Mitochondria Of Infected Cells Research Articles

Dengue virus non-structural protein 3 inhibits mitochondrial respiration by impairing complex I function.

Dengue virus (DENV) infection is a major public health problem worldwide, affecting about 400 million people yearly. Despite its importance, many molecular aspects of dengue pathogenesis remain poorly known. For several years, our group has been investigating DENV-induced metabolic alterations in the host cells, focusing on the bioenergetics of mitochondrial respiration. The results of the present study reveal that the DENV non-structural protein 3 (NS3) is found in the mitochondria of infected cells, impairing mitochondrial respiration by directly targeting one of the components of the electron transport system, the respiratory complex I (CI). NS3 acts as the viral protease during the DENV replication cycle, and its proteolytic activity seems necessary for inhibiting CI function. Our findings uncover new nuances of DENV-induced metabolic alterations, highlighting NS3 as an important player in the modulation of mitochondria function during infection.

Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection.

Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.

Pyruvate Supports RET-Dependent Mitochondrial ROS Production to Control Mycobacterium avium Infection in Human Primary Macrophages.

Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium (M. avium) infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we showed that macrophages infected with M. avium switch to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle volume or changes in the energy charge. Instead, we found that pyruvate import contributes to hyperpolarization of mitochondria in infected cells and increases production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduces the macrophage burden of M. avium. While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments.

A subpopulation of arenavirus nucleoprotein localizes to mitochondria

Viruses need cells for their replication and, therefore, ways to hijack cellular functions. Mitochondria play fundamental roles within the cell in metabolism, immunity and regulation of homeostasis due to which some viruses aim to alter mitochondrial functions. Herein we show that the nucleoprotein (NP) of arenaviruses enters the mitochondria of infected cells, affecting the mitochondrial morphology. Reptarenaviruses cause boid inclusion body disease (BIBD) that is characterized, especially in boas, by the formation of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus NP within the infected cells. We initiated this study after observing electron-dense material reminiscent of IBs within the mitochondria of reptarenavirus infected boid cell cultures in an ultrastructural study. We employed immuno-electron microscopy to confirm that the mitochondrial inclusions indeed contain reptarenavirus NP. Mutations to a putative N-terminal mitochondrial targeting signal (MTS), identified via software predictions in both mamm- and reptarenavirus NPs, did not affect the mitochondrial localization of NP, suggesting that it occurs independently of MTS. In support of MTS-independent translocation, we did not detect cleavage of the putative MTSs of arenavirus NPs in reptilian or mammalian cells. Furthermore, in vitro translated NPs could not enter isolated mitochondria, suggesting that the translocation requires cellular factors or conditions. Our findings suggest that MTS-independent mitochondrial translocation of NP is a shared feature among arenaviruses. We speculate that by targeting the mitochondria arenaviruses aim to alter mitochondrial metabolism and homeostasis or affect the cellular defense.

A Kidnapping Story: How Coxsackievirus B3 and Its Host Cell Interact.

Infections with Coxsackievirus B3 and other members of the enterovirus genus are a common reason for myocarditis and sudden cardiac death in modern society. Despite intensive scientific efforts to cure enterovirus infections, there is still no standardized treatment option. The complexity of Coxsackievirus B3´s effects on the host cell make well defined studies on this topic very challenging. However, recent publications report newly found effects of CVB3´s structural and non-structural proteins on infected cells. For the first time, the viral capsid protein VP1 was shown to have direct influence on the viral life-cycle. By shortening the G0 and the G2 phase and simultaneously prolonging the G1 and G1-S phase, the translation of viral proteins is enhanced and the production of viable CVB3 particles is promoted. Coxsackievirus B3´s viroporin, protein 2B, was recently studied in more detail as well. Structural and physiological analyses identified two hydrophilic α-helices in the structure of 2B, enabling it to insert into cellular membranes of host cells. As main target of 2B the endoplasmatic reticulum was identified. The insertion of 2B into the ER membranes leads to an uncontrolled calcium outflow into the cytoplasm. Additional insertion of 2B into the cell membrane leads to host cell destabilization and in the end to release of viral progeny. The importance of the Coxsackievirus B3´s proteases 2A and 3C in pathogenicity is observed since years. Recently, DAP5 and eIf4G were identified as new cleavage targets for protease 2A. Cleavage of DAP-5 into DAP5-N and DAP5-C changes the gene expression of the host cell and promotes cell death. Additionally, protease 3C targets and cleaves procaspase 8 promoting the mitochondrial apoptosis pathway and cell death. Recent studies identified significant effects of CVB3 on mitochondria of infected cells. Mouse cardiomyocytes showed decreased activities of respiratory chain complexes I-III and changed transcription of important subunits of the complexes I-IV. A disrupted energy metabolism may be one of the main causes of cardiac insufficiency and death in CVB3 infected patients. In addition to a modified energy metabolism, CVB3 affects cardiac ion channels, KCNQ1 in particular. SGK1, which is an important mediator in KCNQ1 membrane insertions, is highly upregulated during CVB3 infections. This results in an increased insertion of KCNQ1 into the cell membrane of cardiac cells. Under stress conditions, this KCNQ1 overshoot may lead to a disturbed cardiac action potential and therefore to sudden cardiac death, as it is often observed in CVB3 infected persons.

Mitochondrial fragmentation affects neither the sensitivity to TNF\u03b1-induced apoptosis of Brucella-infected cells nor the intracellular replication of the bacteria

Mitochondria are complex organelles that participate in many cellular functions, ranging from ATP production to immune responses against viruses and bacteria. This integration of a plethora of functions within a single organelle makes mitochondria a very attractive target to manipulate for intracellular pathogens. We characterised the crosstalk that exists between Brucella abortus, the causative agent of brucellosis, and the mitochondria of infected cells. Brucella replicates in a compartment derived from the endoplasmic reticulum (ER) and modulates ER functionality by activating the unfolded protein response. However, the impact of Brucella on the mitochondrial population of infected cells still requires a systematic study. We observed physical contacts between Brucella containing vacuoles and mitochondria. We also found that B. abortus replication is independent of mitochondrial oxidative phosphorylation and that mitochondrial reactive oxygen species do not participate to the control of B. abortus infection in vitro. We demonstrated that B. abortus and B. melitensis induce a drastic mitochondrial fragmentation at 48 hours post-infection in different cell types, including myeloid and non-myeloid cells. This fragmentation is DRP1-independent and might be caused by a deficit of mitochondrial fusion. However, mitochondrial fragmentation does not change neither Brucella replication efficiency, nor the susceptibility of infected cells to TNFα-induced apoptosis.

Toxoplasma gondii Infection Is Associated with Mitochondrial Dysfunction in-Vitro.

Upon invasion of host cells, the ubiquitous pathogen Toxoplasma gondii manipulates several host processes, including re-organization of host organelles, to create a replicative niche. Host mitochondrial association to T. gondii parasitophorous vacuoles is rapid and has roles in modulating host immune responses. Here gene expression profiling of T. gondii infected cells reveals enrichment of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial dysfunction 6 h post-infection. We identified 11 hub genes (HIF-1α, CASP8, FN1, POU5F1, CD44, ISG15, HNRNPA1, MDM2, RPL35, VHL, and NUPR1) and 10 predicted upstream regulators, including 4 endogenous regulators RICTOR, KDM5A, RB1, and D-glucose. We characterized a number of mitochondrial parameters in T. gondii infected human foreskin fibroblast cells over a 36 h time-course. In addition to the usual rapid recruitment and apparent enlargement of mitochondria around the parasitophorous vacuole we observed fragmented host mitochondria in infected cells, not linked to cellular apoptosis, from 24 h post-infection. An increase in mitochondrial superoxide levels in T. gondii infected cells was observed that required active parasite invasion and peaked at 30 h post-infection. Measurement of OXPHOS proteins showed decreased expression of Complex IV in infected cells at 24 h post-infection, followed by decreased expression of Complexes I and II at 36 h post-infection. No change occurred in Complex V. No difference in host mitochondrial membrane potential between infected and mock-infected cells was observed at any time. Our results show perturbation of host mitochondrial function following T. gondii infection that likely impacts on pathogenesis of disease.

Reactive oxygen species activate NFκB (p65) and p53 and induce apoptosis in RVFV infected liver cells

Rift Valley fever virus (RVFV) infection is often associated with pronounced liver damage. Previously, our studies revealed altered host phospho-signaling responses (NFκB, MAPK and DNA damage responses) in RVFV infected epithelial cells that correlated with a cellular stress response. Here, we report that RVFV infection of liver cells leads to an increase in reactive oxygen species (ROS). Our data suggests the presence of the viral protein NSs in the mitochondria of infected cells, hence contributing to early increase in ROS. Increased ROS levels correlated with activation of NFκB (p65) and p53 responses, which in conjunction with infection, was also reflected as macromolecular rearrangements observed using size fractionation of protein lysates. Additionally, we documented an increase in cytokine expression and pro-apoptotic gene expression with infection, which was reversed with antioxidant treatment. Collectively, we identified ROS and oxidative stress as critical contributors to apoptosis of liver cells during RVFV infection.

Mitochondrial proteomic analysis of human host cells infected with H3N2 swine influenza virus

Swine influenza viruses (SIV) are zoonotic pathogens that pose a potential threat to human health. In this study, we analyzed the differential mitochondrial proteomes of H3N2 SIV-infected human lung A549 cells using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) analysis. In the comparative analysis, 24 altered proteins (13 upregulated and 11 downregulated) were identified in the mitochondria of H3N2 SIV-infected cells; these proteins were involved in cell-to-cell signaling and interaction, cellular movement, and post-translational modification. Moreover, the transcriptional profiles of 16 genes corresponding to the identified proteins were estimated by real time RT-PCR. IPA analysis suggested that the differentially expressed proteins were clustered primarily into the mammalian target of rapamycin (mTOR) and d-glucose signaling pathways. In addition, oxidative phosphorylation and integrin signaling appeared to be major pathways modulated in the mitochondria of infected cells. We further demonstrated that apolipoprotein L2 was upregulated in the cytoplasm and translocated to mitochondria during virus infection. These results were verified by Western blot analysis coupled with confocal microscopy. Collectively, the mitochondrial proteome data provide insights to further understand the underlying mechanisms of H3N2 SIV cross-species infection. In recent years, proteomics has emerged as an indispensable tool to unveil the complex molecular events in virology. we firstly perform mitochondrial proteomic profiles of human cells infected with H3N2 subtype SIV to understand virus-host interactions, and 24 differentially expressed proteins in mitochondrial proteomes were identified in SIV-infected cells. The proteins that were identified to have differential expression were involved in cell-to-cell signaling and interaction, post-translational modification, cell morphology, cellular assembly, cell death, and energy production. Furthermore, Western blot analysis and a confocal assay further demonstrated that the cellular protein APOL2 partially co-localized with mitochondria after virus infection. This is a very important discovery in the underlying replication and pathogenesis of SIV which provides a potential target clue for the design of anti-SIV drugs. Our results will inspire basic study on SIV infection and drive the understanding for replication and pathogenesis of SIV to control this disease.

Phosphorylation of the influenza A virus protein PB1-F2 by PKC is crucial for apoptosis promoting functions in monocytes

The 11(th) influenza A virus (IAV) protein PB1-F2 is encoded by an alternative reading frame of the PB1 polymerase gene and found in the nucleus, cytosol and at the mitochondria of infected cells, the latter is consistent with experimental evidence for its pro-apoptotic function. Here, the function of PB1-F2 as a phosphoprotein was characterized. PB1-F2 derived from isolate IAV(PR8) and synthetic fragments thereof were phosphorylated in vitro by purified protein kinase C (PKC) and cellular extract. Constitutively active PKCalpha interacts with PB1-F2 in yeast two-hybrid assays. (32)P radiolabelling of transfected 293T cells revealed that phosphorylation of PB1-F2 is sensitive to inhibitors of PKC and could be increased by the PKC activator PMA. ESI-MS analysis and cellular expression of PB1-F2 mutants identified the positions Ser-35 as the major and the Thr-27 as an alternative PKC phosphorylation site. Infection of MDCK cells with recombinant IAV(PR8) lacking these PKC sites abrogated phosphorylation of PB1-F2 in vivo. Furthermore, infection of primary human monocytes with mutant viruses lacking these PB1-F2 phosphorylation sites resulted in impaired caspase 3 activation and reduced progeny virus titres, indicating that the integrity of the identified phosphorylation sites is crucial for a cell-specific function of PB1-F2 during virus replication.

Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores

The human CMV UL37x1-encoded protein, also known as the viral mitochondria-localized inhibitor of apoptosis, traffics to the endoplasmic reticulum and mitochondria of infected cells. It induces the fragmentation of mitochondria and blocks apoptosis. We demonstrate that UL37x1 protein mobilizes Ca(2+) from the endoplasmic reticulum into the cytosol. This release is accompanied by cell rounding, cell swelling, and reorganization of the actin cytoskeleton, and these morphological changes can be substantially blocked by a Ca(2+) chelating agent. The UL37x1-mediated release of Ca(2+) from the endoplasmic reticulum likely has multiple consequences, including induction of the unfolded protein response, modulation of mitochondrial function, induction of mitochondrial fission, and protection against apoptotic stimuli.

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells.

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria-host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6+, F1+) and JRS145 (M6-, F1+ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6+, F1- mutant) or SAM2 (M6-, F1- mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-xL were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.

Functional expression of a fragment of human dihydroorotate dehydrogenase by means of the baculovirus expression vector system, and kinetic investigation of the purified recombinant enzyme.

Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus that contained the human cDNA fragment for this protein. After virus infection and protein expression in Trichoplusia ni cells (BTI-Tn-5B1-4), the subcellular distribution of the recombinant dihydroorotate dehydrogenase was determined by two distinct enzyme-activity assays and by Western blot analysis with anti-(dihydroorotate dehydrogenase) Ig. The targeting of the recombinant protein to the mitochondria of the insect cells was verified. The activity of the recombinant enzyme in the mitochondria of infected cells was about 740-fold above the level of dihydroorotate dehydrogenase in human liver mitochondria. In a three-step procedure, dihydroorotate dehydrogenase was purified to a specific activity of greater than 50 U/mg. Size-exclusion chromatography showed a molecular mass of 42 kDa and confirmed the existence of the fully active enzyme as a monomeric species. Fluorimetric cofactor analysis revealed the presence of FMN in recombinant dihydroorotate dehydrogenase. By kinetics analysis, Km values for dihydroorotate and ubiquinone-50 were found to be 4 microM and 9.9 microM, respectively, while Km values for dihydroorotate and decylubiquinone were 9.4 microM and 13.7 microM, respectively. The applied expression system will allow preparation of large quantities of the enzyme for structure and function studies. Purified recombinant human dihytdroorotate dehydrogenase was tested for its sensitivity to a reported inhibitor A77 1726 (2-hydroxyethyliden-cyanoacetic acid 4-trifluoromethyl anilide), which is the active metabolite of the isoxazole derivative leflunomide [5-methyl-N-(4-trifluoromethyl-phenyl)-4-isoxazole carboximide]. An IC50 value of 1 microM was determined for A77 1726. Detailed kinetics experiments revealed uncompetitive inhibition with respect to dihydroorotate (Kiu = 0.94 microM) and non-competitive inhibition with respect to decylubiquinone (Kic = 1.09 microM, Kiu = 1.05 microM). These results suggest that the immunomodulating agent A77 1726 (currently in clinical phase III studies for the treatment of rheumatoid arthritis) is a very good inhibitor of human dihydroorotate dehydrogenase.

Localization of HIV RNA in mitochondria of infected cells: potential role in cytopathogenicity.

The intracellular distribution of HIV-1 RNA transcripts in infected cells was studied using in situ hybridization detected by electron microscopy and cellular fractionation. Although viral RNA and core protein could be detected throughout the cytoplasm and nucleus, viral RNA was found in significantly increased amounts in mitochondria relative to the cytoplasm and nucleus. In contrast, cellular poly(A) RNA or viral gag proteins were not increased in the mitochondria. A cell line containing an integrated latent genome that could be induced to express viral RNA after phorbol ester stimulation showed an increase in viral RNA accumulation in mitochondria parallel with the increase in HIV expression levels. Concomitant with HIV expression, there was a decrease in mitochondrial viability. Using immunofluorescent markers to detect probes to HIV RNA transcripts and antibodies to mitochondrial proteins simultaneously in single cells, there was an inverse relationship between the amount of viral RNA and mitochondrial integrity. High levels of viral RNA in mitochondria were found in acutely (but not chronically) infected cells. We propose that HIV RNA import into mitochondria can compromise mitochondrial function.

Particle Morphology and Cytopathology of Galinsoga Mosaic Virus

SUMMARY Galinsoga mosaic virus (GMV) has isometric particles about 34 nm in diameter when examined in preparations negatively stained with uranyl acetate. They are similar to the particles of viruses belonging to the tombusvirus and dianthovirus groups but not to those of the sobemovirus group. GMV induced the development of large multivesicular bodies from the mitochondria in infected cells which only superficially resembled those observed in cells infected by tombusviruses. The possible taxonomic and functional significance of the multivesicular bodies is discussed and it is concluded that GMV does not readily fit into any of the established virus taxonomic groups.

Stimulation of mitochondrial DNA synthesis as an early function of herpes simplex virus

Infection of HeLa cells by herpes simplex virus type 1 (HSV) results in an enhancement of thymidine incorporation into mitochondrial DNA during the initial period post infectionem (p.i.) [l] whereas that into cellular DNA of nuclear origin is progressively inhibited [2]. Since earlier findings did not exclude a possible involvement of virus-induced nucleotide pool alterations as a basis for the mitochondrial reaction [3] thymidine-kinase-less (tk-) cells were employed in the present study to examine this passibility in a suitable cell culture system. Furthermore, pertinent to the analysis of the phenomenon, was to clarify whether enhancement of mitochondrial DNA synthesis in virus-infected cells could proceed in the absence of viral DNA replication. Our results show an enhanced thymidine incorporation into mitochondrial DNA as compared to appropriate controls by HSV-infected tkmouse cells and isolated mitochondria of infected cells as well. In addition, whereas the effect of virus infection on mitochondrial DNA synthesis could not be

Role of Mitochondria in the Formation of X-bodies in Cells of Nicotiana clevelandii infected by Tobacco Rattle Viruses

SUMMARY Non-crystalline inclusions, referred to as X-bodies, developed in leaf-hair cells of Nicotiana clevelandii infected with a RNA-producing defective isolate (cam/df) of tobacco rattle virus but not in those infected with the nucleo- protein particle-producing strain (cam) from which cam/df was derived. The X-bodies appeared 3 days after inoculation; some persisted for at least 10 weeks. They were digested by pronase and were rich in RNA, which was protected by proteinaceous material from digestion by pancreatic ribo- nuclease; they did not contain virus coat-protein detectable by fluorescent antibody tests. Electron microscopy of thin sections showed that mitochondria in infected cells developed two abnormalities—peripheral membranous sacs and membrane bounded vacuoles—and then aggregated, together with ribosomes and material containing small darkly staining granules, to form small X- bodies. These fused to form larger X-bodies, in which the mitochondria were further modified and eventually became barely recognizable. Finally, the contents of the X-bodies became increasingly amorphous, and the bodies disintegrated. X-bodies also developed in leaf-hair cells infected with the virus particle- producing strain prn, but were less common and mostly persisted for only a few days. These X-bodies were formed in the same way as those produced by cam/df, but contained in addition small aggregates of virus particles resembling the aggregates found in the cytoplasm in other parts of the same cells. The frequency of formation, and persistence, of X-bodies induced by these tobacco rattle viruses paralleled the severity and persistence of their macroscopic effects on N. clevelandii. The possibility that mitochondria are sites of synthesis of tobacco rattle virus RNA is discussed.