Studies of multi-component complexes by NMR: application to viral mRNA export

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

The cells of higher organisms including humans store the genetic information in a form of DNA in a central compartment known as nucleus. In a process called transcription selected parts of DNA are read and information is subsequently coded into messenger RNA (mRNA) molecules. These molecules are subject to further processing and re-arrangements (splicing), consequently providing a blueprint for synthesis of an enormous variety of different proteins. These blueprints are delivered from the nucleus to other parts of the cell, where the cell machinery uses them to produce proteins. Messenger RNA processing, re-arrangement and export from the nucleus are actively performed by various protein components of the splicing and export machinery. These components interact with messenger RNAs and with each other, imposing control so that only mature cellular mRNA is exported. How this control system works is not completely clear. Some viruses produce proteins which make use of this cellular export machinery, tricking it into carrying viral genetic information from the nucleus, and by-passing cellular control mechanisms. In this way the viruses support their own life cycle at the cell's expense; how exactly this happens still needs further research. Better understanding of how the cellular mRNA export system functions (or how internal control mechanisms are by-passed during viral infection) requires characterisation of 3D structural, dynamic and binding properties of its individual protein components, and investigation of how they behave when binding with each other. The proposed research aims at studying molecular basis of interactions between fragments of viral proteins and key native cellular proteins responsible for mRNA export, an area immediately related to our recent successful research. Our preliminary results show that in multi-component molecular complex the viral mRNA may be transferred from one protein molecule to another: how this is achieved will be revealed in the proposed study. The new technique pioneered in the applicant's laboratory will be used to look in detail at how different multiple molecular components of the system interact together, and how different molecular regions operate in the process. Revealing the structural mechanism by which viral proteins hijack the cellular mRNA export system potentially can lead to development of new anti-viral drugs. The methodological aspect of the project also has wider implications. In general, protein-protein interactions play a critical role in virtually all aspects of biological processes. Within a cell, many proteins participate in multi-protein complexes, either transiently or stably, resulting in complex protein interaction networks. The molecular mechanisms behind the precise ordering of binding and dissociation events in multi-protein complexes remain unclear and are generally difficult to study, but provide a topic of utmost interest and importance. The future generations of specific drugs are likely to target key protein-protein interactions, either inducing or suppressing them. All this in turn boosts the interest in new methods by which to analyze complex multi-protein interactions reconstructed in sample tube, and in creating assays where such interactions can be detected, probed and characterized. The novel strategy of investigation of complex interactions proposed in the current project addresses this issue and will be applicable to other multi-protein interaction systems, and is expected to boost the research in the whole field of protein interactions studies, including nanotechnology and drug design.

Technical Summary

The splicing of pre-mRNA, transforming it into the mature mRNA and export from the nucleus to the cytoplasm are fundamental and essential steps in gene expression for all eukaryotes. Only fully-processed and spliced mRNA is exported, and the whole process is subject to tight control. Majority of herpes viruses have evolved mechanisms that use cellular nuclear export pathway to export intronless viral mRNA, thus bypassing the cellular control mechanisms. Signature proteins responsible for viral mRNA export via TAP pathway has been identified in all classes of herpes viruses. These proteins interact with REF, a molecular adaptor normally marking cellular mRNA destined for export. The 3D structure, as well as RNA and TAP binding regions of REF2-I have been recently characterized by the applicant and co-workers in detail. The aim of the proposed project is to reveal how the signature viral proteins interact with REF2-I, and affect its structure and RNA binding properties. The standard solution NMR approach will be used to characterise structural and dynamic properties of functional viral protein fragments and establish the 3D structure of their complexes with functional fragment of REF2-I. The unique feature of the project is that the novel approach proposed and developed by the applicant will be used to investigate in vitro the process of multi-component complex assembly between viral protein fragments, RNA, REF2-I and TAP, and to observe the RNA transfer from one polypeptide molecule to another within this complex. Molecular regions involved at different stages of complex lifecycle will be identified, to reveal molecular mechanism by which viral proteins recruit viral mRNA to REF and TAP. The new strategy of investigating the complex protein-protein-ligand(s) interactions suggested in the proposal will be also applicable to studies of protein interaction networks, to reveal molecular mechanisms behind the precise ordering of binding and dissociation events.
 
Description We have mapped the position and functional significance of amino acid residues responsible for binding with the cellular mRNA export factor REF/Aly in both HSV ICP27 and HVS ORF57. We determined the atomic-resolution structures of the ICP27-REF and ORF57-REF complexes, and provided the first molecular details of how herpes viruses access the cellular mRNA export pathway.



The regions of ORF57 (within aa8-120) which interact with specific and non-specific RNA were revealed, and mechanism of transfer of viral RNA between viral and cellular proteins has been characterized.



We have demonstrated the possibility to investigate multi-component binding events using isotopically-discriminated (IDIS) NMR; the new saturation-transfer version of IDIS experiment was developed and tested. This experiments can be used to study complex interactions in fuzzy complexes which lack rigid 3D structure.
Exploitation Route The results of the project improve the fundamental understanding of molecular processes leading to herpesviral infection. This research brings us one step closer to developing efficient antiviral treatments in the future.
Sectors Healthcare

 
Description The findings of this project were of fundamental nature: we are not aware of them being used by Industry yet, however this research laid a foundation for our further research (now funded by the NIH, USA) which may help to identify a druggable protein targets, helping to find therapeutics against herpesviral infection.
First Year Of Impact 2011
Sector Healthcare
Impact Types Societal

 
Description New collaboration started with the group of Prof. R. Sandi-Goldin, University of California Irvine (USA) 
Organisation University of California, Irvine
Country United States 
Sector Academic/University 
PI Contribution Collaboration started with the University of California Irvine
Start Year 2012
 
Description Media coverage following PloS Pathog publication in 2011 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Multiple coverage in the news web sites

Multiple media coverage, including printed journals, such as Pause Sante (March 2011). http://www.bbsrc.ac.uk/news/health/2011/110107-pr-visualise-herpes-virus.aspx

http://www.sciencedaily.com/releases/2011/01/110106192027.htm

www.future-science.com/doi/pdf/10.4155/fmc.11.6

and many others

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
URL http://www.bbsrc.ac.uk/news/health/2011/110107-pr-visualise-herpes-virus.aspx http://www.sciencedai...
 
Description Media coverage following publication in PLoS Pathog (2014) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Features in the BBSRC news and also on many web sites, eg:


http://www.manchester.ac.uk/aboutus/news/display/?id=11585

http://www.medicalnewstoday.com/releases/272675.php

http://medicalxpress.com/news/2014-02-efficient-treatment-closer-cancer-causing-herpes.html

etc http://www.bbsrc.ac.uk/news/health/2014/140213-pr-better-treatment-herpes.aspx

http://www.medicalnewstoday.com/releases/272675.php

http://www.brightsurf.com/news/headlines/93397/Efficient_treatment_a_step_closer_in_the_fight_against_cancer-causing_herpes.html

no actual impacts realised to date
Year(s) Of Engagement Activity 2014
URL http://www.bbsrc.ac.uk/news/health/2014/140213-pr-better-treatment-herpes.aspx