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Musashi Binding Elements in Zika and Related Flavivirus 3'UTRs
Abstract
Emerging and re-emerging arthropod-borne viruses such as Japanese encephalitis (JEV), Dengue (DENV), Yellow fever (YFV), and Chikungunya (CHIKV) viruses are a growing global health threat. Zika virus (ZIKV) is a neurotropic flavivirus (FV) that can cause congenital infection, which can result in microcephaly and fetal demise. Recently, the translational regulator protein Musashi-1 (Msi1) has been attributed to promoting ZIKV replication, neurotropism, and pathology. Msi1 predominantly binds single-stranded UAG motifs in the 3'UTR of RNA.
Here we systematically analyzed the thermodynamic properties of Musashi binding elements (MBEs) in the 3'UTR of 76 arbovirus genomes in silico. Our results indicate that MBEs in the ZIKV 3'UTR occur predominantly in unpaired, single-stranded structural context, thus supporting experimental observations of Msi1 binding affinity with a thermodynamic model of RNA structure formation.
... Tandem RNA structures within DENV 3 UTR are under different selective pressures in alternating hosts, suggesting the idea that duplicated RNA structures differentially evolved to accommodate specific functions in the two hosts [20]. Likewise, there is evidence for evolutionary pressure on maintaining the primary sequence of parts of duplicated RNA elements, as recently shown for flaviviral dumbbell (DB) elements in the context of finding a biophysical model for explaining a possible route for ZIKV-induced neurotropism [21]. Viral RNA genomes are different from procaryotic and eucaryotic mRNA. ...
Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural elements involved in mediating the viral life cycle, including cyclisation, replication, and encapsidation. Here we report on a comparative genomics approach to characterize evolutionarily conserved RNAs in the 3 UTR of tick-borne, insect-specific and no-known-vector flaviviruses in silico. Our data support the wide distribution of previously experimentally characterized exoribonuclease resistant RNAs (xrRNAs) within tick-borne and no-known-vector flaviviruses and provide evidence for the existence of a cascade of duplicated RNA structures within insect-specific flaviviruses. On a broader scale, our findings indicate that viral 3 UTRs represent a flexible scaffold for evolution to come up with novel xrRNAs.
... Differences within ZIKV lineages been observed, behavior of ZIKV UTR region is different from genomic and ZIKV has the lowest opening energy for MBE among all flaviviruses[93]. When performing a comparison between CHIKV and ZIKV, Ifound that most ZIKV sequences have the lowest opening energy. ...
Arboviruses are a grade of viruses carried by arthropods, which have been in the
headlines due to recent epidemics. Members of this grade are the families Flaviviridae
which includes Zika (ZIKV), Dengue (DENV), Yellow Fever (YFV), among other
viruses and Togaviridae, which includes Chikungunya (CHIKV).
Research on some arboviruses has been strong over the past couple of decades.
Other arboviruses have not garnered much attention until lately. For example, ZIKV
has been understudied until 2015. Since the 1950s ZIKV was considered to cause
only a benign infection in humans. ZIKV became well studied only after the recent
outbreaks of the virus in the Pacific, Americas, and South-East Asia, was found to
be related to severe neuropathology, which includes the development of neurological
defects such as microcephaly on the fetus and Guillain-Barré Syndrome in adults.
CHIKV is another arbovirus that although been circulating for a long time in Africa
and Asia, has been recently introduced into the Americas in 2013, causing recurring
outbreaks in South and Central American naïve populations.
YFV, which been known to be endemic and thought to be controlled in South
America, has re-emerged in Brazil beginning in December 2016. This outbreak, although
restricted to transmission by the sylvatic mosquito Haemagogus leococelaenus,
raised questions among researchers regarding the potential for spread to the United
States due to the presence of the urban vectors Aedes aegypti and Aedes albopictus
and a naïve, largely unvaccinated population. Another question that still remains is
whether YFV will ever reach the Asian continent?
Today, the time it takes for awareness of the health organizations, to convince
the funding agencies, and to work on vaccine development is much more than the
iv
time needed for the disease to change from a local outbreak to a global epidemic.
The overall objective of this work is to provide the grounds for a viral surveillance
system based on evolution, utilizing the current ZIKV and CHIKV outbreaks and
other arboviruses as case studies.
Utilizing phylogenetic and molecular sequence alignment tools I developed a pipeline
to evaluate the genomic changes of viruses on CHIKV and ZIKV. I also created a
pipeline to generate pathogen transmission networks and compare different disease
networks utilizing different network centrality metrics. CHIKV, DENV, YFV and
ZIKV were utilized as case studies. The strategies utilized in this work will enable
better abatement and management strategies of viral outbreaks.
My findings indicate that changes in the coding sequence does not seem to be the
main reason why ZIKV has changed its behavior in terms of pathogenicity. In CHIKV
there is an insertion on the UTR region of a group of sequences and change of virulence
has been associated with UTR sizes in different CHIKV strains. Upon analyzing
viral 3’ and 5’ UTRs, a trinuncleotide motif, known as Musashi Binding Element
was identified in both CHIKV and ZIKV, its presence and availability on ZIKV may
explain a preference to human cells, in CHIKV the motif is present but not available.
Although both CHIKV and ZIKV coexist and have spread in the same regions in a
short period of time, their spread seems to be from independent events. When looking
at transmission networks, there is a high correlation between the different centrality
metrics utilized to measure all four DENV serotypes transmission networks, CHIKV,
YFV and ZIKV have lower correlation, thus, distinct patterns.
Zika virus (ZIKV) infection remains a worldwide concern, and currently no effective treatments or vaccines are available. Novel therapeutics are an avenue of interest that could probe viral RNA-human protein communication to stop viral replication. One specific RNA structure, G-quadruplexes (G4s), possess various roles in viruses and all domains of life, including transcription and translation regulation and genome stability, and serves as nucleation points for RNA liquid-liquid phase separation. Previous G4 studies on ZIKV using a quadruplex forming G-rich sequences Mapper located a potential G-quadruplex sequence in the 3′ terminal region (TR) and was validated structurally using a 25-mer oligo. It is currently unknown if this structure is conserved and maintained in a large ZIKV RNA transcript and its specific roles in viral replication. Using bioinformatic analysis and biochemical assays, we demonstrate that the ZIKV 3′ TR G4 is conserved across all ZIKV isolates and maintains its structure in a 3′ TR full-length transcript. We further established the G4 formation using pyridostatin and the BG4 G4-recognizing antibody binding assays. Our study also demonstrates that the human DEAD-box helicases, DDX3X132-607 and DDX17135-555, bind to the 3′ TR and that DDX17135-555 unfolds the G4 present in the 3′ TR. These findings provide a path forward in potential therapeutic targeting of DDX3X or DDX17’s binding to the 3′ TR G4 region for novel treatments against ZIKV.
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