Structural and spatial analysis of vRNP clustering and virus assembly in Orthomyxoviruses

• Applicant: Dr. Petr Chlanda
• Subject Area: Structural Biology ; Virology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 537419712

Orthomyxoviruses are pleomorphic, membrane-enveloped viruses, which package their RNA genome into several physically separated complexes so-called viral ribonucleoproteins (vRNPs). Viral genome segmentation is a strategy that allows for genome reassortment upon co-infection, leading to antigenic shift, i.e. an increase in genetic diversity without the insertion of mutation into the genome. However, it poses a conundrum on how the vRNPs are sorted and effectively incorporated into budding virions. In addition, spatial and temporal analysis of vRNP reassortment in infected cells remains to be addressed. The family Orthomyxoviridae includes important human pathogens such as the influenza A virus (IAV) that causes annual epidemics and is responsible for several pandemics recorded in modern human history. Our aim is to apply state-of-the-art cellular cryo-electron tomography and cryo-correlative light and electron microscopy to shed light on influenza A virus infection and unravel the vRNP clustering, trafficking, and incorporation into assembling virions at nanometer resolution directly in infected cells. Although previous electron microscopy studies showed vRNP trafficking on membrane compartments they are limited by poor structural preservation. To shed light on these processes high-resolution imaging techniques in combination with virology tools are needed and have not yet been applied. Our preliminary data revealed novel insights into IAV infection - hemagglutinin (HA), the IAV surface glycoprotein, drives membrane remodeling, forming organelles that interact with vRNPs in Rab11 dependent manner. We aim to determine the interaction between HA and vRNPs and deliver a vRNP spatial distribution in cells infected by different members of Orthomyxoviridae including avian and bat influenza A viruses as well as influenza C virus and Thogotoviruses. There is a growing body of evidence that vRNP complexes directly interact with Rab11 via the tripartite polymerase complex and this interaction is critical for vRNP trafficking to the plasma membrane and assembly of the new viral progeny. Hence Rab11-mediated vRNP trafficking is central to the understanding of vRNP clustering and we propose targeting Rab11 as of novel broad-spectrum strategy against IAVs. In addition, we plan to structurally characterize the vRNP incorporation into budding virions; and elucidate the inhibitory mechanism of MxA, an important IAV restriction factor, which constitutes a barrier against zoonotic transmissions and controls influenza virus susceptibility in humans.

• DFG Programme: Research Grants