Proving causality of liquid-liquid phase separation for the acquisition of nuclear-like functions by Giant Viruses Viral Factories

Introduction

Giant Viruses (GVs) encode thousands of ORFan genes, but their study has been nearly impossible due to the lack of genetic tools for reverse genetics. The aims of ViDaMa and the tools I designed will shed light into this Viral protein Dark Matter. Particularly, I will study how new genes from Mimivirus were engineered during evolution to acquire nuclear-like function for their Viral Factories (VFs). VFs physically separate viral DNA replication and transcription from translation and likely segregate DNA into active and silent. How these functions are accomplished remains elusive.

Objectives

I will demonstrate causality of liquid-liquid phase separation (LLPS) for the acquisition of nuclear-like functions of the Viral Factories (VFs) of Mimivirus. To do so, I will:

1. Generate genome-wide loss-of-function screens for the identification of gene function and proteome-wide localization of virtually all Mimivirus and its host Acanthamoeba proteins.
2. Combine biochemistry and cell biology to dissect the nature, functions, and components of the VFs of Mimivirus.
3. Strip down VFs to their minimal components in order to utilize them to improve production efficiency and purity of mRNA during in vitro transcription.

Impact

ViDaMa will empower the GVs scientific community with genome-wide and proteome-wide data of gene function and protein localization. It will transform the field from mostly descriptive to allow the dissection of the molecular mechanisms behind viral phenotypes.

Research Significance

ViDaMa will also address the molecular mechanisms that allow the VFs to acquire nuclear-like functions, directly tackling the viral eukaryogenesis theory. The new classification of VFs as membrane-less organelles and the study of the molecular mechanism behind their biogenesis and functions will shed light into the general mechanism of LLPS.

Future Applications

The generation of in vitro VFs promises an optimization of in vitro transcription systems with tremendous impact on mRNA therapeutics at the development and production level.