Project Details
Projekt
Revealing the molecular mechanism of vaccinia virus mRNA release
Subject Area
Structural Biology
Virology
Cell Biology
Virology
Cell Biology
Term
since 2023
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 515013236
The poxvirus vaccinia virus (VACV) was used as life vaccine to eradicate smallpox. Among the poxviridae VACV has been most extensively studied, morphologically, biochemically and genetically. Poxviruses have recently regained interest due to rising monkeypox cases in Europe and throughout the world. This application focusses on the structurally ill-defined process of VACV early transcription, that can be mimicked in vitro from membrane-free cores. When such cores are incubated with NTPs, the viral transcription machinery packed within the virion produces roughly 100mRNAs. These core-associated mRNAs are subsequently released from the intact core in a manner that is not understood. The existence of pores within the core wall have been postulated but not unequivocally demonstrated. The focus of this application is to decipher the release mechanism of the VACV early mRNAs, both in vitro and in situ. In vitro membrane-free cores, subjected to transcription or not, are imaged by cryo-electron tomography (cryoET) and the images are processed by sub-tomogram averaging (STA). Our preliminary data using BrUTP, anti-BrU labelling and negative staining electron microscopy (EM), confirm that such cores are transcription competent. In addition, on the membrane-free core, cryoET and STA is able to resolve structures not previously analyzed. In situ we plan to use cryo-correlative light- and electron microscopy (cryoCLEM) to study early transcription in infected cells. Colour-separation upon infection with virions triple-labelled in their membrane, core and DNA is used to identify intracellular cores by cryo-light microscopy followed by cryoET of positions of interest. By specifically blocking viral transcription or uncoating, intracellular cores that undergo early transcription or not are accumulated, targeted by cryoCLEM and analysed by cryoET and STA. Parallel LM experiments will be conducted to assay for viral early transcription or its inhibition, feeding infected cells with bromo-uridine (BrU) or 5-ethynyl uridine (EU), followed by anti-BrU- or click-labeling. The strength of this application is the ability to combine in vitro with in situ data. Together these will shed light on the molecular mechanism of VACV mRNA release. The data will be compared to viral and cellular structures with similar RNA-release functions and may form the basis for the development of anti-poxvirus compounds that block the early infection. Finally, the cryoCLEM workflow can be applied to in situ studies of other virus-host systems, such as emerging viruses.
DFG Programme
Research Grants
