Research Projects All Projects
Projects

Quick links

Structural and functional studies of Ryegrass mottle virus-encoded proteins

 

 

Funding: European Regional Development Fund (ERDF) “On Implementation of Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”

Project Title: Structural and functional studies of Ryegrass mottle virus-encoded proteins

Project No.: 1.1.1.2/VIAA/3/19/462

Period: 36 months (1 March 2020 – 28 February 2023)

Project costs: 133 304 57.00 EUR

Project implementer: Dr. biol. Ina Baļķe

 

The aim of the project is to use Ryegrass mottle virus (RGMoV, Sobemovirus) as a model for identification of virus encoded protein structural and functional properties to better understand there roll during virus life cycle. Sobemoviruses are ss (+) RNA viruses that infect both monocotyledons and dicotyledons. Because of their small (4-4.5 bp) genomes and simple structures (encoded by 5 ORFs, not fragmented), they can be used as model objects for viral studies. There genomes are relatively well characterized, but their encoded protein structures and, in particular their role and interactions in the infection process remain to be cleared up. Globalization, the exchange of infected plant material and the introduction of new crop varieties, have contributed to the spread of viruses. Several members of this genus are economically significant pathogens. For virus protein 3D structure determination corresponding protein coding sequences will be cloned in to E.coli expression system vectors and overexpressed, purified and used for X-ray crystallography or Crio-EM, KMR if needed. For protein functional study localization and interaction experiments in protoplasts will be performed for proteins with fluorescent tags (whole protein or split). Fluorescent and confocal microscopy will be used as visualization methods. In turn, by studying the 3D structure of proteins, it is possible to determine the function of an unknown protein from the structural analogues in the databases. This information will spread light upon virus protein roll in virus life cycle and reveal crucial steps in it. That can be used for virus resistant plant development and used for other virus (mammalian) research. Also important information about potential use of virus vector for recombinant protein expression in plants will be inquired. In some way, the project is unique by carrying out studies of protein-protein interactions in protoplasts, as well as obtaining a 3D structure/s for uncharacterised virus proteins, providing high-quality information to the field of virology as a whole.

Information published 02.03.2020.

 

Project progress

 01.03.2020. – 31.05.2020.

Within Activity No.1, the previously created plasmids of RGMoV 3C serine protease catalytic center mutant (Procm) with and without the transmembrane domain (TMD) were expressed in E.coli expression strain C2566. The total lysates of the cells were analyzed on an SDS-PAA gel, as well as the pellet and supernatant after cell disintegration were analyzed to verify the expression level and protein solubility. As both variants of Procm are in the soluble fraction, the preparative purification of both proteins was performed on a Ni2+ affinity chromatography column, as well as additional purification on a gel filtration column to obtain a purer material for 3D structure determination was tested. Optimization of purification conditions has been performed for Procm with TMD. Catalytically active Pro was expressed and purified for functionality studies. Work has also begun on the expression and purification of P1 protein from inclusion bodies (IB).

In Activity No.3 an additional literature analysis was performed to create plasmids for protein-protein interaction studies with split fluorescent proteins (sFP). Work has started on the design of the constructs.

Information published 31.05.2020. 

 

Progress of the project

01.06.2020. – 31.08.2020.

Within Activity No.1, a purification scheme of Procm without TMD has been developed, as well as it has been purified in a preparative amount for the crystallization condition optimization study. Work continues on the optimization of Procm with TMD purification conditions, as well as on the optimal purification method selection for P1 protein purification from IB and on the studies of catalytically active Pro functionality.

During Action No.2, work has been started on testing the conditions for Procm without TMD crystallization and co-crystallization with peptide.

In Activity No.3 work has been started on the development of plasmids for sFP protein interaction studies.

Information published 31.08.2020. 

Progress of the project

01.09.2020. – 30.11.2020.

Within Activity No.1, work continues on the optimization of Procm with TMD purification conditions, as well as on the optimal purification method selection for P1 protein purification from IB. P16 N-terminal domain P10 was expressed in E.coli expression strain C2566. Expression and protein solubility analysis on SDS-PAA gel was performed. Purification of P10 was performed on a Ni2+ affinity chromatography column. P16 expression was performed in E.coli expression strain C2566. Analysis of P16 expression clones on SDS-PAA gel was performed. Work on catalytically active Pro functionality continues.

During Action No.2, 3D structure Procm without TMD and without peptide was determined. Testing of P16 N-terminal domain P10 crystallization conditions has been started. Preparation of a publication on previously obtained crystallization data for catalytically active Pro with and without cofactor was started.

In Activity No.3 constructs for sFP experiments was created. Work has begun on testing the expression of sFP constructs in the E.coli system.

Information published 30.11.2020. 

 

Progress of the project

01.12.2020. – 28.02.2021.

Within Action No.1, the work was continued on the optimization of P1 refolding buffers, as well as on the optimization of Procm with TMD purification conditions. Preparative expression of P16 N-terminal domain P10 in E.coli and purification for crystallization experiments were performed. A new expression vector for catalytically active ∆117Pro with an N-terminal 6 histidine tag was created for faster Pro purification. ∆117Pro was expressed in E.coli expression strain C2566, expression, protein solubility analysis on SDS-PAA gel were performed, as well as ∆117Pro was purified on Ni2+ affinity chromatography column. Work had begun on the construction of the ORFx-encoded protein Px expression vector for E.coli system.

During Action No.2 experiments for optimization of P10 crystallization conditions have been started. Work continues on the preparation of a publication manuscript on previously obtained crystallization data for catalytically active Pro with and without cofactor.

In Activity No.3 continues the work on the expression of sFP constructs in the E.coli system. An analysis of the literature on available protoplast isolation and transfection methods has been performed.

Information published 26.02.2021. 

 

Progress of the project

01.03.2021. – 31.05.2021.

Within Action No.1, the interaction of P1 with RGMoV CP mRNA after refolding with and without Zn2+ was tested using the gel-shift method. The P1 refolding method has been developed. Using this method, P1 was purified in a preparative amount for crystallization optimization experiments. Expression of ORFx-encoded protein Px in E. coli expression strain C2566 was performed. Its expression, solubility was analyzed in SDS-PAA gel, as well as purification of Px on Ni2+ affinity chromatography column was tested. Additional purification experiments of ∆117Pro with N-terminal 6 histidine tag were performed using gel filtration.

During Action No.2 experiments for optimization of P1 crystallization conditions have been started. The experiment of optimizing P10 crystallization conditions continues. Work continues on the preparation of a publication manuscript on previously obtained crystallization data for catalytically active Pro with and without cofactor.

In Activity No.3 has started work on compilation of protoplast isolation and transfection methods described in the literature.

Within Activity No.5, a virtual poster “Application of fluorescent proteins in plant virus research” was created within the framework of “European Scientists Night” (April 30, 2021).

Information published 31.05.2021. 

 

Progress of the project

01.06.2021. – 31.08.2021.

Within Action No.1, work has been started on the optimization of Px purification of the ORFx encoded protein. Work has begun on the expression of VPg-RdRp fusion protein in E. coli expression strain C2566 and purification on a Ni2 + affinity chromatography column.

During Action No.2 the initiated optimization experiments of P1 and P10 crystallization conditions were continued. Work continues on the improvement of the publication manuscript on previously obtained crystallization data for catalytically active Pro with and without cofactor.

In Activity No.3 continues the work on the summarization of protoplast isolation and transfection methods described in the literature.

Information published 31.08.2021. 



Mājas lapas izstrādi finansēja ERAF 2.1.1.2. aktivitātes projekts Nr. 2010/0196/2DP/2.1.1.2.0/10/APIA/VIAA/004 "Latvijas biomedicīnas pētījumu integrācija Eiropas zinātnes telpā".