Structural and functional characterization of MAVS-DDX3-vRNA complex

Project Information

MDR

Grant agreement ID: 844115

DOI

10.3030/844115

Project terminated on 1 February 2021

EC signature date 10 April 2019

Start date 1 September 2019

End date 31 August 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 187 572,48

EU contribution

€ 187 572,48

Coordinated by

ACADEMISCH MEDISCH CENTRUM BIJ DE UNIVERSITEIT VAN AMSTERDAM
Netherlands

Periodic Reporting for period 1 - MDR (Structural and functional characterization of MAVS-DDX3-vRNA complex)

Reporting period: 2019-09-01 to 2021-08-31

Summary of the context and overall objectives of the project

Project MDR-REP-844115-1 – Periodic Report

What is the problem/issue being addressed?

The mitochondrial antiviral signalling (MAVS) adaptor protein is a central signalling hub for host cells to mount an antiviral response following RNA virus infections, which is initiated by the cytosolic receptors that trigger the type-I interferon (INFs) path through the MAVS (Fig1). It has recently been shown that the RNA helicase DDX3 is a novel atypical member of the viral cytosolic receptor pool. It is required to activate the MAVS during the antiviral response. It is currently unknown how the complex partners MAVS-DDX3-viral RNA (MDR) interact for assembly and what is the MDR mechanism of action at the molecular level. Notably, silencing DDX3 or MAVS expression suppress activation of the native immune response, an event that in the physiological condition is at the front line of host defences against RNA viruses such HIV-1. However, understanding how viral RNA triggers the innate immune reaction requires the MDR structure elucidation. Moreover, these findings will exploit MDR molecular features to create a new generation of adjuvants in anti-retroviral therapy.
Our research addressed the structural and functional characterization of the MDR complex by biophysical and cellular biology techniques. This work aimed to understand how cellular protein sensors interact with retroviral RNA to trigger the native immune response and induce the expression of antiviral proteins.


Why is it important for society?

These studies will have fundamental implications for understanding novel virus sensors and their role in innate immunity. Therefore, the central role in triggering antiviral immune response makes MDR a strategic pharmacological target.


What are the overall objectives?

The objectives of the project include 1) to perform FRET experiments by tagging DDX3 and the viral RNA, in order to test the hypothesis that they assemble into a supramolecular signalling platform before interacting with MAVS and triggering the immune response; 2) determination of the 3D structure of the MDR, employing cryo-Electron Microscopy (cryo-EM) and X-ray crystallography as appropriate to shed light on the key interactions involved in the complex assembly; 3) to identify and test mutation sites on DDX3 and MAVS responsible for the complex formation based on the MDR structure, able to lower the HIV-1 infection rate in vitro cells.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first length of the project, the objectives of the WP1 have been fully met, and the single-particle EM analysis described in WP2 has been initiated. Below I list the results obtained on this multidisciplinary work:

1) Cloning of both DDX3 and MAVS genes in Lentivirus expression vectors carrying fluorescent tags to perform FRET, FLIM experiments, aiming to detect the complex formation within the cell.

2) Generation of stable cell lines containing expression vectors reported above, currently used in the host lab to perform Flow Cytometry FRET-based assay to study complex assembly dynamics.

3) Imaging cells carrying the vectors reported above on a fluorescent microscope to assess expression level.

4) Purification of both proteins by affinity and size exclusion chromatography techniques.

5) Negative staining electron microscope imaging of both proteins on a Tecnai microscope T12 operating at 120 electron volts.

This research achieved the production of the key components MAVS and DDX3 of the MDR complex and provided their preliminary functional and biophysical characterization. These results represent the foundation for the structural characterization of the MDR complex to shed light on the key interactions involved in its formation. Further structural findings will be essential to exploit MDR as a pharmacological target.

A review, titled DDX3: where the viral replication meets innate immunity, by Luigi De Colibus and Teunis B H Geijtenbeek, has been submitted to the journal Trends Biochemical Sciences. This review highlights the importance of the structural biology approach to the elucidation of DDX3 key interactions with viral proteins and host factors during the infection, which can be targeted for designing novel drugs to fight viruses.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The further progression of the project would allow the visualization at the atomic level of the MDR complex. It would represent a tremendous scientific achievement given its key role, recently discovered, in the infection process of HIV-1. Moreover, solving the MDR atomic structure will facilitate the design of new immune modulators to help fight the HIV-1 infection. This virus is responsible for the immune deficiency disease still affecting millions of people worldwide, causing one of the higher death rates in developing countries, where it is even more challenging to access advanced treatments. Furthermore, because the MDR complex is involved in binding highly structured viral RNA molecules, it could also interact with regions of the viral RNA genome of SARS-Cov2, responsible for the current pandemic. Therefore, experiments to test the MDR role in the innate immune response against SARS-CoV2 are needed to challenge the hypothesis that MDR could be exploited as a pharmacological target against COVID-19 disease.
In this way, it would be possible to expand the arsenal of weapons at our disposal to fight viral infection by boosting innate immunity, and to tackle viral pandemics to come.

Imaging cells expressing MAVS fused to Monovinus fluorescent protein.

SDS-PAGE of MAVS isoforms.

Agarose gel showing PCR band of DDX3 used for cloning.

Negative stained electron microscopy image of DDX3 protein.

Negative stained electron microscopy image of MAVS fibers.

DDX3-MAVS signaling during HIV-1 infection.

Imaging cells expressing DDX3 fused to mTurquoise fluorescent protein.

Agarose gel showing PCR bands of MAVS isoforms used for cloning.