A Cell-free Infection System to Study Translation, Replication and Phage-particle Production during Infection of E. coli By Bacteriophage Qβ
Author(s): Pernille Skov Rasmussen, Charlotte Rohde Knudsen
Background: Viruses infect all kingdoms of life, and new species are continuously being discovered. The single-stranded (+)-RNA viruses comprise the largest group of viruses, which includes pathogens such as Dengue virus, Corona virus and West Nile virus. Also, the simple bacteriophage Qβ belongs to this group of viruses. Studies of the mechanism of Qβ infection can increase our general understanding of the single-stranded (+)-RNA viruses, which can be exploited in the pursuit to treat and prevent diseases caused by pathogenic (+)-RNA viruses.
Methods: In this study, we have analysed the production of infectious Qβ phage particles in three different cell-free infection systems upon addition of the Qβ genome as a template. The cell-free infection systems were based on cell-free protein expression systems: two commercial systems and one custom-made system. We studied the course of infection by analysing the production of viral RNA, proteins and phage particles produced in the cell-free reactions. The replication of the viral RNA was determined by RT-PCR, while the translation of the viral proteins was examined by radiolabelling, and the production of infectious phage particles was evaluated by double-layered plaque assays.
Results: Bacteriophage Qβ was found to replicate in two of the three tested cell-free infection systems. Specifically, the viral RNA was replicated, the viral proteins were translated, and infectious phage particles were produced in the cell-free infection systems. The pattern of translation regulation of the viral proteins appeared similar to in vivo infection. Infectious Qβ phage particles were produced at yields of 2.5 × 105 PFU/μL reaction and 2.5 × 103 PFU/μL reaction in the commercial and custom-made system, respectively. Importantly, intact Qβ phage particles were shown not to replicate in the cell-free infection systems under the tested conditions.
Conclusion: Cell-free infection systems can support replication of viral RNA, translation of viral proteins and self-assembly of infectious Qβ phage particles. We provide opportunities for further optimisation of the phage particle yield. Cell-free infection systems can be used in the future to study newly discovered viruses, the development of antiviral and antibacterial drugs, and in biotechnology.