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DNA topoisomerase 1 facilitates the transcription and replication of the Ebola virus genome.

ABSTRACT: Ebola virus (EBOV) protein L (EBOL) acts as a viral RNA-dependent RNA polymerase. To better understand the mechanisms underlying the transcription and replication of the EBOV genome, we sought to identify cellular factors involved in these processes via their coimmunoprecipitation with EBOL and by mass spectrometry. Of 65 candidate proteins identified, we focused on DNA topoisomerase 1 (TOP1), which localizes to the nucleus and unwinds helical DNA. We found that in the presence of EBOL, TOP1 colocalizes and interacts with EBOL in the cytoplasm, where transcription and replication of the EBOV genome occur. Knockdown of TOP1 markedly reduced virus replication and viral polymerase activity. We also found that the phosphodiester bridge-cleaving and recombination activities of TOP1 are required for the polymerase activity of EBOL. These results demonstrate that TOP1 is an important cellular factor for the transcription and replication of the EBOV genome and, as such, plays a key role in the EBOV life cycle.

SUBMITTER: Takahashi K

PROVIDER: S-EPMC3754039 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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DNA topoisomerase 1 facilitates the transcription and replication of the Ebola virus genome.

Takahashi Kei K Halfmann Peter P Oyama Masaaki M Kozuka-Hata Hiroko H Noda Takeshi T Kawaoka Yoshihiro Y

Journal of virology 20130508 16

Ebola virus (EBOV) protein L (EBOL) acts as a viral RNA-dependent RNA polymerase. To better understand the mechanisms underlying the transcription and replication of the EBOV genome, we sought to identify cellular factors involved in these processes via their coimmunoprecipitation with EBOL and by mass spectrometry. Of 65 candidate proteins identified, we focused on DNA topoisomerase 1 (TOP1), which localizes to the nucleus and unwinds helical DNA. We found that in the presence of EBOL, TOP1 col ...[more]

PMID: 23658456

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Project description:Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication.IMPORTANCE Ebola virus and other emerging RNA viruses are significant but unpredictable public health threats. Therapeutic approaches with broad-spectrum activity could provide an attractive response to such infections. We describe a novel assay that can identify small molecules that overcome Ebola virus-encoded innate immune evasion mechanisms. This assay identified as hits cancer chemotherapeutic drugs, including doxorubicin. Follow-up studies provide new insight into how doxorubicin induces interferon (IFN) responses, revealing activation of both the DNA damage response kinase ATM and the DNA sensor cGAS and its partner signaling protein STING. The studies further demonstrate that the ATM and cGAS-STING pathways of IFN induction are a point of vulnerability not only for Ebola virus but for other RNA viruses as well, because viral innate immune antagonists consistently fail to block these signals. These studies thereby define a novel avenue for therapeutic intervention against emerging RNA viruses.

Project description:In bacteria the concurrence of DNA replication and transcription leads to potentially deleterious encounters between the two machineries, which can occur in either the head-on (lagging strand genes) or co-directional (leading strand genes) orientations. These conflicts lead to replication fork stalling and can destabilize the genome. Both eukaryotic and prokaryotic cells possess resolution factors that reduce the severity of these encounters. Though Escherichia coli accessory helicases have been implicated in the mitigation of head-on conflicts, direct evidence of these proteins mitigating co-directional conflicts is lacking. Furthermore, the endogenous chromosomal regions where these helicases act, and the mechanism of recruitment, have not been identified. We show that the essential Bacillus subtilis accessory helicase PcrA aids replication progression through protein coding genes of both head-on and co-directional orientations, as well as rRNA and tRNA genes. ChIP-Seq experiments show that co-directional conflicts at highly transcribed rRNA, tRNA, and head-on protein coding genes are major targets of PcrA activity on the chromosome. Partial depletion of PcrA renders cells extremely sensitive to head-on conflicts, linking the essential function of PcrA to conflict resolution. Furthermore, ablating PcrA's ATPase/helicase activity simultaneously increases its association with conflict regions, while incapacitating its ability to mitigate conflicts, and leads to cell death. In contrast, disruption of PcrA's C-terminal RNA polymerase interaction domain does not impact its ability to mitigate conflicts between replication and transcription, its association with conflict regions, or cell survival. Altogether, this work establishes PcrA as an essential factor involved in mitigating transcription-replication conflicts and identifies chromosomal regions where it routinely acts. As both conflicts and accessory helicases are found in all domains of life, these results are broadly relevant.

Dataset Information

DNA topoisomerase 1 facilitates the transcription and replication of the Ebola virus genome.

Publications

DNA topoisomerase 1 facilitates the transcription and replication of the Ebola virus genome.

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