<p>Flaviviruses are enveloped, positive-strand RNA viruses that
are spread by mosquitoes and ticks and can cause serious disease in humans.
Flavivirus virions undergo extensive structural changes during their life
cycle, including during maturation and fusion. Flaviviruses are initially
assembled at the endoplasmic reticulum in a non-infectious, immature state, and
then traffic to the trans-Golgi network, where a pH drop triggers a structural
rearrangement of glycoproteins prM and E on the virus surface from 60 trimers
to 90 dimers. A host protease, furin, then cleaves prM which makes the
transition irreversible. Upon exiting the host cell, pr disassociates from the
virus and the infectious, mature virus is able to enter a new cell. <br></p><p><br></p>
<p> </p>
<p>In Chapter 1, an overview of flaviviruses is presented,
including a brief history of their discovery and interaction with humans,
followed by what is known about their life cycle and the maturation process.
The structure of a mature flavivirus is then described, including the
symmetrical arrangement of glycoproteins on the virion surface, the lipid
membrane, and the nucleocapsid core, followed by an introduction of the
structural proteins that assemble into the virion. The structure of the
immature flavivirus is then described. The chapter concludes with a description
of the dynamics and heterogeneity observed for flaviviruses.</p><p><br></p>
<p> </p>
<p>The conformational rearrangements that occur during
flavivirus maturation remain unclear. The structures of immature and mature
flaviviruses determined with cryo-electron microscopy (cryo-EM) demonstrated
that flaviviruses are icosahedral particles with 180 copies of glycoproteins on
their surface. Icosahedral viruses typically have a quasi-equivalent
arrangement of glycoproteins, but flaviviruses lack quasi-equivalence and
instead the three subunits within an asymmetric unit occupy different chemical
environments. Although the subunits are the same proteins, the unique
environment of each subunit can be exploited for tracking subunits during
conformational rearrangements. For example, the unique labeling of a subunit
can be used to identify it in the immature and mature virion.</p><p><br></p>
<p> </p>
<p>In Chapter 2, the maturation process was studied by
developing tools to differentially label protein subunits and trap potential
intermediates of maturation. The tools included heavy-atom compounds and
antibody Fabs, which were used to probe Kunjin virus (KUNV), an Australian
subtype of West Nile virus (WNV). One heavy-atom compound, potassium
tetranitroplatinate(II), was found to derivatize immature KUNV, likely at sites
on both E and prM. Higher-resolution studies will be required to determine if
the compound differentially labeled the three subunits. The other tool
developed was the E16 Fab. E16 Fab, originally isolated from a mouse immunized
with WNV E and found to bind to two out of three subunits on mature WNV, was
used to differentially label subunits in immature KUNV. Based on poor epitope
accessibility on immature KUNV, E16 Fab was hypothesized to trap an
intermediate state of maturation. In the cryo-EM reconstruction of E16 Fab
bound to immature KUNV it was found that the virion had localized distorted density
and apparent non-uniform binding of the E16 Fab. Based on this result it was
proposed that flaviviruses had imperfect icosahedral symmetry. <br></p><p><br></p>
<p> </p>
<p>The structural asymmetry of immature and mature flaviviruses
was investigated in Chapter 3. Icosahedral symmetry has always been imposed
during cryo-EM reconstructions of flaviviruses, as it led to stable convergence
of orientations. When reconstructions of immature KUNV and ZIKV were performed
without imposing symmetry, the reconstructions showed that the flaviviruses had
an eccentric nucleocapsid core, which was positioned closer to the membrane at
one pole. At the opposite pole, the glycoprotein and inner leaflet densities
were weak and distorted. Furthermore, there were protrusions from the core that
contacted the transmembrane helices of the glycoproteins. In the asymmetric
reconstruction of mature KUNV, the core was positioned concentric with the
glycoprotein shell, in contrast to the immature virion, indicating that
maturation alters the interactions between the core and the glycoproteins. The
asymmetric reconstructions suggested that there is variable contact between the
core and glycoproteins during assembly, which may be due to membrane curvature
restrictions in the budding process. </p>
<p> </p>
<p><br></p><p>In Chapter 4, extracellular vesicles (EVs) that were
released during dengue virus (DENV) infection were characterized by mass
spectrometry. EVs may play a significant role in flavivirus infection, as they
have been shown to transport both viral proteins and infectious RNA. EVs likely
represent alternative modes of virus transmission and aid in immune evasion.
However, previous studies on EVs are controversial because EVs are potential
contaminated during assays by co-purifying virions and other particulates. The
identification of EV biomarkers would greatly reduce contamination because
biomarkers would enable isolation of pure EVs by affinity purification.
Therefore, a strategy was developed to isolate EVs and profile them with
proteomics. The four proteins cystatin-A, filamin B, fibrinogen beta chain, and
endothelin converting enzyme 1 were found to be statistically enriched in the
DENV sample and represent potential EV biomarkers. </p>
<p> </p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/7994219 |
| Date | 15 May 2019 |
| Creators | Matthew D Therkelsen (6589034) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/Structural_Asymmetry_of_Flaviviruses/7994219 |
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