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|Title: ||Viral interferon antagonists and antiviral drugs|
|Authors: ||Nobre, Rita|
|Supervisors: ||Randall, R. E.|
IFN signaling pathway
Cell-based screen assays
Hepatitis B core protein
Hepatitis C core protein
Rabies P protein
|Issue Date: ||30-Nov-2009|
|Abstract: ||For this project, we developed reporter cell lines that express viral proteins with the potential to be used in cell-based screening assays to select chemical candidates for antiviral drugs. The viral proteins expressed in these reporter cell lines (Hepatitis B core and precore, Hepatitis C core (1a and 4a), and Rabies P (BH and SADL16)) were presented in the literature as responsible for interfering with the IFN signaling pathway, specifically for blocking the expression of its key protein STAT1.
We cloned the viral genes into the pdl’SurvpkIB reporter plasmid and, through a lentiviral delivery system, infected the Hep2Mx1TIPSE cells and the A549Luc cells resulting in the Hep2Mx1TIPSEHBVprecore, Hep2Mx1TIPSEHBVcore, Hep2Mx1TIPSEHCVcore (1a and 4a), and A549lucRabiesP (SADL16 and BH) reporter cell lines.
We assessed the obtained viral cell lines according to their ability to block the IFN signaling pathway by using three different assays: an immunoblot targeting the protein STAT1, a phenotypic assay for survival in
the presence of puromycin (in viral Hep2Mx1TIPSE cells), and a quantitative measure of luciferase expression (in viral A549Luc cells).
Concerning the immunoblot targeting STAT1, the results showed that only cell lines expressing the Rabies P protein (namely the A549lucRabiesPSADL16 cell line) were able to decrease the level of expression of STAT1.
The phenotypic assay conducted on the Hep2Mx1TIPSE viral cell lines were intended to show impairment of the IFN signaling pathway through the down-regulation of the IFN stimulated gene Mx1. Normal Hep2Mx1TIPSE cells contain a puromycin resistance gene controlled by the Mx1 promoter.
Therefore, when puromycin is added to these cells in the presence of IFN, the signaling pathway is activated and Mx1 as well as the puromycin resistance gene are expressed resulting in cell survival. Results showed that the cell lines expressing the HCV core and HBV precore proteins also survived puromycin addition. However, the Hep2Mx1TIPSEHBVcore cells died in the presence of
puromycin suggesting that in these cells the HBVcore protein affects Mx1 protein expression.
Since it was expected that all viral cell lines would be able to down- regulate Mx1 by impairing the IFN signaling pathway, it was assumed that the level of viral expression may not have been enough to be detected by this kind of assay and therefore a quantitative study would be crucial for the continuation of this project.
The cell lines expressing the Rabies P protein demonstrated their ability to block STAT1 and contained a luciferase gene under the control of an IFN regulated promoter. These cells were therefore considered the best candidates for the quantitative assay. We compared the difference between luciferase expression in the viral cells in the presence and absence of IFN with A549Luc cells (control cells) and verified that in both cases there was an increase in the amount of luciferase expression upon the addition of IFN, which is concordant with the up-regulation of the IFN signaling pathway. However, this increase was considerably less in cells expressing the viral protein. This result confirms a partial blockage of the IFN signaling pathway in these cells.
This experiment demonstrates a new alternative step in the creation of cell lines that express the Rabies P protein and that can be applied to the manufacturing process of antiviral drugs. However, in order to achieve the successful production of cell lines, it would be essential to improve viral protein expression.|
|Publisher: ||University of St Andrews|
|Appears in Collections:||Biology Theses|
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