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Short Communication |
School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK
Correspondence
Michael J. Carter
m.carter{at}surrey.ac.uk
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ABSTRACT |
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-phase protein synthesis and the inhibition of mitochondrial respiration could be reproduced by ectopic expression of the -phase protein US3. An HHV-1 mutant lacking this protein failed to inhibit oxygen consumption in infected cells relative to controls. It was concluded that US3 was mediating the suppression of mitochondrial respiration following HHV-1 infection. The integrity of the electron-transport chain in HHV-1-infected cells was analysed further and the site of the block in electron transport was located between complexes II and III, a site previously shown to be affected by Poliovirus.
Present address: Cancer Research UK, London EC1M 6BQ, UK. 
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), human T-cell leukemia virus type 1 (D'Agostino et al., 2002) and Rubella virus (Lee et al., 1999), and changes in location are observed in infection by Human herpesvirus 1 (HHV-1, herpes simplex virus 1) and Hepatitis B virus (Murata et al., 2000; Takada et al., 1999). Virus proteins may traffic to mitochondria (Henkler et al., 2001) and affect the function of mitochondrial components (Rahmani et al., 2000). Influenza virus protein PB1-F2 binds to mitochondrial inner membranes, triggering apoptosis (Gibbs et al., 2003; Yamada et al., 2004). However, relatively little is known about how such processes might affect the basic mitochondrial function of energy generation. Norkin (1977) demonstrated that total cellular respiration was reduced by simian virus 40 infection of CV-1 cells, although levels of ATP were maintained. Poliovirus induces a rapid inhibition of host-cell respiration and blocks electron transport between complexes II and III (Koundouris et al., 2000). In this case too, cellular ATP levels remain unaffected. In contrast, ATP levels decline during HHV-1 infection, which has been attributed to mitochondrial dysfunction (Murata et al., 2000). We hypothesized that such effects might be common in virus infection and have tested different viruses. We found that HHV-1 and influenza virus both inhibited the mitochondrial respiratory chain. Further investigation of the HHV-1-induced effect showed that the virus targeted a site between complexes II and III and that this was mediated by protein US3. Cellular respiration was measured by using a Clark-type oxygen electrode (OE) (Hansatech Instruments). Cells were infected with 10 p.f.u. per cell by using the following virus–cell combinations: HHV-1 (strain HFEM, kindly provided by Professor A. C. Minson, Cambridge, UK), Measles virus (Edmonston strain, kindly provided by Professor V. ter Meulen, Würzburg, Germany) and coxsackievirus (strain B3) were grown in HeLa cells; cytomegalovirus (strain Ad 169, kindly provided by Professor A. C. Minson) was cultivated in MRC-5 cells; Feline calicivirus (strain F9) was grown in CRFK cells and influenza virus (strain A/Puerto Rico/8/34, kindly provided by Dr P. Digard, Cambridge, UK) was grown in MDCK cells. Both mock- and virus-infected cells were recovered by scraping and their viability was determined by dye exclusion. Cells (3x106) were washed with Dulbecco's PBS and resuspended in 0.4 ml Dulbecco's PBS for transfer to the OE for measurement of oxygen consumption. Oxygen consumption was attributed entirely to mitochondria, as inhibition of mitochondrial electron transport by antimycin A (3 µg ml–1) abolished measurable uptake completely.
Only HHV-1 and influenza virus (Fig. 1a and b) suppressed cellular respiration. Feline calicivirus, Measles virus, cytomegalovirus and coxsackievirus B4 had no detectable effect (data not shown). HHV-1 was the more potent, reducing oxygen consumption rate by 31 % at 6 h post-infection (p.i.), 54 % at 12 h p.i. and 60 % at 18 h p.i. (Fig. 1a). Influenza virus reduced consumption by 30 % at 6 h p.i. and by 45 % at 12 h p.i. (Fig. 1b). The viabilities of infected and uninfected cells were not significantly different at these times. Inhibition of cell respiration occurred well before the onset of cytopathic effect.
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), which may be responsible for the effect observed. However, the genesis of this effect by HHV-1 was unknown. Therefore, we analysed the integrity of the electron-transport chain in infected cells to locate the site of any blockage. HHV-1-infected (12 h p.i.) or mock-infected HeLa cells were harvested as above, washed twice with mitochondrial respiration buffer (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5 mM HEPES, pH 7.1), resuspended in 0.4 ml mitochondrial respiration buffer and transferred into the OE. Digitonin (0.375 mg ml–1) was added to permeabilize the plasma membranes. Respiration was monitored following the addition of substrate electron donors feeding electrons into known points in the electron-transport chain. When appropriate, electron transport through specific complexes was subsequently blocked by using specific inhibitors to confirm observations. Substrates/inhibitors used were: pyruvate (5 mM) plus malate (5 mM), rotenone (3 µg ml–1), succinate (5 mM), antimycin A (3 µg ml–1), tetramethyl-p-phenylenediamine (TMPD) (0.5 mM) plus ascorbate (1 mM), and sodium azide (5 mM). The results are presented in Fig. 2. Complex I was assessed by the addition of pyruvate plus malate (P/M) to donate electrons to complex I and subsequently by the addition of rotenone to prevent their onward transmission to complex II. We detected no increase in oxygen consumption following pyruvate and malate addition, even in the controls, probably because endogenous substrates were present in excess, saturating flow through this complex. Assessment of complex II by the addition of succinate clearly stimulated oxygen consumption in mock-infected cells. This was blocked by antimycin A (Fig. 2a), demonstrating that electron transfer from complex II to oxygen via complexes III and IV was responsible. This stimulation was absent in HHV-1-infected cells (Fig. 2b) and antimycin A had no inhibitory effect. This confirmed that electrons donated by the succinate were not able to flow on to complex III and located an infection-specific respiratory-chain block between complexes II and III. We then investigated electron flow post-cytochrome c to complex IV by using TMPD/ascorbate. Following the addition of TMPD/ascorbate, both control and infected cells showed an increased rate of oxygen consumption (Fig. 2a, b) and thus electrons were able to flow from cytochrome c to complex IV. As expected, in all cases, sodium azide blocked respiration completely. These data confirmed that complex IV was functional in both infected and mock-infected cells. We concluded that the most likely site of the virus-induced electron-transport block was between complexes II and III, although an additional block at complex I could not be excluded.
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-protein US3 is known to traffic to the mitochondria (Calton et al., 2004) and work by Reynolds et al. (2002) has demonstrated that the equivalent protein made by HHV-1 concentrates in the perinuclear area, the same site to which mitochondria are known to migrate in the infected cell (Murata et al., 2000). Suspicion thus fell on the HHV-1 US3 protein and this was also consistent with the time of onset of the effect, which in our hands occurred during the phase, well before the onset of synthesis of the gamma-phase marker protein glycoprotein D. Consequently, we sought to examine the role of US3 directly by specific expression of this protein inside uninfected cells. In order to avoid effects caused by any carrier or polymerase-expressing viruses, we decided against vaccinia T7-based or baculovirus expression systems and selected instead direct transfection using mRNA produced in vitro. For this purpose, HeLa cells were grown in 75 cm2 tissue-culture flasks, infected with HHV-1 at an m.o.i. of 10 p.f.u. per cell and harvested when a cytopathic effect was visible. Total RNA was extracted by using RNAzol B (Biotecx Laboratories) and the US3 coding sequence was produced by RT-PCR amplification from total RNA. The following primers were designed to introduce unique restriction sites at each end (HindIII and XbaI) for ease of subsequent cloning (underlined): reverse (antisense) primer, 3'-GTCAGTCTAGATCATTTCTGTTGAAACAGCGG-5', and forward (sense) primer, 5'-CGAAGCTTCGAATGGCCTGTCGTAAGTTTT-3'.
The US3 PCR-amplification products were cloned into pGEM-T Easy vector (Promega), verified by sequencing and subsequently excised from the vector by using HindIII and SpeI for forced-orientation ligation into the pSP64 Poly(A) vector (Promega) from which mRNA transcripts could be prepared in vitro using an Ambion mMESSAGE mMACHINE SP6 transcription kit. The gene for luciferase was similarly cloned into pSP64 Poly(A) for use as a control to determine both transfection efficiency and the optimum time of protein expression following transfection. Both transcripts were verified as active by translation of the mRNA to be used in transfection in vitro. In both cases, products of the expected size were derived (data not shown). HeLa cells were transfected with 2 µg mRNA prepared in vitro by using Lipofectin (Invitrogen). Expression of transfected mRNA in the cells was confirmed by reference to the control luciferase, detected by luminometry. Peak expression occurred after 6 h. Transformation efficiency was determined as 80 % by immunostaining of transfected cells with anti-luciferase antibody (Promega). In order to determine the effect of the US3 gene product on mitochondrial respiration, we used both mRNAs in separate transfections and compared these with a ‘no RNA’ control. At 6 h post-transfection, we measured total HeLa cell respiration in all samples in the OE. Transfection with US3 mRNA affected mitochondrial respiration profoundly, resulting in a decrease in total cell respiration of 47 % in comparison with cells transfected with luciferase mRNA, which were indistinguishable from the mock-transfected cells (Fig. 3a).
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). This mutant expresses only the first 68 residues of US3, with aa 69–357 deleted, including all motifs associated with protein kinase function; residues beyond the deletion are frame-shifted. This mutant (termed R7041 in the original report) lacks detectable protein kinase activity, a phenotype that is reversed when the deletion is reverted (Purves et al., 1987). R7041 was constructed in HHV-1 (strain F) virus and it was necessary first to establish that this strain also exhibited the inhibition of mitochondrial respiration that we had observed in strain HFEM. We found that the time of onset of cytopathic effect in both wild-type and mutant virus was indistinguishable from that observed using strain HFEM, and HHV-1 strain F wild-type virus clearly induced an identical effect on respiration, establishing a 49 % reduction by 12 h p.i. In comparison, the US3 deletion mutant failed to inhibit oxygen consumption and respiration was indistinguishable from that of mock-infected cells (Fig. 3b
). As the function of US3 had already been established, it was not necessary to consider the revertant virus in this experiment.
US3 clearly has a role in pre- and post-mitochondrial modulation of apoptosis in HHV-1-infected cells (Asano et al., 1999, 2000; Benetti & Roizman, 2004; Cartier et al., 2003; Geenen et al., 2005; Goshima et al., 1998; Jerome et al., 1999; Leopardi et al., 1997; Munger & Roizman, 2001; Ogg et al., 2004). Here, we have also shown an effect on the mitochondria themselves. It is becoming increasingly apparent that many viruses interact with mitochondria in the infected cell, possibly in an attempt to modulate the induction of apoptosis in the host. Such protein interactions may have an effect on mitochondrial function, whether or not this is their primary role. Mitochondrial dysfunction may be relatively insignificant in acute infection unless it reduces energy supply sufficiently to impair virus replication. However, effects such as these may gain in importance in the context of longer-term persistent or chronic infections, where the cell's energy balance may be subtly altered, perhaps affecting cell function. For example, following infection with Hepatitis C virus, oxidative injury occurs through the generation of reactive oxygen species as a direct result of mitochondrial dysfunction (Okuda et al., 2002). Hepatitis C virus core protein is known to traffic to mitochondria (Schwer et al., 2004) and persistent expression of this protein in transgenic mice leads to alterations in mitochondrial appearance and, ultimately, damage to heart muscle and cardiomyopathy (Omura et al., 2005). Similar effects might provide a common pathway for host-cell functional impairment operated by diverse viruses in different host cells and should be considered where virological explanations for cellular dysfunction are suspected, but evidence for causation by one particular type of virus is weak. This may be relevant for instance in suspected virological aetiology of chronic fatigue syndrome.
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Received 14 February 2006;
accepted 30 March 2006.
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, Mock-infected cells; 
, virus-infected cells. (b) Influenza virus infection of MDCK cells. Filled columns, mock-infected cells; shaded columns, virus-infected cells. Asterisks indicate significant divergence from the control values: *, P<0.05; **, P<0.01; ***, P<0.001.
