Host Factors Screen and Mechanistic Study for Zika Virus in a Novel Chinese Tree Shrew Cell Line Model

Abstract

The recent Zika virus (ZIKV) outbreak has been declared a global public health emergency. Zika virus infection has significantly threatened human health globally. Conventional animal models, e.g. rodent model, of ZIKV infection have provided valuable insights on ZIKV virology. However, due to significant genetic differences, the knowledge obtained from rodent models are not always applicable to understanding human infections. The Chinese tree shrew has a close genetic relationship with primates and human and thus could be a superior animal model for ZIKV infection. Furthermore, recently the Chinese tree shrew was shown to be highly susceptible to ZIKV infection and has emerged as a new model for studying ZIKV. In this study, we have developed a stable Chinese tree shrew breast cancer cell line model (TSBC) from a spontaneous breast cancer tumor of an animal. TSBS is highly susceptible to ZIKV infection, and we thoroughly tested this system as a new model for investigating the cell biology of ZIKV infection.

Small interference RNAs (siRNA), which function in the RNA interference (RNAi) pathway, have been widely used for the investigation of gene functions. In RNAi, mRNA is degraded by siRNA with a complementary sequence, resulting in the silencing of the target gene. We used a RNAi screen method to identify essential genes and host factors of ZIKV in TSBC. Conventional siRNA libraries are chemically synthesized and there is no existing siRNA library for the Chinese tree shrew. We developed a new approach using bacteria to produce sequence specific prokaryotic siRNA (pro-siRNA) and pro-siRNA-based transcriptome-wide RNAi libraries. The pro-siRNA library represents the exact isoforms of expressed mRNAs and can be customized to any species.

We produced a TSBC specific pro-siRNA library and performed two RNAi screens on TSBC. The first siRNA screen identified essential genes required for the survival of TSBC, including STT3 Oligosaccharyltransferase Complex Catalytic Subunit A (STT3A), Adipogenesis Regulatory Factor (ADIRF), Secreted Protein Acidic and Cysteine Rich (SPARC), and Nicastrin (NCSTN). NCSTN has shown potential roles in cell cycle regulation and induction of cell apoptosis. The second siRNA screen is to identify host factors of ZIKV infection in TSBC. We identified and studied three novel host factors including Aspartyl-TRNA Synthetase (DARS), Kruppel Like Factor 6 (KLF6), and Tubulin Folding Cofactor D (TBCD), which are involved in ZIKV entry and replication processes. We found that TBCD is involved in the functions of endoplasmic reticulum, Golgi apparatus and intracellular pH regulation during the ZIKV infection process. Clinically approved drugs, such as Halofuginone, Compound Glycyrrhizin Acid Injection (CGI), Brefeldin A and Bafilomycin A1, were tested for their therapeutic potentials in ZIKV infection. Our study established a novel cell line infection model for ZIKV and revealed novel mechanisms of ZIKV infection.

Date of Award	7 Feb 2022
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Xin DENG (Supervisor) & Linfeng HUANG (External Co-Supervisor)