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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.

SUBMITTER: Chang CW

PROVIDER: S-EPMC2741444 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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

Chang Chia-Wei CW Li Hui-Chun HC Hsu Che-Fang CF Chang Chiao-Yen CY Lo Shih-Yen SY

Journal of biomedical science 20090902

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 ...[more]

PMID: 19725950

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Project description:Apicomplexan parasites actively invade host cells using a mechanism predicted to be powered by a parasite actin-dependent myosin motor. In the model apicomplexan Toxoplasma gondii, inducible knockout of the actin gene, ACT1, was recently demonstrated to limit but not completely abolish invasion. This observation has led to the provocative suggestion that T. gondii possesses alternative, ACT1-independent invasion pathways. Here, we dissected the residual invasive ability of ?act1 parasites. Surprisingly, we were able to detect residual ACT1 protein in inducible ?act1 parasites as long as 5 days after ACT1 deletion. We further found that the longer ?act1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed. Both findings are consistent with the quantity of residual ACT1 retained in ?act1 parasites being responsible for their invasive ability. Furthermore, invasion by the ?act1 parasites was also sensitive to the actin polymerization inhibitor cytochalasin D. Finally, there was no clear defect in attachment to host cells or moving junction formation by ?act1 parasites. However, ?act1 parasites often exhibited delayed entry into host cells, suggesting a defect specific to the penetration stage of invasion. Overall, our results support a model where residual ACT1 protein retained in inducible ?act1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion.The prevailing model for apicomplexan invasion has recently been suggested to require major revision, based on studies where core components of the invasion machinery were genetically disrupted using a Cre-Lox-based inducible knockout system. For the myosin component of the motor thought to power invasion, an alternative parasite myosin was recently demonstrated to functionally compensate for loss of the primary myosin involved in invasion. Here, we highlight a second mechanism that can account for the surprising ability of parasites to invade after genetic disruption of core invasion machinery. Specifically, residual actin protein present in inducible knockout parasites appears able to support their limited invasion of host cells. Our results have important implications for the interpretation of the apicomplexan invasion model and also highlight significant considerations when analyzing the phenotypes of inducible knockout parasites generated using Cre-Lox technology.

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

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

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