Mihin Ebolavirus tarvitse isäntäsolun DNA topoisomerasia?

J Virol. 2013 Aug;87(16):8862-9. doi: 10.1128/JVI.03544-12. Epub 2013 May 8.

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

Takahashi K¹, Halfmann P, Oyama M, Kozuka-Hata H, Noda T, Kawaoka Y.

Tiivistelmä, 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.

PMID:

23658456

[PubMed - indexed for MEDLINE]

PMCID:

PMC3754039

Free PMC Article

• Topoisomeraasi avustaa  replikaation pidentämisesä 

 

EBOV-interaktomitutkimus vahvisti DNA topoisomeraasin TOP1:n cofaktoriosan EBOLreplikaatiossa

22 lokakuuta 2014 
http://acceleratingscience.com/proteomics/mass-spectrometry-analysis-for-co-factors-involved-in-ebola-virus-replication/

 LC-MS/MS analysis identified 65 candidate proteins that co-precipitated as the EBOL interactome. Of these, the researchers chose nuclear protein DNA topoisomerase 1 (TOP1) for further investigation because previous studies had implicated this enzyme in RNA viral replication. TOP1 first unwinds the nucleic acid helical structure to allow transcription and replication by cleaving phosphodiester bridges, and it then repairs the cut ends. To begin, Takahashi and co-authors used Western blotting to confirm that TOP1 co-precipitated with EBOL. They also revealed cellular location with immunofluorescence—demonstrating that, following treatment with Venus-EBOL, TOP1 co-localized in the cytoplasm as well as in the nucleus.

To study the effect of TOP1 on viral replication, the team infected cells with an EBOV mutant in the presence or absence of small interfering TOP1 RNA (siTOP1). They found that treatment with siTOP1 downregulated TOP1 and reduced viral replication. Treatment did not, however, affect in vitro infection using two other viruses, vesicular stomatitis virus (VSV) or influenza virus, suggesting the response was EBOV-specific. Furthermore, using a mini-genome assay to investigate RNA-dependent polymerase activity, the scientists found that knockdown of TOP1 reduced EBOV enzyme activity but not that of the influenza virus.
In order to understand and further confirm the interaction between EBOL and the host cell protein, Takahashi et al. next examined whether TOP1’s DNA and RNA phosphodiester bridge-cleaving action was involved in promoting viral replication. Using mutant TOP1 constructs, the researchers found that following siTOP1 downregulation of cellular EBOL polymerase, transfection with a TOP1 mutant containing the phosphodiester bridge-cleaving activity restored activity of the viral enzyme. For further confirmation of the EBOL co-factor in EBOV replication, the researchers saw that treating infected HEK293 cells with irinotecan (CPT-11) and topotecan, inhibitors of TOP1, also reduced specific polymerase activity.
The authors propose that information arising from the characterization of the EBOV interactome in host cells will aid in drug discovery for the treatment of this deadly disease.