusion