Author: Susmita Shrestha
Shrestha, Susmita, 2025 Generation and Characterisation of Viral Protease-Dependent Reporter Systems to Monitor Orthoflavivirus Infections in Living Cells, Flinders University, College of Medicine and Public Health
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In recent years, pathogenic orthoflaviviruses such as Dengue Virus (DENV), West Nile Virus (WNV), and Zika Virus (ZIKV) have affected millions of people worldwide, resulting thousands of death globally. Despite an urgent need, there are no approved antiviral therapies for these viruses. While live cell imaging represents a powerful approach in antiviral drug discovery and testing, current approaches are limited due to difficulties in manipulation of viral genomes and challenges in generation of reliable reporter systems. To overcome these limitations and address knowledge gaps in our understanding of virus-host interactions and antiviral drug efficacy, we sought to generate and characterise viral protease-dependent reporter systems. These systems comprised expression of a green fluorescent protein (GFP ‘mStayGold’) anchored to endoplasmic reticulum (ER) via a Sec61β transmembrane domain and featuring a nuclear localization signal (NLS) separated by specific cleavage peptide sequences that are cleaved by the virally encoded NS2B/NS3 proteases of DENV, ZIKV and WNV. Accordingly, viral infection and cleavage of the reporter construct by the viral NS2B/3 protease was expected to result in nuclear translocation of mStayGold (GFP), allowing detection of infected cells by live cell imaging. We aimed to generate these reporter cell lines via cloning of lentiviral reporter plasmids specific to DENV, WNV, and ZIKV protease (pr), and preparation of replication-defective lentiviral vectors. Despite successful cloning and transfection, generation of stable reporter cell lines for WNVpr and ZIKVpr reporter constructs could not be achieved. However, we sought to validate the plasmid constructs via transient transfection of HeLa cells using the WNVpr and ZIKVpr reporter plasmids. Additionally, we were able to improve the properties of a previously generated stable reporter cell line for DENVpr via Fluorescence Activated Cell Sorting (FACS) to obtain stronger and more homogenous expression of the fluorescent protein component of the reporter, mStayGold. Virus infection experiments were performed in the DENVpr, WNVpr, and ZIKVpr reporter cell lines and to further test the functionality and reliability of the reporter cell line, studies of the DENVpr system were performed using the antiviral compound JNJ-A07. The analysis of live cell imaging data for transiently expressed WNVpr and ZIKVpr reporter constructs following infection with the respective viruses (24h.p.i, MOI=2) revealed no significant differences in levels of early nuclear translocation of GFP for, WNV (Kunjin strain) infection compared to uninfected cells. However, ZIKVpr data showed a significant difference (*, p<0.01) between levels of nuclear GFP translocation for ZIKV uninfected and infected cells at 24h.p.i, assuming comparable infection levels and reporter expression levels, this result suggests Zika Virus NS2B/NS3 protease cleavage efficiency is higher than West Nile Virus NS2B/NS3 protease for early detection (24h.p.i) of infection using equivalent reporter constructs. Notably, the DENVpr reporter system enabled reliable analysis of JNJ-A07 antiviral activity in dose-response studies. Similar functioning of our reporter systems through Western blot analysis using previously generated eGFP tagged DENVpr and ZIKVpr reporter construct cell lines showed the expected viral protease-mediated cleavage of the reporter proteins in DENV- and ZIKV-infected cells. Overall, we have successfully developed reporter systems and supportive data towards achieving our aim of detection of virus infection in living cells via viral protease-dependent fluorescent reporter cleavage events. In spite of the challenges, we propose that our reporter systems might have broad applications in antiviral drug development and analysis of viral replication cycles.
Keywords: Viral Protease, Reporter Systems, Orthoflavivirus, Monitoring Infections, West Nile Virus, Zika Virus, Dengue Virus
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Dr. Nicholas Eyre