Development of Therapeutic Agents Against ss (+) RNA Virus Infection and Mechanistic Study of the Interplay between Enterovirus 71 and the Host Secretory Pathways

抗正鏈RNA病毒感染的治療劑的開發以及腸病毒71型與宿主分泌途徑之間相互作用的機理研究

Student thesis: Doctoral Thesis

View graph of relations

Author(s)

Related Research Unit(s)

Detail(s)

Awarding Institution
Supervisors/Advisors
Award date13 May 2020

Abstract

Positive-sense single-stranded RNA (ss(+)RNA) viruses, including coronaviruses such as severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, Flaviviruses such as Japanese encephalitis virus, Zika virus and Dengue virus, and enteroviruses such as Enterovirus 71, are among the leading causes of human and animal infectious diseases in the world, and the morbidity and mortality of diseases caused by these viruses have been increasing every year. To date, no effective antiviral agents are available for treating the infections of these causatives. Moreover, the current global outbreak of coronavirus highlights the urgent need for effective vaccines and antiviral drugs. The characterization of how viruses manipulate host secretory pathways should help us understand the life cycle of viral infections and provide us with knowledge for developing antiviral agents. In this thesis, we assessed the antiviral effects of autophagy inhibitors (chapter 3), mechanically studied how EV71 hijacked the host secretory pathway to facilitate viral amplification (chapter 4), and assessed Ca2+ channel blocker—berbamine’s antiviral effects on several ss(+)RNA viruses and dissected the underlying mechanism (chapter 5).

First, our findings show that flaviviruses, including JEV and ZIKV, induce autophagy to promote replication, whereas autophagy-related protein 5 knockdown or treatment of cells with autophagy inhibitors suppressed flavivirues propagation. Using a high-content image screening, we identified enanderinanin J, an ent-kauranoid dimer, as a late-stage autophagy inhibitor, manifested by its ability to increase LC3-II accumulation, lysosomal pH and inhibit the fusion between autophagosome and lysosome. We then evaluated the antiviral activity of enanderinanin J on DENV, JEV, ZIKV and EV71. The results show that enanderinanin J potently inhibited these viruses, with relatively low cytotoxicity.

Second, we focused on the post-entry life cycle of EV71, studying how EV71 manipulates secretory pathways, including autophagic machinery, to facilitate RNA replication, assembly and release. Our findings show that EV71 reorganized the membranous structures of the trans Golgi network to generate superior vesicle packets for genome RNA replication. We also found that autophagosome membranes were not related to EV71 replication organelles (ROs) because only some autophagy related proteins were involved in EV71 replication and some essential autophagy related proteins are not required for replication. These components of autophagic machinery were beneficial for viral RNA replication in an autophagy-independent manner. Followed by replication, the newly synthesized vRNAs were either transported to ER through retrograde transport to translate viral proteins, or translocated to endosomal vesicles for assembly, where finally the packaged virions were secreted to extracellular space through lysosomal vesicles. Our findings show that knockout of Rab7A or VPS33B blocked the endosomal trafficking, leading to a remarkable increase of viral proteins and viral particles within the cells, and less virions secreted outside the cells. Moreover, the depletion of VPS33B markedly induced cell death in EV71 infected cells. These data indicate that EV71 takes full advantage of the secretory pathway for viral assembly and release, and suppresses cell apoptosis caused by viral lytic release to enable further viral amplification within the cells.

Lastly, Ca2+ signalling plays important roles in almost every aspect of viral life cycles. In our studies, we found that chelation of intracellular Ca2+ with BAPTA-AM blocked flavivirus infection. We found that berbamine and tetrandrine, two bis-benzylisoquinoline alkaloids isolated from herbs and known calcium channel or signalling inhibitors, potently inhibited flaviviruses JEV, ZIKV and DENV. Three other analogues of berbamine, fangchinoline, isotetrandrine and E6 berbamine, impaired flavivirus infections as well. Through comparing the selectivity index of these compounds, we found that berbamine has more potential as an antiviral therapeutic. Moreover, we found that berbamine inhibited EV-71, MERS-CoV and SARS-CoV-2 infection of host cells, suggesting that berbamine can be developed as a broad-spectrum antiviral agent. In vivo study shows that berbamine can protect mice from lethal challenge of JEV. We further found that berbamine inhibited TRPMLs (endo-lysosomal Ca2+ channels) by compromising the endolysosomal trafficking of plasma membrane receptors, including membrane receptors of flaviviruses. This led to the increased secretion of these receptors via extracellular vesicles and the concomitant decrease of receptor levels at the plasma membrane, thereby preventing flavivirus from entering host cells. Berbamine is an over-the-counter (OTC) drug, which is considered safe and effective for use by the general public without a prescription. In summary, our findings indicate that berbamine has great potential as a therapeutic against a variety of viral infections.

    Research areas

  • therapeutic agents, viral infection, flavivirus, enterovirus, enanderinanin J, berbamine, autophagy, antiviral agent, secretory pathway