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Characterization of regulatory Flavivirus RNA structure elements

Abstract

Flaviviruses (FVs) are small, single stranded positive-sense RNA viruses of 10-12kb length with highly structured untranslated regions (UTRs). The FV genus includes human pathogens such as Yellow fever virus (YFV), West nile virus (WNV), Japanese encephalitis virus (JEV), Dengue virus (DENV) and the recently emerging Zika virus (ZIKV). The heavily structured UTRs are crucial for regulation of the viral life cycle, inducing processes such as genome circularization, viral replication, packaging, and triggering pathogenicity. Here, we present a study for computational identification and characterization of conserved structural elements in the UTRs of mosquito-borne flaviviruses (MBFV) using covariance models (CMs).
Characterization of regulatory Flavivirus RNA structure elements
Roman Ochsenreiter1, Andrea Tanzer1, Ivo L. Hofacker1,2, Michael T. Wolnger1,3*
1Department of Theoretical Chemistry, University of Vienna, Währingerstraße 17, 1090 Wien, Austria
2Bioinformatics and Computational Biology Research Group, University of Vienna, Währingerstraße 17, 1090 Wien, Austria
3Center for Anatomy and Cell Biology, Medical Unversity of Vienna, Währingerstraße 13, 1090 Wien, Austria
[1] Villordo, S.M., Carballeda, J.M.,Filomatori, C.V., Garmarnik, A.V. (2016), RNA Structure Duplications and Flavivirus Host Adaptation, Trends Microbiol
[2] Pijlman, G. P. et al. (2008), A Highly Structured, Nucleas-Resistant, Noncoding RNA Produced by Flaviviruses Is Required for Pathogenicity, Cell
[3] Chapman, E. et al. (2014), RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA, eLife
[4] Rauscher S. et al. (1997), Secondary structure of the 3’-noncoding region of avivirus genomes: comparative analysis of base pairing probabilities, RNA
[5] Lorenz, R., Bernhart, S.H., Höner zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P.F., Hofacker, I.L. (2011), ViennaRNA Package 2.0, Algorithms for Molecular Biology
[6] Nawrocki, E.P., Eddy S.R., Infernal 1.1: 100-fold faster RNA homology searches (2013), Bioinformatics
Contact: roman.ochsenreiter@univie.ac.at - http://www.tbi.univie.ac.at
1. Motivation
6. Acknowledgements
This work was partly funded by the Austrian Science
Fund FWF projects "RNA regulation of the transcrip-
tome" (F43), "mRNAs von Viren: Evolution und
Struktur-Funktionsbeziehungen" (FWF-I-1303) and the
Austrian/French project "RNA-Lands" (FWF-I-1804-
N28 and ANR-14-CE34-0011).
Flaviviruses (FVs) are small, single stranded positive-sense RNA viruses of 10-12kb length
with highly structured untranslated regions (UTRs) [1]. The FV genus includes human
pathogens such as Yellow fever virus (YFV), West nile virus (WNV), Japanese encephalitis
virus (JEV), Dengue virus (DENV) and the recently emerging Zika virus (ZIKV).
The heavily structured UTRs are crucial for regulation of the viral life cycle, inducing
processes such as genome circularization, viral replication, packaging, and triggering
pathogenicity [2]. Here, we present a study for computational identication and
characterization of conserved structural elements in the UTRs of mosquito-borne
aviviruses (MBFV) using covariance models (CMs).
Upon FV infection, accumulation of
stable long non-coding viral RNAs,
termed subgenomic aviviral RNAs
(sfRNAs) is observed. Intact sfRNAs
are essential for FV survival and
pathogenesis [3].
sfRNAs are produced by the host
exoribonuclease Xrn1, which degrades
the viral genome in 5' to 3' direction.
During degradation, Xrn1 is eectively
stopped at highly stable structure
elements in the 3'UTR, termed xrRNA
(Xrn1-resistant RNA elements).
FVs typically have several xrRNA
elements, each of which possessing
dierent capacity to stop Xrn1, and
thus giving rise to sfRNAs of dierent
lengths. Most important are the so
called 'stem-loop' (SL) and 'dumb-
bell' (DB) elements [4].
2. sfRNA
3. SL/DB RNA families
The Rfam Database currently contains
CMs for both SL-II and DB elements.
Initial screens of UTRs of all FV species
could not reliably annotate elements in
most species, due to the models'
specicity for WNV and JEV.
We manually built seed alignments for
all SL and DB elements in DENV, YFV
and ZIKV. Next, we iteratively rened
the models by scanning all FV UTRs
with our CMs using cmsearch [6] and
manually aligned strong hits to our
seed alignment. This procedure was
repeated until no more new signicant
hits were obtained.
The resulting SL and DB seed
alignments reveal strong structural
heterogeneity, not only among ele-
ments, but also within individual virus
families. While SL/DB elements share
common functionality, they cannot be
properly aligned. We therefore suggest
to consider them as RNA clans, rather
than RNA families.
4. Filtering CMs
We use a Self-Organizing-Map (SOM) for
clustering and visualization of all putative
xrRNAs for quality assessment of CMs
in our clans. Redundant or unspecic CMs
can be quckly identied by frequent co-
localization of descriptors, indicating low
model specicity.
5. Results
We screened all available FV 3'UTRs and
were able to characterize considerably
more xrRNA elements, compared to
screens based on existing Rfam families.
Moreover, we found evidence for pre-
viously unknown elements (DB-II in YFV)
or potentially important events in sfRNA
evolution, such as rearrangement of ZIKV
SL elements compared to DENV.
5'
RCS2
3'
Table 1. Detection ratios for SL/DB elements in various
FVs. An 'X' indicates that a particular species has no
element of this type.
Detection Ratio
Virus Sequences SL-II SL-IV DB-I DB-II SL-Rfam DB-Rfam
DENV-1 1613 1 0.99 1 0.99 0 0.99
DENV-2 1100 1 1 1 0.98 0 0.99
DENV-3 862 0.99 0.95 1 0.91 0 0.99
DENV-4 153 0.99 0 1 1 0 0.99
J EV 239 1 1 1 1 0.91 0.89
WNV 993 1 0.76 1 0.94 1 0.94
ZIKV 83 1 1 1 X 0 0.73
YFV 57 1 0.19 1 0.12 0 0.95
TMUV 35 1 1 1 1 1 0
USUV 90 1 1 1 1 0.99 0
C
A
G
G
C
CAGA
A
A
A
A
___C C U G
CCACCG
G
A
AGUUG
A
G
U
A
G
A
C
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U
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C
U
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C
C
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A
C
A
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C
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_
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A
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_
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CG
C
C
A
G
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AA_AA
G
C
C
A
C
C
U
G
A
U__CC
G
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A
A
G
G
U
G
C
U
G
C
C
U
G
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A
C
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C
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C
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AAA
G
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A
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A
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C
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C
U
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A
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GA
U
C
A
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_
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AGC
C
AUAGU
A
CGGA
A
A
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C
U
A
U
G
C
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A
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UCAGCCACAGCUU
GGG
G
A
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C
U
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A
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GC
UA
C
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A
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C
A
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C
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A
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DENV
ZIKV
C
A
G
_
_
_
_
____
C
C
C
A
U
C
A
U
AA
UGAUGCCAUGGCU
AAGCU
G
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G
A
G
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C
C
A
U
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C
U
G
G
C
U
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A
U
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A
JEV
SL-IISL-I
AROV/KOKV YFV
DB-IIDB-I
G
G
G
A
G
G
C
C
A
C
A
A
A
C
C
A
U
G
G
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A
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C
UGUA
C
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CAUGGCGU
A
GCAG
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A
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C
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C
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A
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G
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C
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_
A
A
C
C
A
A
G
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A
A
U
G
G
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___GU
G
A
CCCAGGG_GGA
A
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A
C
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A
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_
A
X X
X
Fig. 2 Consensus RNA structures of each RNA family of the
SL-I/II and DB-I/II clans. An 'X' indicates that a species has
no element of this type. Consensus structure prediction has
been computed with RNAalifold [5].
DB.DENV.1
DB.DENV.2
DB.JEV.1
DB.JEV.2
DB.KOKV.1
DB.KOKV.2
DB.YF
DB.ZIKV.1
Fig. 3 Self-Organizing-Map. Each circle represents a
cluster of similar DB elements. The segment sizes reflect
the strength of a particular descriptor (here: cmsearch E-
value of a CM) in this cluster. Frequent co-localization of
two or more descriptors indicate redundant CMs (brown
and green segments).
5'3'
CDS
Xrn1
SL-I SL-II DB-I DB-II 3'-SL
5'3'
3'
Fig. 1 Architecture of MBFV 3'UTRs. RNA elements attri-
buted to halting Xrn1 are shown in red and blue. SL1 and SL2
correspond to previously described SL-II and SL-IV in West
nile virus (WNV).
A U
--
P3
P2
P1
C
A
5' 3'
C
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