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.
Subject(s)
Chaperonin 10/metabolism , Chaperonin 60/metabolism , Ectromelia virus/physiology , Ectromelia, Infectious/immunology , Fibroblasts/physiology , Heat-Shock Response/immunology , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Animals , Apoptosis , Cell Line , Fibroblasts/virology , Gene Expression Regulation , Immune Evasion , Mice , Protein Transport , Proto-Oncogene Proteins c-bcl-2/metabolism , Virulence , Virus ReplicationABSTRACT
Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fissionâ»fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis.
Subject(s)
Ectromelia virus/physiology , Fibroblasts/metabolism , Fibroblasts/virology , Macrophages/metabolism , Macrophages/virology , Mitochondria/physiology , Mitochondria/ultrastructure , Animals , Autophagy/physiology , Dynamins/metabolism , Ectromelia virus/ultrastructure , Fibroblasts/pathology , GTP Phosphohydrolases/metabolism , L Cells , Macrophages/pathology , Membrane Potential, Mitochondrial/physiology , Mice , Microtubule-Organizing Center/metabolism , Microtubule-Organizing Center/virology , Mitochondria/metabolism , Mitochondria/virology , Mitochondrial Proteins/metabolism , RAW 264.7 Cells , Reactive Oxygen Species/analysis , Tubulin/metabolism , Virion/metabolism , Virus ReplicationABSTRACT
Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF-derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.
Subject(s)
Bone Marrow Cells/drug effects , Bone Marrow Cells/virology , Ectromelia virus/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Animals , Antigens/immunology , Apoptosis/drug effects , Bone Marrow Cells/metabolism , Cell Line , Cells, Cultured , Cytokines/metabolism , Endocytosis/drug effects , Endocytosis/immunology , Immunophenotyping , Interferon Type I/metabolism , Leukocytes/immunology , Leukocytes/metabolism , Leukocytes/virology , Lymphocyte Activation/immunology , Male , Mice , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/metabolism , Virus ReplicationABSTRACT
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.
Subject(s)
Cytoskeleton/metabolism , Ectromelia virus/growth & development , Fibroblasts/metabolism , Animals , Cell Line , Ectromelia virus/metabolism , Humans , MiceABSTRACT
During mousepox in resistant (C57BL/6) or susceptible (BALB/c) strains of mice, stimulation of Th1 or Th2 cytokine immune response, respectively, is observed. Because mechanisms of different polarization of T cells remain elusive, in this study, we quantitatively assessed the phenotype of antigen-presenting cells (APCs) involved in ectromelia virus (ECTV) antigen presentation and cluster formation with effector cells in secondary lymphoid organs of BALB/c and C57BL/6 mice. We showed that both strains of mice display similar dynamics and kinetics of viral antigen presentation by CD11c(+) , CD11b(+) , and CD19(+) cells. CD11c(+) and CD11b(+) cells highly participated in viral antigen presentation during all stages of mousepox, whereas CD19(+) cells presented viral peptides later in infection. The main population of dendritic cells (DCs) engaged in ECTV antigen presentation and cell junction formation with effector cells was a population of myeloid CD11b(+) DCs (mDCs). We suggest that, on the one hand, ECTV may differentially affect the functions of APCs depending on the strain of mice. On the other hand, we suggest that some types of APCs, such as mDCs or other DCs subsets, have different abilities to direct the shape of immune response depending on the host resistance to mousepox.
Subject(s)
Antigen-Presenting Cells/classification , Antigen-Presenting Cells/virology , Ectromelia virus/immunology , Immunophenotyping , Animals , Antigen-Presenting Cells/chemistry , Antigens, CD19/analysis , CD11b Antigen/analysis , CD11c Antigen/analysis , Dendritic Cells/immunology , Lymph Nodes/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Spleen/immunologyABSTRACT
Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.
Subject(s)
Apoptosis Regulatory Proteins/immunology , Apoptosis/immunology , Autophagy/immunology , Ectromelia virus/physiology , Genes, bcl-2/immunology , Microtubule-Associated Proteins/immunology , Animals , Apoptosis Regulatory Proteins/antagonists & inhibitors , Beclin-1 , Cell Line , Chlorocebus aethiops , DNA Replication , Ectromelia, Infectious/immunology , Ectromelia, Infectious/physiopathology , Flow Cytometry , Mice , Microscopy, Fluorescence , RNA, Small Interfering/pharmacology , Vero Cells , Virus ReplicationABSTRACT
Newborn higher vertebrates are largely immuno-incompetent and generally survive infections--including poxviruses--by maternal antibody protection. Here, we show that mice survived epidemics as adults only if exposed to lethal orthopoxvirus infections during infancy under the umbrella of maternal protective antibodies. This implies that both the absence of exposure to infection during early infancy or of effective vaccination renders the population highly susceptible to new or old re-emerging pathogens.