DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Molecular basis of rhabdovirus entry into the host cell – MoBaRhE

How do rhabdoviruses enter the cells they infect?

Rhabdoviruses, which include many human deadly viruses such as rabies virus, possess a transmembrane glycoprotein (G) that is essential for the entry of the virus into its host cell. The project aims to understand how this viral key opens the different cellular locks.

Determination of the molecular bases of rhabdoviruses entry into the host cell

The objective of this project is to characterize the molecular bases of the rhabdoviruses entry into their host cell. It comprises two axes aimed at: <br />1) Investigating the interaction between vesicular stomatitis virus (VSV) and rabies virus (RAV) glycoprotein and their identified receptors (LDL-LLDL receptor for VSV, low growth factor affinity receptor Nerve-p75NTR- and neural adhesion molecule -NCAM- for RAV). <br />2) Elucidating how G catalyses the fusion of viral and cellular membranes; i.e. identifying the intermediate structures adopted by G during its fusogenic structural transition and understanding how the glycoproteins cooperate with each other on the viral surface during the fusion process. <br />Specifically, we want to: <br />1) Determine the structure of VSV G ectodomain in complex with the LDLR module it recognizes. <br />2) Determine the structure of G ectodomain for other rhabdoviruses in particular for rabies virus. <br />3) Characterize the intermediate structures formed by G during the structural transition.

For this project, we use several structural biology approaches :
1) First, we try to crystallize the glycoproteins of several rhabdoviruses. This implies that we are able to express them in great quantity. For this, we use either the virus itself or an expression system using another virus: the baculovirus, which allows the expression of the glycoprotein in lepidopterans cells. Once the crystals are obtained, we solve their structure and, therefore, that of the glycoprotein by X-ray diffraction.
2) We also use electron microscopy to characterize the supramolecular structure and organization of glycoproteins on the surface of the viral particle. This can be done after staining the particle with heavy salts or after high-speed freezing in amorphous ice.
3) Finally, one of the original aspects of the project is to use high-speed atomic force microscopy (HS-AFM) to gain access to the dynamics of G structural transition at the surface of the virus under conditions which are very close to its natural environment.

We have determined the structure of intermediate conformations of the Chandipura virus glycoprotein (another vesiculovirus close to VSV). We mainly validated the crystallographic structures that we had already determined during a previous ANR project. Particularly, the crystal structure exhibited an unexpected dimeric arrangement. Therefore, we constructed a series of mutants carrying mutations at this dimeric interface, verified that the fusion properties were abolished and finally selected compensatory mutations.
We have also finely characterized the interaction between VSV G and its receptor, the «Low density Lipoprotein Receptor« (LDLR). LDL R contains 7 domains rich in cysteine ??(CR). We showed that G specifically recognizes some CR domains of LDLR and we were able to determine the crystal structure of G in complex with two of these domains. The two structures revealed that the two CR domains bind G in a similar manner. The residues involved in the interaction between G and each of these two CR domains have been identified.
In the course of this work, we have also demonstrated that the only receptors of the virus are all of the LDLR family.
This work definitively solves the VSV receptor problem: the glycoprotein of this virus has evolved to specifically recognize the CR domains of a family of cellular receptors. This opens up interesting prospects for a protein and a virus which is extremely used in biotechnology.

We are currently expressing several other rhabdovirus glycoproteins. Our objective is still to determine their structure in order to have a global vision of their functioning but also of the constraints that weigh on their evolution.
The determination of the structure of VSV G in complex with its receptor opens interesting prospects in biotechnology. Indeed, it is a first step for the construction of mutants recognizing other specific receptors.

1) Baquero E, Albertini AA, Raux H, Abou-Hamdan A, Boeri-Erba E, Ouldali M, Buonocore L, Rose JK, Lepault J, Bressanelli S, Gaudin Y. Structural intermediates in the fusion-associated transition of vesiculovirus glycoprotein. EMBO J. 2017 36(5):679-692.

Rhabdoviruses are enveloped viruses whose genome is a single strand RNA molecule of negative polarity. The best studied rhabdoviruses are vesicular stomatitis virus (VSV) and rabies virus (RAV) which still causes 50,000 human deaths annually. Other rhabdoviruses (such as Chandipura virus, CHAV) are deadly to humans or potentially cause significant economic damages.
Rhabdoviruses have a transmembrane glycoprotein G, which is essential for viral entry. First, G recognizes a receptor on the cell surface. Then, after endocytosis of the virion, in the acidic environment of the endosome, G undergoes a huge structural transition. This low-pH-induced transition from a pre- to a post-fusion state catalyzes the fusion between the viral and endosomal membranes. During this conformational change, G exposes hydrophobic motifs that interact with and destabilize the target membrane.
G is the prototype of class III viral fusion glycoproteins which also encompasses the glycoproteins gB of herpesviruses and gp64 of baculovirus. Among all the structures that have been determined for viral glycoproteins of this class, the only pre-fusion structure that is known is that of VSV G.
This project gathers three teams, one of biochemists and virologists specialists of rhabdoviruses, another expert in electron microscopy (EM) and a third one with in depth expertise in atomic force microscopy (AFM). Our objective is to characterize the molecular basis of rhabdovirus entry into their host cell. It comprises two axes that aim to:
1) Study the interaction between VSV and RAV G and their identified receptors (low density lipoprotein receptor -LDLR- for VSV, low affinity nerve growth factor receptor -p75NTR- and neural cell adhesion molecule -NCAM- for RAV).
2) Elucidate how G catalyzes membrane fusion; i.e. identify intermediate structures adopted by G during the structural transition and how glycoproteins cooperate with each other at the surface of the virus during the process. These questions arise for all viral fusion glycoproteins, beyond that of rhabdovirus.
Specifically, we will work:
1) On the determination of the structure of VSV G ectodomain in complex with the LDLR module it recognizes.
2) On the determination of the structure of the ectodomain of other rhabdovirus G. We will work on the ectodomains of the two, structural (G) and non-structural (GNS), glycoproteins of bovine ephemeral fever virus (these glycoproteins are homologous but the non-structural one has lost its fusion properties) and on RAV G. We will try to crystallize them alone or, in the case of RAV G, in complex with soluble forms of their cellular receptors.
3) On the characterization of the intermediate structures adopted by G during the structural transition.
Concomitantly, the structure of the glycoproteins in their natural environment (i.e. at the surface of viral particles) will also be examined by EM and high-speed atomic force microscopy (HS-AFM). Using EM, we hope to see fusion intermediates that we can trap in vitro. Using HS-AFM, we hope to gain access to the dynamics of both the structural transition of G and its reorganization into a regular network at low pH.
Finally, construction of mutants and characterization of their fusion properties will validate hypotheses arising from structural analysis.
Although this collaborative project is essentially fundamental research, there are several aspects which may have applied impacts in the medical field, for instance, in the rational design of new antiviral molecules and of wide spectrum vaccines against RAV related viruses.

Project coordinator

Monsieur Yves GAUDIN (Institut de Biologie Cellulaire Intégrative)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

UMR9198 CEA/CNRS/PSUD Institut de Biologie Cellulaire Intégrative
U1006 INSERM Bio Atomic Force Microscopy Laboratory
UMR9198 CEA/CNRS/PSUD Institut de Biologie Cellulaire Intégrative

Help of the ANR 490,990 euros
Beginning and duration of the scientific project: September 2015 - 36 Months

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