CONSTRUCTION AND CHARACTERIZATION OF A CYAN FLUORESCENT-EXPRESSING POLIOVIRUS

Open Access
Author:
Yang, Ziwei
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Craig Eugene Cameron, Thesis Supervisor
  • Craig Eugene Cameron, Honors Advisor
  • Joyce Jose, Faculty Reader
Keywords:
  • Virology
  • Biochemistry
  • Molecular Biology
  • Microbiology
  • Poliovirus
Abstract:
Positive-strand RNA viruses are well known agents that cause diseases in humans, animals and plants.1 This includes West Nile virus, Severe Acute Respiratory Syndrome (SARS) Virus, Chikungunya virus and most recently Zika virus.2 Due to concerns regarding the potential threat to the public health posed by these viruses, production of effective antiviral therapeutics against RNA viruses has become an increasingly hot topic. However, this requires us to have a comprehensive understanding of the characteristics and mechanisms by which viruses replicate in cells. One particular aspect of this is the cell-to-cell variability of infection by RNA viruses due to viral genetic variation. To uncover this unique feature of RNA virus infection, our laboratory uses poliovirus (PV) as a model RNA virus to investigate and characterize the kinetics of viral replication. My goal is to unravel the distinct trait of viral replication in different individual cells due to the intrinsic diversity of each virus population. All positive-strand RNA viruses exist as a population of genetically distinct variants that leads to different outcomes of infection in individual cells of a tissue or organ.3 However, few laboratories characterize this between-cell variability of infection.32, 33 To do this, a single cell platform must be employed, which requires the use of a fluorescent reporter virus that can be utilized for live-cell imaging.11 Here, I will explain in detail how a recombinant cyan fluorescent protein-expressing PV, CFP-PV, was designed and produced for such purpose. Initial characterizations of the virus using fluorescent imaging suggest that the engineered virus is able to generate robust cyan fluorescent signal upon infection in HeLa cells. In addition, according to the plaque assay results, the CFP-PV demonstrates growth properties stronger than the currently available GFP-PV reporter virus in the Cameron lab by one log order, indicating that the reporter gene engineered into the viral construct does not affect the virus’s infectivity at large. This shows that the reporter virus produced in this work functions well as expected and is ready to be used for single-cell analysis, which aims to characterize the between-cell variability of infection by observing each infection event individually in a population of infected cells.