Increased ATP generation in the host cell is required for efficient vaccinia virus production

Abstract

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 microM 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.

Figure 1

Western blotting analysis of ND4 expression in vaccinia virus infected cells. (A) The HeLa cells were infected with MOI = 1 (or 5 or 20) of vaccinia virus for various time periods, as indicated. ND4 expression (marked by an arrow) was analyzed by Western blotting. ERK2 protein was served as a loading control. Occasionally, two non-specific bands (one larger and one smaller than ND4, indicated by thin lines) were detected in the samples. (B) ND4 was also up-regulated in HuH7 cells after vaccinia virus infection. The ND4 protein is marked by an arrow while a non-specific band is indicated by a thin line.

Figure 2

Western blotting analysis of drug effects on ND4 expression in VV infected cells. (A) HeLa cells were infected with VV (MOI = 1) for the indicated times in the presence or absence of 45 μM apigenin. Actin was used as a loading control. (B) The ND4 up-regulation by vaccinia virus infection (MOI = 1) was inhibited by the DNA polymerase inhibitor, ara C. Cells were incubated with ara C (40 μg/ml), an inhibitor of poxviral DNA synthesis, 30 min before VV infection and throughout the infection. The ND4 protein is indicated with an arrow while non-specific bands are indicated by thin lines. ERK2 protein was used as a loading control.

Figure 3

Analysis of several nuclear genes encoding mitochondrial electron transport chain proteins after VV infection by Western blotting analysis. The Roman numbers in parentheses represent the respiratory complex in which the gene participates. ERK2 protein was used as a loading control.

Figure 4

The intracellular ATP concentration was significantly increased after VV infection. Intracellular ATP concentration was measured at the indicated times (1, 4, 8, 10, 12, and 21 hr) after vaccinia virus infection in HeLa cells (MOI = 1 in Fig. 4A and MOI = 5 in Fig. 4B). Experiments were performed three times in duplicate.

Figure 5

(A) The intracellular ATP concentration of vaccinia virus infected (MOI = 1) and oligomycin-treated cells. ATP concentration was measured 13 hrs after vaccinia virus infection in the presence of various concentration of oligomycin. Experiments were performed three times in duplicate. (B) Western blotting analysis of Vaccinia viral protein expression (A type inclusion protein and IMV heparin binding surface protein) in the presence of 4.5 μM oligomycin. ERK2 protein was used as a loading control. (C) Both the intracellular and the extracellular vaccinia viruses were reduced 13 hr after virus infection in the presence of 4.5 μM oligomycin. Experiments of plague assay were performed three times in triplicate.

Figure 6

(A) The expression of Bcl-2 was not elevated during vaccinia virus infection. HeLa cells were infected with VV (MOI = 1) for the indicated times, then cell lysates were analyzed by Western blotting. ERK2 protein was used as a loading control. (B) Western blotting analysis of Bcl-2 in HeLa cells stably transfected with different shRNA clones against Bcl-2. HeLa cells with shRNA against the Luciferase gene were used as a control. ERK2 protein was used as a loading control.

Figure 7

(A) Western blotting analysis of ND4 protein in vaccinia virus-infected HeLa cells with shRNAs against Luc or Bcl-2. ERK2 protein was used as a loading control. (B) Intracellular ATP concentration was measured at the indicated times (1, 4, 10, and 21 hr) after vaccinia infection of HeLa cells (MOI = 1). Experiments were performed three times in duplicate.