Ectromelia Virus Affects the Formation and Spatial Organization of Adhesive Structures in Murine Dendritic Cells In Vitro.

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs' immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs' organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.

Deficiency of Selected Cathepsins Does Not Affect the Inhibitory Action of ECTV on Immune Properties of Dendritic Cells.

Ectromelia virus (ECTV), an orthopoxvirus, undergoes productive replication in conventional dendritic cells (cDCs), resulting in the inhibition of their innate and adaptive immune functions. ECTV replication rate in cDCs is increased due to downregulation of the expression of cathepsins - cystein proteases that orchestrate several steps during DC maturation. Therefore, this study was aimed to determine if downregulation of cathepsins, such as B, L or S, disrupts cDC capacity to induce activating signals in T cells or whether infection of cDCs with ECTV further weakens their functions as antigen-presenting cells. Our results showed that cDCs treated with siRNA against cathepsin B, L and S synthesize similar amounts of pro-inflammatory cytokines and exhibit comparable ability to mature and stimulate alloreactive CD4+ T cells, as untreated wild type (WT) cells. Moreover, ECTV inhibitory effect on cDC innate and adaptive immune functions, observed especially after LPS treatment, was comparable in both cathepsin-silenced and WT cells. Taken together, the absence of cathepsins B, L and S has minimal, if any, impact on the inhibitory effect of ECTV on cDC immune functions. We assume that the virus-mediated inhibition of cathepsin expression in cDCs represents more a survival mechanism than an immune evasion strategy.

Ectromelia virus suppresses expression of cathepsins and cystatins in conventional dendritic cells to efficiently execute the replication process.

BACKGROUND: Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors - cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxvirus infection on host cells, we investigated the influence of ectromelia virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxvirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs. RESULTS: Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV. CONCLUSIONS: Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the virus to replicate effectively in infected cells.

Ectromelia virus induces tubulin cytoskeletal rearrangement in immune cells accompanied by a loss of the microtubule organizing center and increased α-tubulin acetylation.

Ectromelia virus (ECTV) is an orthopoxvirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.

ECTV Abolishes the Ability of GM-BM Cells to Stimulate Allogeneic CD4 T Cells in a Mouse Strain-Independent Manner.

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4+ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4+ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4+ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.

Long actin-based cellular protrusions as novel evidence of the cytopathic effect induced in immune cells infected by the ectromelia virus.

The aim of the study was to evaluate the influence of ectromelia virus (ECTV) infection on actin cytoskeleton rearrangement in immune cells, such as macrophages and dendritic cells (DCs). Using scanning electron and fluorescence microscopy analysis we observed the presence of long actin-based cellular extensions, formed by both types of immune cells at later stages of infection with ECTV. Such extensions contained straight tubulin filaments and numerous punctuate mitochondria. Moreover, these long cellular projections extended to a certain length and formed convex structures termed "cytoplasmic packets". These structures contained numerous viral particles and presumably were sites of progeny virions' release via budding. Further, discrete mitochondria and separated tubulin filaments that formed a scaffold for accumulated mitochondria were visible within cytoplasmic packets. ECTV-induced long actin-based protrusions resemble "cytoplasmic corridors" and probably participate in virus dissemination. Our data demonstrate the incredible capacity for adaptation of ECTV to its natural host immune cells, in which it can survive, replicate and induce effective mechanisms for viral spread and dissemination.

[MAVS protein and its interactions with hepatitis A, B and C viruses]. / Bialko MAVS i jego interakcje z wirusami zapalenia watroby typu A, B i C.

Mitochondrial antiviral signaling protein (MAVS) transmits activation signal of type I interferon (IFN) gene transcription in the molecular intracellular pathway, which depends on the protein encoded by retinoic acid inducible gene I (RIG-I) or melanoma differentiation-associated protein-5 (MDA-5). MAVS, as a signal molecule, performs an essential function in the development of an antiviral immune response. The molecule of MAVS consists of two domains: the N-terminal domain and the C-terminal domain. The N-terminal end of MAVS contains the caspase activation and recruitment domain (CARD). CARD is responsible for MAVS interaction with RIG-I and MDA-5, which act as cytosolic sensors detecting foreign viral genetic material in the host cell. After binding to viral RNA, RIG-I or MDA-5 activates MAVS and transmits the signal of IFN type I gene expression. The C-terminal transmembrane domain (TM) of MAVS anchors the protein to the outer mitochondrial membrane. In this paper interactions between MAVS and hepatitis virus type A (HAV), type B (HBV) and type C (HCV) are presented. Mechanisms of indirect activation of MAVS by viral DNA and RNA, as well as the strategies of HAV, HBV and HCV for blocking of the intracellular signaling pathway at the level of MAVS, are described.

Strategies of NF-κB signaling modulation by ectromelia virus in BALB/3T3 murine fibroblasts.

Nuclear factor κB (NF-κB) is a pleiotropic transcription factor that regulates the expression of immune response genes. NF-κB signaling can be disrupted by pathogens that prevent host immune response. In this work, we examined the influence of ectromelia (mousepox) virus (ECTV) on NF-κB signaling in murine BALB/3T3 fibroblasts. Activation of NF-κB via tumor necrosis factor (TNF) receptor 1 (TNFR1) in these cells induces proinflammatory cytokine secretion. We show that ECTV does not recruit NF-κB to viral factories or induce NF-κB nuclear translocation in BALB/3T3 cells. Additionally, ECTV counteracts TNF-α-induced p65 NF-κB nuclear translocation during the course of infection. Inhibition of TNF-α-induced p65 nuclear translocation was also observed in neighboring cells that underwent fusion with ECTV-infected cells. ECTV inhibits the key step of NF-κB activation, i.e. Ser32 phosphorylation and degradation of inhibitor κBα (IκBα) induced by TNF-α. We also observed that ECTV prevents TNF-α-induced Ser536 of p65 phosphorylation in BALB/3T3 cells. Studying TNFR1 signaling provides information about regulation of inflammatory response and cell survival. Unraveling poxviral immunomodulatory strategies may be helpful in drug target identification as well as in vaccine development.

Modulation of proinflammatory NF-κB signaling by ectromelia virus in RAW 264.7 murine macrophages.

Macrophages are antigen-presenting cells (APCs) that play a crucial role in the innate immune response and may be involved in both clearance and spread of viruses. Stimulation of macrophages via Toll-like receptors (TLRs) results in activation of nuclear factor κB (NF-κB) and synthesis of proinflammatory cytokines. In this work, we show modulation of proinflammatory NF-κB signaling by a member of the family Poxviridae, genus Orthopoxvirus--ectromelia virus (ECTV)--in RAW 264.7 murine macrophages. ECTV interfered with p65 NF-κB nuclear translocation induced by TLR ligands such as lipopolysaccharide (LPS) (TLR4), polyinosinic-polycytidylic acid (poly(I:C)) (TLR3) and diacylated lipopeptide Pam2CSK4 (TLR2/6). We observed that ECTV modulates phosphorylation of Ser32 of inhibitor of κB (IκBα) and Ser536 of p65. Interference of ECTV with TLR signaling pathways implied that proinflammatory cytokine synthesis was inhibited. Our studies provide new insights into the strategies of proinflammatory signaling modulation by orthopoxviruses during their replication cycle in immune cells. Understanding important immune interactions between viral pathogens and APCs might contribute to the identification of drug targets and the development of vaccines.

[Participation of heat shock proteins in modulation of NF-кB transcription factor activation during bacterial infections]. / Udzial bialek szoku cieplnego w modulacji aktywacji czynnika transkrypcyjnego NF-кB podczas zakazen bakteryjnych.

Nuclear factor kappa-light-chain enhancer of activated B-cells (NF-кB) is a pleiotropic transcription factor, which regulates processes of immune response and inflammation. NF-кB can undergo activation as a result of bacterial infections via Toll-like receptors (TLR), which recognize pathogen-associated molecular patterns (PAMP), such as lipopolysaccharides (LPS). Stimulation of the cells results in phosphorylation of inhibitor кB (IкB) and the translocation of NF-кB to the nucleus, where the transcription of genes encoding molecules, such as proinflammatory cytokines and chemokines takes place. Activation of NF-кB undergoes modulation upon heat shock, which induces the expression of heat shock proteins (HSP). NF-кB, in turn, is involved in the regulation of transcription of genes encoding HSP, while members of HSP family are modulators of NF-кB activation, which occurs as a result bacterial infections and leads to the development of inflammation. HSP90 is a major chaperone, which is associated with IкB kinase (IKK) subunits. HSP90 inhibitors enable dissociation of such complexes, thus blocking NF-кB and inflammatory process during bacterial infections. HSP72 and HSP70, in turn, modulate the expression of NF-кB controlled genes during sepsis and play a protective role, whereas exogenous HSP70 may enhance the inflammatory response. Bacterial HSP, such as HSP60 of Chlamydia pneumophila and Helicobacter pylori, or GroL of Porphyromonas gingivalis, as well as HSP65 and HSP70 of Mycobacterium tuberculosis and DnaK of Francisella tularensis activate NF-κB and inflammation. Knowledge of these interactions is extremely helpful in the development of therapeutic strategies.

Modulation of NF-кB transcription factor activation by Molluscum contagiosum virus proteins.

Molluscum contagiosum virus is a human and animal dermatotropic pathogen, which causes a severe disease in immunocompromised individuals. MCV belongs to the Poxviridae family whose members exert immunomodulatory effects on the host antiviral response. Poxviruses interfere with cell signaling pathways that lead to the activation of nuclear factor кB, a pleiotropic transcription factor which is crucial for regulation of the immune response, the cell cycle and apoptosis. In resting cells, NF-κB is present in the cytoplasm, where it is associated with inhibitor κB. Upon stimulation by activators, such as proinflammatory cytokines and bacterial or viral products, the inhibitory protein undergoes phosphorylation and proteasomal degradation. NF-κB, in turn, translocates to the nucleus, where it regulates the transcription of various genes that are essential for processes mentioned above. Since poxviruses replicate exclusively in the cell cytoplasm, NF-кB became a good target for poxviral immunomodulation. MCV encodes various proteins which interfere with the signaling pathways that lead to the activation of NF-κB. Ligand inhibitor encoded by MCV, MC54, binds interleukin-18 and inhibits interferon-γ production. Other MCV proteins, MC159 and MC160, belong to intracellular inhibitors of NF-κB and are members of viral FLICE-inhibitory proteins (vFLIPs). MC159 protein encoded by MCV was shown to inhibit apoptosis of virus-infected cells. Such interactions serve immune evasion and are responsible for the persistence of MCV.

[Cooperation between heat shock proteins in organizing of proteins spatial structure]. / Wspóldzialanie bialek szoku cieplnego w organizowaniu struktury przestrzennej bialek.

Heat shock proteins (Hsps) are a class of proteins with highly conserved amino acid sequences. They are widespread in nature; they are found in archeons, true bacteria and eukaryotic organisms. Hsps from various families, commonly interact to execute essential cellular tasks, such as molecular regulation of newly synthesized protein-folding or restoration of the appropriate conformation of denatured and aggregated proteins. In this review we discuss mechanisms of spatial organization of protein structure mediated by Hsp10, Hsp40, Hsp60, Hsp70, Hsp104 (Hsp100) and Hsp110. Interactions between Hsps of different molecular weights are described.

Orthopoxvirus Zoonoses-Do We Still Remember and Are Ready to Fight?

The eradication of smallpox was an enormous achievement due to the global vaccination program launched by World Health Organization. The cessation of the vaccination program led to steadily declining herd immunity against smallpox, causing a health emergency of global concern. The smallpox vaccines induced strong, humoral, and cell-mediated immune responses, protecting for decades after immunization, not only against smallpox but also against other zoonotic orthopoxviruses that now represent a significant threat to public health. Here we review the major aspects regarding orthopoxviruses' zoonotic infections, factors responsible for viral transmissions, as well as the emerging problem of the increased number of monkeypox cases recently reported. The development of prophylactic measures against poxvirus infections, especially the current threat caused by the monkeypox virus, requires a profound understanding of poxvirus immunobiology. The utilization of animal and cell line models has provided good insight into host antiviral defenses as well as orthopoxvirus evasion mechanisms. To survive within a host, orthopoxviruses encode a large number of proteins that subvert inflammatory and immune pathways. The circumvention of viral evasion strategies and the enhancement of major host defenses are key in designing novel, safer vaccines, and should become the targets of antiviral therapies in treating poxvirus infections.

Differential Activation of Splenic cDC1 and cDC2 Cell Subsets following Poxvirus Infection of BALB/c and C57BL/6 Mice.

Conventional dendritic cells (cDCs) are innate immune cells that play a pivotal role in inducing antiviral adaptive immune responses due to their extraordinary ability to prime and polarize naïve T cells into different effector T helper (Th) subsets. The two major subpopulations of cDCs, cDC1 (CD8α+ in mice and CD141+ in human) and cDC2 (CD11b+ in mice and CD1c+ in human), can preferentially polarize T cells toward a Th1 and Th2 phenotype, respectively. During infection with ectromelia virus (ECTV), an orthopoxvirus from the Poxviridae family, the timing and activation of an appropriate Th immune response contributes to the resistance (Th1) or susceptibility (Th2) of inbred mouse strains to the lethal form of mousepox. Due to the high plasticity and diverse properties of cDC subpopulations in regulating the quality of a specific immune response, in the present study we compared the ability of splenic cDC1 and cDC2 originating from different ECTV-infected mouse strains to mature, activate, and polarize the Th immune response during mousepox. Our results demonstrated that during early stages of mousepox, both cDC subsets from resistant C57BL/6 and susceptible BALB/c mice were activated upon in vivo ECTV infection. These cells exhibited elevated levels of surface MHC class I and II, and co-stimulatory molecules and showed enhanced potential to produce cytokines. However, both cDC subsets from BALB/c mice displayed a higher maturation status than that of their counterparts from C57BL/6 mice. Despite their higher activation status, cDC1 and cDC2 from susceptible mice produced low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, and the ability of these cells to stimulate the proliferation and Th1 polarization of allogeneic CD4+ T cells was severely compromised. In contrast, both cDC subsets from resistant mice produced significant amounts of Th1-polarizing cytokines and demonstrated greater capability in differentiating allogeneic T cells into Th1 cells compared to cDCs from BALB/c mice. Collectively, our results indicate that in the early stages of mousepox, splenic cDC subpopulations from the resistant mouse strain can better elicit a Th1 cell-mediated response than the susceptible strain can, probably contributing to the induction of the protective immune responses necessary for the control of virus dissemination and for survival from ECTV challenge.

Integrity of the Intestinal Barrier: The Involvement of Epithelial Cells and Microbiota-A Mutual Relationship.

The gastrointestinal tract, which is constantly exposed to a multitude of stimuli, is considered responsible for maintaining the homeostasis of the host. It is inhabited by billions of microorganisms, the gut microbiota, which form a mutualistic relationship with the host. Although the microbiota is generally recognized as beneficial, at the same time, together with pathogens, they are a permanent threat to the host. Various populations of epithelial cells provide the first line of chemical and physical defense against external factors acting as the interface between luminal microorganisms and immunocompetent cells in lamina propria. In this review, we focus on some essential, innate mechanisms protecting mucosal integrity, thus responsible for maintaining intestine homeostasis. The characteristics of decisive cell populations involved in maintaining the barrier arrangement, based on mucus secretion, formation of intercellular junctions as well as production of antimicrobial peptides, responsible for shaping the gut microbiota, are presented. We emphasize the importance of cross-talk between gut microbiota and epithelial cells as a factor vital for the maintenance of the homeostasis of the GI tract. Finally, we discuss how the imbalance of these regulations leads to the compromised barrier integrity and dysbiosis considered to contribute to inflammatory disorders and metabolic diseases.

NF-κB as an Important Factor in Optimizing Poxvirus-Based Vaccines against Viral Infections.

Poxviruses are large dsDNA viruses that are regarded as good candidates for vaccine vectors. Because the members of the Poxviridae family encode numerous immunomodulatory proteins in their genomes, it is necessary to carry out certain modifications in poxviral candidates for vaccine vectors to improve the vaccine. Currently, several poxvirus-based vaccines targeted at viral infections are under development. One of the important aspects of the influence of poxviruses on the immune system is that they encode a large array of inhibitors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is the key element of both innate and adaptive immunity. Importantly, the NF-κB transcription factor induces the mechanisms associated with adaptive immunological memory involving the activation of effector and memory T cells upon vaccination. Since poxviruses encode various NF-κB inhibitor proteins, before the use of poxviral vaccine vectors, modifications that influence NF-κB activation and consequently affect the immunogenicity of the vaccine should be carried out. This review focuses on NF-κB as an essential factor in the optimization of poxviral vaccines against viral infections.

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

Macrophages are the first encounters of invading bacteria and are responsible for engulfing and digesting pathogens through phagocytosis leading to initiation of the innate inflammatory response. Intracellular digestion occurs through a close relationship between phagocytic/endocytic and lysosomal pathways, in which proteolytic enzymes, such as cathepsins, are involved. The presence of cathepsins in the endo-lysosomal compartment permits direct interaction with and killing of bacteria, and may contribute to processing of bacterial antigens for presentation, an event necessary for the induction of antibacterial adaptive immune response. Therefore, it is not surprising that bacteria can control the expression and proteolytic activity of cathepsins, including their inhibitors - cystatins, to favor their own intracellular survival in macrophages. In this review, we summarize recent developments in defining the role of cathepsins in bacteria-macrophage interaction and describe important strategies engaged by bacteria to manipulate cathepsin expression and activity in macrophages. Particularly, we focus on specific bacterial species due to their clinical relevance to humans and animal health, i.e., Mycobacterium, Mycoplasma, Staphylococcus, Streptococcus, Salmonella, Shigella, Francisella, Chlamydia, Listeria, Brucella, Helicobacter, Neisseria, and other genera.

First Insight into the Modulation of Noncanonical NF-κB Signaling Components by Poxviruses in Established Immune-Derived Cell Lines: An In Vitro Model of Ectromelia Virus Infection.

Dendritic cells (DCs) and macrophages are the first line of antiviral immunity. Viral pathogens exploit these cell populations for their efficient replication and dissemination via the modulation of intracellular signaling pathways. Disruption of the noncanonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling has frequently been observed in lymphoid cells upon infection with oncogenic viruses. However, several nononcogenic viruses have been shown to manipulate the noncanonical NF-κB signaling in different cell types. This study demonstrates the modulating effect of ectromelia virus (ECTV) on the components of the noncanonical NF-κB signaling pathway in established murine cell lines: JAWS II DCs and RAW 264.7 macrophages. ECTV affected the activation of TRAF2, cIAP1, RelB, and p100 upon cell treatment with both canonical and noncanonical NF-κB stimuli and thus impeded DNA binding by RelB and p52. ECTV also inhibited the expression of numerous genes related to the noncanonical NF-κB pathway and RelB-dependent gene expression in the cells treated with canonical and noncanonical NF-κB activators. Thus, our data strongly suggest that ECTV influenced the noncanonical NF-κB signaling components in the in vitro models. These findings provide new insights into the noncanonical NF-κB signaling components and their manipulation by poxviruses in vitro.

Manipulation of Non-canonical NF-κB Signaling by Non-oncogenic Viruses.

Nuclear factor (NF)-κB is a major regulator of antiviral response. Viral pathogens exploit NF-κB activation pathways to avoid cellular mechanisms that eliminate the infection. Canonical (classical) NF-κB signaling, which regulates innate immune response, cell survival and inflammation, is often manipulated by viral pathogens that can counteract antiviral response. Oncogenic viruses can modulate not only canonical, but also non-canonical (alternative) NF-κB activation pathways. The non-canonical NF-κB signaling is responsible for adaptive immunity and plays a role in lymphoid organogenesis, B cell development, as well as bone metabolism. Thus, non-canonical NF-κB activation has been linked to lymphoid malignancies. However, some data strongly suggest that the non-canonical NF-κB activation pathway may also function in innate immunity and is modulated by certain non-oncogenic viruses. Collectively, these findings show the importance of studying the impact of different groups of viral pathogens on alternative NF-κB activation. This mini-review focuses on the influence of non-oncogenic viruses on the components of non-canonical NF-κB signaling.

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

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.