Phase separations and transitions by Henipavirus V and W proteins: molecular mechanisms and implications for functions and pathogenesis – HENIPHASE

The Nipah and Hendra viruses (NiV & HeV) (Henipavirus genus) are zoonotic pathogens responsible for severe encephalitis in humans. Their natural reservoir are fruit bats. Inter-human transmission of NiV and circulation of Henipaviruses in bat species from a growing number of countries, constitute a serious threat to human health. Although an efficient vaccine against HeV in horses is available, neither vaccines nor therapeutic treatments are available in humans against Henipaviruses. The high pathogenicity, wide host range and interspecies transmission of NiV & HeV led to their classification as BSL4 pathogens and potential bio-terrorism agents. The prevention and/or the containment of present and future epidemics will depend on our capacity to conceive effective strategies to combat these viruses. The long-term goal of this project is to shed light onto the molecular mechanisms of Henipavirus pathogenesis as a prerequisite for the rational design of future therapeutic approaches.
Their V and W proteins are key players in the evasion of the host innate immune and inflammatory response. V and W share an N-terminal intrinsically disordered (ID) domain (NTD) and have distinct C-terminal domains. A region of HeV NTD was found to confer to V the ability to undergo a liquid to gel phase transition accompanied by the formation of amyloid-like fibrils. Congo red staining of transfected or infected cells suggests fibril formation also in cellula, and fluorescence microscopy showed that W forms condensates in the nuclei of transfected cells.
Our hypothesis is that the phase-separated condensates formed by the V/W proteins may sequester key cell proteins involved in the cell innate immune and inflammatory response thereby contributing to the high pathogenicity of these viruses. Our aim is to further investigate the abilities of these proteins to phase separate and fibrillate and to shed light onto their functional implications.
We will combine in vitro (Partner 1) and in cellula (Partners 2 & 3) studies. In vitro studies will use purified wt and mutated proteins and will enable deciphering the molecular and sequence determinants governing the ability of V/W proteins to phase separate and fibrillate. In cellula studies will imply V/W transient expression and infection experiments followed by quantitative cell imaging, interaction studies with cellular proteins of the innate immune pathway and measurements of IFN-stimulated genes & chemokines responses. V/W transient expression and infection studies will also use bat cells, which will enable unveiling possible differences between human and bat cells, thus potentially providing hints on the mechanisms by which bats efficiently control Henipavirus infection. We will also investigate the ability of well-known inhibitors of protein fibrillation to block Henipavirus V and W fibrillation and possibly hamper the adverse effects of V/W proteins on cell functions.
Partner 1 is an expert in the structure-function relationships of ID regions from paramyxoviral proteins, Partner 2 is an expert of Henipavirus infection, bat cells engineering & innate immune response, and Partner 3 developed innovative approaches to image and quantify viral amyloids in infected cells.
The strength of this project lies in (i) its feasibility (availability of multiple preliminary data & tools already generated by the partners), (ii) the expertise and complementarity of the partners, (iii) its originality (very few studies have investigated the impact of phase separation by viral proteins on host functions and even fewer have described amyloidogenic viral proteins), (iv) its potential to unveil a new molecular mechanism underlying Henipavirus pathogenesis, (v) to shed light on the molecular basis by which bats control Henipavirus infection, (vi) to illuminate the relationships between intrinsic disorder, phase transitions and amyloid formation and (vii) to set the stage for the development of new antiviral strategies.