Identification of proviral and host restriction factors that impact the flaviviriclae lifecycle
Date
2021
Authors
Shue, Byron
Editors
Advisors
Beard, Michael
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Thesis
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Abstract
The complex interplay between virus and host is an often overlooked key determinant
to the outcome of infection. Host factors are instrumental in supporting the entire virus
lifecycle, where a majority have been implicated in the virus replication, especially for
the formation of replication organelles. Simultaneously, the innate immune response
confers a rapid non-specific defence against invading flaviviruses, where interferon
(IFNs) and subsequently interferon stimulated genes (ISGs) are upregulated to directly
inhibit virus replication and induction of an antiviral state within a cellular population.
Although numerous host factors have been identified and characterised to be critical
for the flaviviridae lifecycle through the usage of both RNAi and CRISPR genome-wide
screens, limitations with RNAi technology combined with modifications of screening
methodology may allow elucidation of novel host factors for virus infection. This thesis
aims to identify and characterise novel host factors which positively and negatively
influence the flaviviridae lifecycle, using in part genome wide CRISPR knockout (KO)
technology.
Hepatitis C Virus (HCV) is a medically important member of the flaviviridae family and
thus chosen as a model virus to search for pan-flaviviridae novel host factors. The first
aim explores the generation of multiple HCV sub-genomic (SGR) cell lines with
mCherry, GFP and thymidine kinase (TK) reporter genes and their compatibility with
our chosen screening platform, the GeCKO LentiCRISPRv2 library. Proof of concept
experiments with the LentiCRISPRv2 system were performed via simplistic KO of
EGFP or Cluster of Differentiation 81 (CD81), demonstrating the effectiveness of
CRISPR in targeting both exogenous and endogenous genes. Furthermore, issues
which surface upon attempting the genome-wide CRISPR screen with the HCV SGR
are addressed and future directions proposed.
The second aim repurposes the optimised GeCKO LentiCRISPRv2 library with an
altered screening methodology to identify novel host factors important for ZIKV
infection which may also be important for cell survival. Our top hits include previously
identified host factors from the endoplasmic reticulum membrane protein complex
(EMC) complex in addition to novel host factors Bcl-2-associated X protein 2 (BAX2)
and Receptor for Activated C Kinase 1 (RACK1). We show that RACK1 is an important pro-viral host factor for both mosquito and tick-borne flaviviruses. Furthermore, it plays
a critical role in the construction of replication organelles early in the virus lifecycle.
Furthermore, flavivirus non-structural protein 1 (NS1) which is important for the
biogenesis of vesicle packets (VPs) is able to interact with RACK1 within the ER lumen.
Collectively this aim reinforces the utility of CRISPR/Cas9 genome-wide KO screens
in the identification of viral host dependency factors and identifies RACK1 as a scaffold
protein for the recruitment of viral NS proteins that are essential to the biogenesis of
the replication complex.
The third aim investigates the cellular innate immune response to Zika Virus (ZIKV)
infection. We show that biologically relevant transformed and primary cells infected
with ZIKV have abrogated upregulation of IFN-b and associated ISGs. In addition, we
have identified that ZIKV attenuates the RIG-I-like Receptors (RLR) signalling pathway,
required for the efficient induction of ISGs. Expression of the highly characterised
multifunctional ISG viperin inhibits the ZIKV lifecycle and this observation is
complemented by the usage of ZIKV infected viperin-/- mouse embryonic fibroblasts
(MEFs). Finally, we show that the C-terminus of viperin is critical for ZIKV antiviral
activity, an observation which is supported in future publications by collaborators.
Collectively, this thesis not only enhances our understanding of the flavivirus lifecycle
and their complex relationship with the host but also may guide towards the
development of effective novel therapeutics against flavivirus infection.
School/Discipline
School of Biological Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2021
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