Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Biochemistry
Non Technical Summary
Animal coronaviruses are responsible for significant pathogenesis in animal livestock including pigs (transmissible gastroenteritis virus and porcine epidemic diarrhea virus) and chickens (infectious bronchitis virus). Many efforts to develop antiviral drugs to treat coronavirus disease have focused on using a class of drugs call nucleotide analogues. These nucleotide analogue drugs are incorporated into the viral RNA genome as the virus generates new copies of the genome. This can result in viral mutagenesis or a halt to genome replication causing fewer viral progeny to be produced from an infected cell. However, coronaviruses possess an enzyme activity that removes many nucleotide analogue drugs that have been incorporated into viral genomes. This proofreading function provides coronaviruses with a natural resistance to many such drugs and creates a challenge for the development of effective therapeutics.This study uses recombinant protein expression to produce viral proteins for in vitro study. We will test the activity of the viral enzymes responsible for copying viral RNA genomes and removing nucleotide analogues using gel electrophoresis and mass spectrometry. We will also examine the structure of the multi-protein complex using single-particle cryo-electron microscopy to obtain an atomic-level view of how these proteins interact to bring about RNA proofreading.These data will produce methods and a structural template for testing and understanding the mechanisms of potential coronavirus antiviral drugs. These molecular insights are crucial for the development of effective therapeutics to combat coronavirus disease in animal livestock.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The major goal of this project is to understand the molecular mechanisms of RNA proofreading by the coronavirus RNA replication machinery. The objectives are to determine structures of animal coronavirus replication complexes using single-particle cryo-electron microscopy and establish assays for measuring the extent of RNA proofreading using biochemical assays in vitro. A clear understanding of coronavirus RNA replication, particularly the mechanisms by which these viruses are resistant to broad RNA antiviral therapeutics is essential for the management and treatment of coronavirus disease in livestock.
Project Methods
Our studies will address longstanding questions in the coronavirus field with answers having the strong potential to impact not only the ongoing coronavirus outbreaks in livestock, but also future emerging coronaviruses. The proposed studies undertake a breadth of science to uncover basic enzyme mechanisms and interactions, evaluate existing antiviral candidates and discover novel antiviral agents. Given the large number of co-factors and enzymes that participate in coronavirus RNA synthesis, it is only possible to understand the complicated process of viral RNA synthesis by including all of the players. The proposed work stands apart from much ongoing coronavirus research in exploring new avenues, testing hypotheses and expanding therapeutic possibilities for combating coronavirus infections. We have already successfully expressed and purified coronavirus proteins.Coronavirus proteins will be recombinantly expressed in bacterial and insect cell expression systems. The proteins will be purified using affinity chromatography and size exclusion. These proteins form the basis for in vitro biochemistry and structural biology. Biochemistry experiments will focus on extending RNA primers using the coronavirus polymerase in the presence of the viral exonuclease and analyzing the results by gel electrophoresis and mass spectrometry. We anticipate that nucleotide analogues incorporated during primer extension, mimicking base-pair mismatches, will be removed by the editing exonuclease. We will also combine viral enzymes and co-factors and examine the structure of the viral proofreading complex using single-particle cryo-electron microscopy. This imaging technique collects many thousands of views of individual proteins enabling 3D reconstruction of protein structure. This structure will be used as a template for understanding how this complex works and for designing novel antiviral drugs.