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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. 



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".