Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line

Abstract

Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc.

Functional paralysis of GM-CSF–derived bone marrow cells productively infected with ectromelia virus

“…Infected cells exhibited multiple irregular long cellular extensions, similar to those previously observed in ECTV-infected L929 fibroblasts [27]. Although VACV and CPXV have been shown to induce morphological changes of DCs, these viruses do not stimulate formation of long cytoskeletal protrusions in infected cells [22,38].…”

“…At the late stage (24 hpi) of infection with live-ECTV, majority of the infected cells had larger, but still regular viral factories and numerous progeny virions located near the viral factories and within cellular extensions, formed extensively by infected cells (Fig 1A). Presence of multiple cellular extensions gave the cells a unique, multifaceted morphology, previously observed in ECTV-infected L929 fibroblasts [27]. Moreover, at 24 hpi viral factories in infected GM-BM were strongly stained with anti-ECTV pAbs, suggesting that the quantity of antigens recognized by those antibodies increased together with the progress of virus replication.…”

“…After washing with 0.1% Triton X-100 in PBS, cells were incubated with anti-rabbit IgG-Rhodamine RedX (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) secondary antibody diluted in blocking solution. ECTV antigens were stained for 60 min with FITC-conjugated pAbs, obtained as previously described [27]. DNA was stained with 1 μg/ml Hoechst 33342 (Calbiochem, San Diego, CA, USA) for 2.5 min.…”

The in Vitro Inhibitory Effect of Ectromelia Virus Infection on Innate and Adaptive Immune Properties of GM-CSF-Derived Bone Marrow Cells Is Mouse Strain-Independent

“…The following mAbs were used for cytokine detection: anti-tumor necrosis factor (TNF)-FITC (MP6-XT22, rat IgG1), anti-IL-12(p40/p70)-APC (C15.6, rat IgG1; both from BD Biosciences) and anti-CCL3 [macrophage inflammatory protein 1 alpha (MIP-1α)]-PE (DNT3CC; rat IgG2a; eBioscience, San Diego, CA, United States). In some experiments, cells were stained intracellularly for the presence of ECTV antigens using a Cytofix/Cytoperm kit and rabbit polyclonal Abs anti-ECTV-FITC, obtained as previously described ( Szulc-Dabrowska et al, 2016 ). The staining procedure also included appropriate isotype controls obtained from BD Biosciences and eBioscience.…”

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line

“…This unique feature requires intracellular reorganization of the cytoskeleton and organelles, including mitochondria, endoplasmic reticulum, lysosomes, endosomes, and Golgi apparatus [ 22 ]. Our previous study indicated that ECTV infection leads to cytoskeletal rearrangement and alterations in mitochondrial network morphology and distribution in established cell lines [ 23 , 24 , 25 ]. In the present study, we asked how ECTV infection affects mitochondrial network morphology and physiology in permissive cells.…”