Increased ATP generation in the host cell is required for efficient vaccinia virus production
© Chang et al; licensee BioMed Central Ltd. 2009
Received: 25 December 2008
Accepted: 2 September 2009
Published: 2 September 2009
To search for cellular genes up-regulated by vaccinia virus (VV) infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR) assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.
Vaccinia virus (VV), a member of the Poxviridae family, is an enveloped, DNA virus with a genome of 192 kb encoding about 200 proteins . Various cell lines can be infected by VV, including HeLa, CV-1, mouse L, and chicken CEF cells [2, 3]. VV causes major changes in host cell machinery shortly after infection, and cytopathic effects (CPE) are observed several hours after infection with VV [2–4]. VV infection modulates host cell gene expression: several previous studies have shown that mRNA synthesis in the host cells was inhibited immediately after VV infection [5, 6]. Microarray analysis showed that around 90% of the host genes were down-regulated after VV infection, including genes involved in DNA replication, transcription, translation, apoptosis, and the proteasome-ubiquitin degradation pathway [7, 8]. Only a smaller fraction of host genes were up-regulated after VV infection, including WASP protein, and genes implicated in immune responses [7, 8].
Several viral factors of VV utilize ATP and several steps in viral multiplication of VV require ATP [9–14]. ATP is also required for DNA packaging and capsid maturation of herpes simplex virus, for capsid assembly and release of type D retrovirus, for capsid assembly of human immunodeficiency virus, and for budding of influenza virus [15–18]. Therefore, it was expected that viral factors would modulate cellular energetics to benefit the virus, though this area is understudied .
In this study, the possible up-regulation of host cell genes after VV infection was analyzed by differential display-reverse transcriptase-polymerase chain reaction (ddRT-PCR), a simple technique with high sensitivity and specificity http://www.seegene.com. Two mitochondrial genes involved in the electron transport chain (ND4 and COII) to generate ATP were found to be up-regulated after VV infection using this assay.
Materials and methods
HeLa cells, MDCK cells, HuH7 cells and Con1 cells with full-length HCV genome were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS),100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, USA) . HCV sub-genomic replicon cells were cultured in DMEM with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 400 μg/ml G418 . HepG2 and 1.3 × ES2 HepG2 (HBV) were cultured in DMEM containing 10% FBS, 100 U/ml penicillin,100 μg/ml streptomycin and 1% non-essential amino acids (Gibco, USA) . All cultured cells were maintained at 37°C with 5% CO2.
Vaccinia virus WR strain was used to infect HeLa cells in this study, following previously published procedures for virus amplification and plaque assay [23, 24]. Cytosine arabosinide (ara C), where used, was added to the cells at a concentration of 40 μg/ml .
Influenza A virus WSN33 was used to infect MDCK cells following previously published procedures for virus amplification and plaque assay .
Plasmid construction and DNA transfection
To clone the DNA fragment for N1L gene coding region, vaccinia genomic DNA was used as template and forward and reverse PCR primers (5'-CGGAATTCATGAGGACTCTACTTAT-3' and 5'-TGCTCTAGATTTTTCACCATATAGATC-3') were used to amplify the gene fragment. After PCR, the DNA fragment was digested by restriction enzymes (Eco RI/Xba I) and cloned into the expression vector pcDNA3.1-V5-His A (linearized by Eco RI/Xba I). This expression plasmid was verified by sequencing. An Exgen 500 kit (Fermentas, USA) was used to transfect DNA into HeLa cells following the manufacturer's instructions.
RNA extraction and ddRT-PCR
Total RNAs were extracted from HeLa cells 21 hr after VV infection (MOI = 1) using an RNeasy Mini kit (Qiagen, Germany) following the manufacturer's instructions. The ddRT-PCR assay was performed using a GeneFishing DEG Premix kit (Seegene, Korea), following the manufacturer's instructions.
Western blotting analysis
Our previous procedures were followed for Western blotting analysis [27, 28]. A rabbit polyclonal antibody against ERK-2, a mouse monoclonal antibody against Bcl-2 and goat antibodies against ND4 and COII were purchased from Santa Cruz Biotechnology (USA). Antibodies against SDHA, ATP5O, SDHB, COVc were purchased from Abcam company (UK). Rabbit antibodies against vaccinia viral proteins (A type inclusion protein and IMV heparin binding surface protein) were generated in the lab.
Measurement of ATP production
Ten hours after 2 × 105 HeLa cells were seeded in one 35-mm culture dish, cells were infected with VV (MOI = 1 or 5). Intracellular ATP was then analyzed at different time points (virus-infected cells versus non-infected cells) using the ATP bioluminescence assay kit HS II (Roche, Germany) to determine ATP.
RNAi experiments were performed using the lentiviral expressing system http://rnai.genmed.sinica.edu.tw, following the manufacturer's instructions. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica.
Elevated expression of mitochondrial genes ND4 and COII after vaccinia virus infection
Apigenin and AraC blocked ND4 induction after vaccinia virus infection
Analysis of several nuclear genes encoding mitochondrial electron transport chain proteins after VV infection
Intracellular ATP generation increased after vaccinia virus infection
Oligomycin, an inhibitor of ATP generation, reduced the production of vaccinia virus
Vaccinia virus infection induces ND4 expression through a Bcl-2 independent pathway
Vaccinia virus N1L protein was reported to have a similar secondary (though not primary) structure to Bcl-2 protein . To determine whether N1L may be responsible for the up-regulation of ND4 and COII after VV infection, the N1L gene fragment was cloned and expressed in the cells. No up-regulation of ND4 or COII was detected in the presence of N1L protein (Additional file 6).
In this study, at least some of the genes involved in ATP generation were found to be up-regulated after VV infection (Fig. 1). Only two viral genes were detected in our ddRT-PCR assay among about 200 proteins vaccinia virus encoded (Additional file 1). Thus, only a small portion of differentially genes was identified by this assay. Therefore, it is not surprising that only ND4 and COII genes rather than all 12 mRNA molecules in the same polycistronic transcript were identified . Host cell genes up-regulated after vaccinia virus infection found in this study were not the same as those found in previous microarray assays, which suggests that ddRT-PCR and microarray assays should be used in tandem for a more complete analysis of differential gene expression between mock-infected and virus-infected cells [7, 8].
Our results demonstrate that ATP generation did increase after vaccinia virus infection (Fig. 4). Our results (Figs. 1, 3, and 4) also indicate that increased ATP generation did not require the up-regulation of all the proteins involved in mitochondrial electron transport chain. Up-regulation of mitochondrial-encoded rather than nuclear-encoded proteins involved in mitochondrial electron transport chain could increase ATP generation. This may suggest that mitochondrial-encoded proteins rather than nuclear-encoded ones involved in electron transport chain are rate-determining proteins in ATP generation.
Our results demonstrate that increased ATP production is essential for efficient VV production (Fig. 5). It is not surprising that viral factors would modulate cellular energetics to benefit the virus, though this area is understudied . To our knowledge, this study is the first report to demonstrate that virus infection could up-regulate the expression of genes involved in ATP generation and increase ATP production. It is interesting to note that the intracellular mature virions were more sensitive than the extracellular enveloped virions to oligomycin treatment (Fig. 5C). This may suggest that the assembly of intracellular mature virions is more ATP-dependent .
ATP is required for the budding of influenza virus . Similar up-regulation of ND4 expression after VV infection was not detected after replication of hepatitis B and C viruses or infection with influenza A virus (Additional file 2). One possibility is that there was no need to increase ATP generation because there was already enough existing energy source for multiplication of these viruses. Alternatively, increased ATP generation in cells may still occur here after infection with these viruses though the up-regulation of ND4 is smaller than that caused by VV infection.
No amplification of mitochondrial DNA after vaccinia virus infection was observed (data not shown). Therefore, the up-regulation of ND4 after virus infection is possibly through transcriptional regulation. However, little is known about the regulation of mitochondrial transcription . Bcl-2 family proteins are implicated in regulating cellular bioenergetics, perhaps by regulating the availability of mitochondrially produced ATP . However, the amount of Bcl-2 protein in the host cell did not increase after VV infection (Fig. 6A). Induction of ND4 protein and increased ATP generation are still occurred after VV infection in HeLa cells with reduced amount of Bcl-2 protein (Fig. 7A and 7B). VV N1L protein has been reported to have similar secondary structure as Bcl-2 protein, but not primary structure, and may influence ATP levels in vivo [35, 36]. However, neither host ND4 nor COII genes were up-regulated by N1L protein (Additional file 6), which is consistent with a previous report showed that N1L did not interact with mitochondria . These results indicate that increased ATP generation after VV infection is a Bcl-2 independent event. Further studies are needed to clarify the mechanism(s) of the up-regulation of genes involved in ATP production after VV infection.
The up-regulation of ND4 expression was reduced by apigenin and ara C (Fig. 2), suggesting that event(s) occurring after viral DNA replication are responsible for the up-regulation of ND4 expression. These results are in agreement with the observation that neither ND4 nor COII gene was up-regulated by N1L protein (Additional file 6) since N1L is an early gene product .
In summary, at least some of genes involved in ATP generation were up-regulated by VV infection, and in turn, cellular ATP generation was increased. This increased ATP generation was required for efficient VV production.
We thank Dr. Wen Chang for providing Vaccinia virus WR strain, Dr. Charles M. Rice for providing the Con1 cells, Dr. J.-H. Ou for providing the HCV sub-genomic RNA cells, Dr. King-Song Jeng for providing the 1.3 × ES2 HepG2 (HBV) cells, and Dr. George G. Brownlee for providing 12 plasmids to generate influenza A virus. We thank Dr Harry Wilson of Academia Sinica for manuscript editing. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, supported by grants from the NSC National Research Program for Genomic Medicine (NSC 94-3112-B-001-003 and NSC 94-3112-B-001-018-Y. This work was supported by grants from the National Science Council of Taiwan (NSC953112B320001-02) and from the Tzu Chi University (TCIRP96004-05) to Dr. Shih-Yen Lo.
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