RNAi Screen for Host Factors Required to Zika Virus in Mosquito and Biochemical Study of the p19 Protein

Abstract

RNA interference (RNAi) is a gene regulation mechanism that occurs in many eukaryotic organisms. RNAi machinery can degrade messenger RNA (mRNA) in a sequence-specific manner mediated by small interfering RNA (siRNA). Due to the ability of designing sequence-specific siRNAs, RNAi technology can be used to study gene functions in any biological pathways. RNAi-based forward genetic screen can be applied to uncover novel genes involved in important diseases. However, traditional RNAi screens are expensive and laborious. The invention of the pro-siRNA (for ‘prokaryotic siRNA’) technology has significantly reduced the cost of producing siRNAs for individual genes and siRNA libraries for any organism.

To uncover important host factors of ZIKa Virus (ZIKV) in its natural host mosquito, I established a pro-siRNA-based RNAi screen using both Vero(E6) cell line and C6/36 cell line. Pro-siRNA libraries for the specific cell lines were produced using a high-throughput and cost-effective method developed in our lab. A Zika virus infection and detection system has been established for mosquito cells using an antibody against a viral protein and a high-content screen system to track the infected cells. The pro-siRNA screens have identified several candidate genes that are potential regulators of Zika viral infection process.

To further improve the pro-siRNA technology, the biochemistry of p19 protein was investigated especially on its binding affinity to double-stranded RNAs (dsRNAs). The Tombusvirus-encoded p19 was considered an obsolete identification at the time of its discovery, until it was recognized as an important viral pathogen a decade later. Research eventually revealed that p19 protein could sequester siRNA and thus inhibit RNAi, which made it a useful biotechnical tool for plants and animals. However, all the recombinant p19 proteins purified in previous studies would have carried some small RNAs from the bacterial cells. To address this issue, I aimed to improve the purity of recombinant p19 proteins produced in Escherichia coli (E. coli). Firstly, I established the expression and purification method for holo p19 proteins without carrying siRNAs using RNase III-deficient E. coli strain HT115(DE3). I unexpectedly found that holo p19 proteins could form not only dimer, as known previously, but also polymers, which have not been reported. A series of biochemical assays using purified holo p19 proteins showed that p19 formed dimer only when 21-nt siRNAs were bound to the p19 proteins. The polymer forms of p19 proteins could bind to dsRNAs shorter or longer than 21 nt. Gel filtration and negative-stain electron microscopy techniques confirmed the formation of p19 polymers. This project uncovered a hidden complexity about the dsRNA affinity of p19 and novel structural conformations of p19 proteins. The process of polymerization and the underappreciated binding capacity of p19 to long dsRNAs could help understand the biological roles of p19 and resolve a few key issues for its application in biotechnology.

Date of Award	16 May 2023
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Xin DENG (Supervisor), Linfeng HUANG (External Co-Supervisor) & Hon Yeung CHEUNG (Co-supervisor)