An in vitro RNA synthesis assay to measure the fidelity of the measles virus polymerase
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Date
2020-01-20
Authors
Thorington, Robyn
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Abstract
In the year 2000, the World Health Organization began its efforts to eradicate the measles virus (MeV) that lead to both a drastic decrease in the number of cases of measles globally and a decrease in the variability of the MeV genome, which has shown remarkable stability for an RNA virus. While this apparent stability may be due to selective pressure, I hypothesize that there is a high-fidelity mechanism in the genome replication by the MeV RNA-dependent RNA polymerase (RdRp) complex.
Replication of MeV requires three proteins; the nucleoprotein, the phosphoprotein, and the large protein. The large protein is the enzymatic subunit of the RdRp, and it is responsible for all enzymatic functions related to transcription and replication. The nucleoprotein’s role is to protect the genome and act as a guide for the RdRp, while the phosphoprotein acts as a bridge between the other two proteins.
In order to examine the lack of variability in the MeV genome, I worked to establish an in vitro RNA synthesis assay using the MeV RdRp, which can be used to shed more light on the molecular mechanism of MeV replication and to determine the cause of the virus’s genetic stability.
To accomplish this, I have taken two approaches, one using endogenous replicative proteins isolated using a cell lysate. The other is the use of recombinant proteins, which involves cloning the three proteins into expression vectors and then purifying the proteins from either a bacterial or baculovirus expression system. The two approaches were taken in order to account for the many different variables that affect the success of the assay. The functionality of the assay and rate of incorporation for the RdRp was monitored using P32 -labelled nucleotides. This assay can be used for further exploration of the dynamics and interactions that drive MeV transcription and replication. The lack of host cellular elements allows for control over experimental variables, ensuring isolation of the components that are driving each interaction. This system can also be used for high-throughput drug screening, as the MeV RdRp is an ideal candidate as a drug target as RdRps are not found in humans. There is no current treatment other than supportive care for MeV and related viruses, including Mumps virus, Nipah virus, and Hendra virus.
This was the first study to express and purify the MeV L protein. This protein can be used in the future for mutagenesis studies of the protein to determine domain functionality and for Cryo-electron microscopy studies to explore the structure of this under-studied protein.
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Keywords
Measles virus, RdRp, Viral polymerase, Recombinant protein expression, in vitro RNA synthesis, Measles large protein, Measles nucleoprotein, Measles phosphoprotein