Bunyavirus Late Penetration and Autophagy

• Applicant: Dr. Pierre-Yves Lozach, Ph.D.
• Subject Area: Virology ; Cell Biology
• Term: from 2017 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 361190725

The Bunyaviridae is the largest family of RNA viruses, many members of which cause severe diseases in domestic animals and humans, such as fatal hepatitis, encephalitis, and hemorrhagic fever. With an increasing number of outbreaks, bunyaviruses are listed as high-priority emerging pathogens by the World Health Organization.Ideally, preventing bunyavirus infection requires approaches targeting early virus-host cell interactions, before the release of the virus genome into the cytosol. Our group has established that bunyaviruses are late-penetrating viruses, a large group of viruses that share dependence on late endosomal maturation for productive penetration. We have recently analyzed small interfering RNAs against the whole human genome for their ability to block entry and infection by Uukuniemi (UUKV), a prototype bunyavirus. This screen revealed that the v-SNARE membrane fusion protein VAMP3, which is thought to promote autophagosome-endosome fusion, is critical for the virus late penetration.Many open questions and conceptual uncertainties remain regarding bunyavirus entry. While UUKV can be confidently considered as a late-penetrating virus, infectious entry does not require Rab7, the only small Rab GTPase documented thus far as critical for the maturation of classical late endosomes. Our screens identified six Rabs with a potential role in UUKV infection. Though VAMP3 promotes the autophagosomal degradation process, the role of autophagy in UUKV penetration and infection remains to be explored. Several of our screen hits have a function related to the autophagosomal maturation, and after 30 min internalization, we observed UUKV in autophagic vesicles.With this proposal, we aim to elucidate the cellular mechanisms governing bunyavirus intracellular trafficking and penetration. Our central hypothesis is that UUKV makes use of a non-classical late endocytic process to penetrate cells. To test our working hypothesis, we will analyze the dynamics of the small Rab GTPases in UUKV entry. On the other hand, we will clarify the link between autophagy and UUKV penetration. To this end, we will take advantage of the reliable and accurate fluorescence-based assays that our group developed to study bunyavirus infection, in combination with a large panel of perturbants of both endocytic and autophagic routes.Overall, we expect to get a detailed view of the Rabs and autophagic factors involved in UUKV late penetration. The information gleaned may provide valuable clues regarding hard-to-investigate, basic cellular mechanisms that control the endo-lysosomal and autophagic pathways. Together, our results will provide a handle to develop specific treatments against these emerging pathogenic viruses.

• DFG Programme: Research Grants