Effect of measles virus (MV) on mitochondrial respiration.
Background & objectives: Studies of interaction between viruses
and mitochondria have shown that viruses can affect the mitochondria.
Also linkage between human diseases and mitochondrial dysfunction has
been revealed. We studied the effect of measles virus (MV) on cell
respiration in MV infected Hela cells to see any possible changes in
Methods: Total cell respiration (TCR) of MV infected (10 pfu/cell) HeLa cells was measured using oxygen electrode (OE). Cell lines were grown in growth medium. Virus titration was done in VERO cell line using plaque assay method.
Results: TCR of HeLa cells was not significantly changed post MV infection and was similar to most infected cells.
Interpretation & conclusion: Infection with measles virus did not reveal any significant effect on mitochondrial respiration in HeLa cells. Further studies need to be done using advanced techniques to throw more light on this aspect.
Key words Apoptosis--measles virus--mitochondria--respiration
Measles virus (Physiological aspects)
Measles virus (Research)
Mitochondria (Physiological aspects)
|Publication:||Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2010 Indian Council of Medical Research ISSN: 0971-5916|
|Issue:||Date: Jan, 2010 Source Volume: 131 Source Issue: 1|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: Iran Geographic Code: 7IRAN Iran|
Many studies have shown that viruses and viral proteins may affect
mitochondrial function, induce mitochondrial abnormalities or affect
apoptosis, sometimes by effects mediated at the mitochondria (1-11).
Mitochondria play a crucial role in apoptotic process (12), which can be
viewed as part of the host cell response to infection. Since cell
suicide would reduce the yield of any infecting virus it would be widely
applicable as a defence mechanism (13). Apoptosis also plays an
essential role in pathogenesis of many diseases caused by viruses.
Several viruses are also known to affect apoptotic pathways which permit
the maintenance of latent viral infections or enhance the efficiency of
viral replication (14). The basic hypothesis for this study was that
viruses may play an important key role in aetiology of diseases in which
energy generation is impaired. One example of such a situation might be
provided by chronic fatigue syndrome (CFS). CFS remains a poorly
understood and controversial disease, because no causal agents have been
identified. Diagnostic laboratory tests have poor sensitivity and
specificity (15). The aetiology and pathogenesis of CFS still remains
unknown thus CFS could be a state of viral infections, most probably
persistent infections. Measles virus (MV) is an extremely contagious
agent inducing acute illness (16), belongs to the genus morbillivirus
within the family Paramyxoviridae with a non segmented single-stranded
RNA molecule of negative sense (17). Several studies have revealed that
MV can induce apoptosis in infected cells (18-23). This might well
proceed by mitochondrial interaction and therefore the purpose of this
study was to determine any possible change/s in cellular oxygen
consumption post MV infection in Hela cells to determine any
mitochondrial effect of this virus.
Material & Methods
Measles virus (Edmonston strain, kindly provided by Prof. V. ter Meulen, Wu'rzburg, Germany) was grown in HeLa (human cervical epithelial carcinoma) cells. VERO (African green monkey kidney) cell line was used for viral titration (24). All cell lines were obtained from European Collection of Cell Cultures (ECACC, UK). Cell lines were grown in growth medium [Foetal bovine serum heat inactivated (FBS), penicillin/ streptomycin (P/S), non essential amino acid (NEA) and minimal essential medium (MEM), (all obtained from Gibco, USA)] and were incubated at 37[degrees]C in a humidified incubator in an atmosphere of 5 per cent C[O.sub.2] until confluent. For harvesting of the cells, monolayers were detached by rinsing in PBS (Invitrogen) and then in 5 ml trypsin-versin solution (Invitrogen, UK). Most of this solution was then poured off and the cells allowed standing in the residual liquid until detached. The cells were added into 10 per cent FBS medium and collected by centrifugation for 5 min at 1500 x g before re-suspension in fresh medium. Cells were transferred to fresh flasks achieving a subculture ratio of 1/10 and were grown as above. Confluent monolayers of cell in 75 [cm.sup.2] tissue culture flasks were rinsed with PBS and infected with 1 ml virus inoculum (10 pfu/cell). The infected flasks were kept at room temperature for 1 h. Cells were incubated in their respective media supplemented with 2 per cent FBS at 37[degrees]C in incubator until cytopathic effect (CPE) was observed (3 days for measles). Cell associated virus was released by one cycle of freeze-thaw: flasks were placed at -70[degrees]C for 1 h to freeze and allowed to thaw on the bench at room temperature. Cell debris was thoroughly re-suspended using a pipette and clarified at 377 x g for 5 min. Supernatant for inoculation was collected and snap frozen for storage at -70[degrees]C (2.5 [cm.sup.2]). Dishes were seeded with cells, incubated overnight at 37[degrees]C, and checked for confluence. The plaque assay method (25) was used for titration of the virus. For oxygen measurement procedure, the cells were infected with MV (10 pfu/cell) and allowed to adsorb for 1 h at room temperature with gentle rocking. Inocula were replaced with MEM supplemented by 2 per cent FB S and incubated for the desired time periods (6, 12, 24, 48 h). At each time point infected and control cell sheets were gently detached from the flasks using a scraper, re-suspended in 0.4 ml PBS and transferred into the oxygen electrode (OE) chamber. The rate of oxygen consumption or total cell respiration (TCR) was measured by a Clark-type OE according to manufacturer's Instruction (Oxyg32, Hansatech Instruments Ltd, UK).
Cultures were assessed visually for the appearance of CPE. Typical MV-induced CPE was seen as cell fusion yielding syncytia (Fig. 1). The CPE changes commenced after 60 h post-infection and considerable CPE appeared after 3 days post-infection. The respiratory rate was measured after 6, 12, 24 and 48 h post MV infection, when no CPE was evident in the cultures (Fig. 2). Basal respiration in mock-infected and infected HeLa cells was not significantly different.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Studies done on viral effects on cell respiration in vitro (26,21) showed that poliovirus and human herpes virus type one (HHV-1) infections of the cells caused a rapid decrease in total cellular respiration, which was related to mitochondrial dysfunction. Probably such effects will be of limited significance within the context of an active, productive and lytic infection, where the host cell has no long-term future. However, infected cells clearly survive in the context of persistent infections; this must indicate that apoptosis is blocked in such cases and in these instances; long-term reduction in a cell's ability to generate energy might adversely affect cell function. Thus, the hypothesis for present study was that similar effects might be induced by diverse viruses and may underlie conditions where no single virus aetiology has been proven. This study was done to determine whether viruses can indeed affect mitochondrial energy generation. It has been shown that MV induces mitochondrial structural abnormalities in dendrites during persistent infection of the brain termed subacute sclerosing panencephalitis (SSPE)28 and also has been associated with apoptosis. Results of the present study showed that MV infection did not decrease total cellular respiration in HeLa cells. Further studies need to be done to clarify this issue.
Author thanks Mashhad University of Medical Sciences (MUMS), Iran, for financial support and Drs M.J. Carter and G.E.N. Kass from University of Surrey, UK for scientific consultation.
Received June 6, 2008
(1.) Wang Y, Lau SH, Sham JS, Wu MC, Wang T, Guan XY. Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellular carcinogenesis. Oncogene 2004; 23 : 142-8.
(2.) Sharp TV, Wang HW, Koumi A, Hollyman D, Endo Y, Ye HT, et al. K15 protein of Kaposi's sarcoma-associated herpesvirus is latently expressed and binds to HAX-1, a protein with antiapoptotic function. J Virol 2002; 76 : 802-16.
(3.) Lefebvre L, Ciminale V, Vanderplasschen A, D'Agostino D, Burny A, Willems L, et al. Subcellular localization of the bovine leukemia virus R3 and G4 accessory proteins. J Virol 2002; 76 : 7843-54.
(4.) Nudson WA, Rovnak J, Buechner M, Quackenbush SL. Walleye dermal sarcoma virus Orf C is targeted to the mitochondria. J Gen Virol 2003; 84 : 375-81.
(5.) Liu J, Wei T, Kwang J. Avian encephalomyelitis virus induces apoptosis via major structural protein VP3. Virology 2002; 300 : 39-49.
(6.) Liu J, Wei T, Kwang J. Avian encephalomyelitis virus nonstructural protein 2C induces apoptosis by activating cytochrome c/caspase-9 pathway. Virology 2004; 318 : 169-82.
(7.) Erdtmann L, Franck N, Lerat H, Le Seyec J, Gilot D, Cannie I, et al. The hepatitis C virus NS2 protein is an inhibitor of CIDE-B-induced apoptosis. J Biol Chem 2003; 278 : 18256-64.
(8.) Chanturiya AN, Basanez G, Schubert U, Henklein P, Yewdell JW, Zimmerberg J. PB1-F2, an influenza A virus-encoded pro apoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes. J Virol 2004; 78 : 6304-12.
(9.) Chen W, Calvo P.A, Malide D, Gibbs J, Schubert U, Bacik I, et al. A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 2001; 7 : 1306-12.
(10.) Zander K, Sherman MP, Tessmer U, Bruns K, Wray V, Prechtel AT, et al. Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression. J Biol Chem 2003; 278 : 43202-13.
(11.) Everett H, Barry M, Lee SF, Sun X, Graham K, Stone J, et al. M11L: a novel mitochondria-localized protein of myxoma virus that blocks apoptosis of infected leukocytes. J Exp Med 2000; 191 : 1487-98.
(12.) Mayer B, Oberbauer R. Mitochondrial regulation of apoptosis. News Physiol Sci 2003; 18 : 89-94.
(13.) McFadden G, Lalani A, Everett H, Nash P, Xu X. Virus-encoded receptors for cytokines and chemokines. Semin Cell Dev Biol 1998; 9 : 359-68.
(14.) Roulston A, Marcellus RC, Branton PE. Viruses and apoptosis. Annu Rev Microbiol 1999; 53 : 577-628.
(15.) Holmes GP, Kaplan JE, Gantz NM, Komaroff AL, Schonberger LB, Straus SE, et al. Chronic fatigue syndrome: a working case definition. Ann Intern Med 1988; 108 : 387-9.
(16.) Bedford H. Measles: the disease and its prevention. Nurs Stand 2003; 17 : 46-52.
(17.) Bellini WJ, Rota JS, Rota PA. Virology of measles virus. J Infect Dis 1994; 170 (Suppl 1) : S15-23.
(18.) Vuorinen T, Peri P, Vainionpaa R. Measles virus induces apoptosis in uninfected bystander T cells and leads to granzyme B and caspase activation in peripheral blood mononuclear cell cultures. Eur J Clin Invest 2003; 33 : 434-42.
(19.) Servet-Delprat C, Vidalain PO, Azocar O, Le Deiser F, Fischer A, Rabourdin-Combe C. Consequences of Fas-mediated human dendritic cell apoptosis induced by measles virus. J Virol 2000; 74 : 4387-93.
(20.) McQuaid S, McMahon J, Herron B, Cosby SL. Apoptosis in measles virus-infected human central nervous system tissues. Neuropathol Appl Neurobiol 1997; 23 : 218-24.
(21.) Ito M, Yamamoto T, Watanabe M, Ihara T, Kamiya H, Sakurai M. Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA. FEMS Immunol Med Microbiol 1996; 15 : 115-22.
(22.) Auwaerter PG, Kaneshima H, McCune JM, Wiegand G, Griffin DE. Measles virus infection of thymic epithelium in the SCID-hu mouse leads to thymocyte apoptosis. J Virol 1996; 70 : 3734-40.
(23.) Esolen LM, Park SW, Hardwick JM, Griffin DE. Apoptosis as a cause of death in measles virus-infected cells. J Virol 1995; 69 : 3955-8.
(24.) Shishido A, Yamanouchi K, Hikita M, Sato T, Fukuda A, Kobune F. Development of a cell culture system susceptible to measle, canine distemper, and rinderpest viruses. Arch Gesamte Virusforsch 1967; 22 : 364-80.
(25.) Dulbecco R, Vogt M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med 1954; 99 : 167-82.
(26.) Koundouris A, Kass GE, Johnson CR, Boxall A, Sanders PG, Carter MJ. Poliovirus induces an early impairment of mitochondrial function by inhibiting succinate dehydrogenase activity. Biochem Biophys Res Commun 2000; 271 : 610-4.
(27.) Derakhshan M, Willcocks MM, Salako MA, Kass GE, Carter MJ. Human herpesvirus 1 protein US3 induces an inhibition of mitochondrial electron transport. J Gen Virol 2006; 87 : 2155-9.
(28.) Paula-Barbosa MM, Tavares MA, Borges MM. Mitochondrial abnormalities in cortical dendrites from patients with early forms of subacute sclerosing panencephalitis (SSPE). Acta Neuropathol 1984; 63 : 117-22.
Reprint requests: Dr Mohammad Derakhshan, Assistant Professor of Clinical Virology, Microbiology & Virology Research Centre Avicenna Research Institute & Department of Clinical Bacteriology & Virology, Ghaem University Hospital Faculty of Medicine, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran e-mail: email@example.com, firstname.lastname@example.org
Microbiology & Virology Research Centre, Avicenna Research Institute & Department of Clinical Bacteriology & Virology, Ghaem University Hospital, Faculty of Medicine, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran
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