Flavivirus-host interactions

on RNA and protein level

Mgr. Hana Pejšová
Supervisor: RNDr. Ján Štěrba, PhD.
 Flaviviruses
• Member of the Flaviviridae family with more than 70 related viruses

• Mosquito-borne, tick-borne, insect-specific and no-known-vector flaviviruses

• Similar genome organisation and conserved non-structural protein

patterns

• Tick-borne encephalitis virus, dengue virus, zika virus etc.

(Kleinert et al., 2019)

Tick-borne encephalitis virus

• Arthropod-borne virus of the Flaviviridae family

• Transmitted by Ixodes ticks

• The reservoir of the virus are small rodents, deer, moles etc.

• 3 serologic subtypes - European subtype

- Siberian subtype
- Far eastern subtype
 Tick-borne encephalitis virus
• Virions of approximately 50 nm

• Genome: +ss RNA (11 kb)

• 1 open reading frame - 3 structural proteins (C, M, E)

- 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B a NS5)

• 5‘ and 3‘ untranslated regions

(Mandl, 2005)

(Owolabi et al., 2016)

 Subgenomic flaviviral RNA

• Subgenomic flaviviral RNA is a product of incomplete degradation of viral genomic RNA by

XRN-1

• Stemloops and pseudoknots has been demonstrated in mosquito-borne flaviviruses such as

WNV, YFV, DENV-2, ZIKV, MVEV but also in TBEV recently

ZIKV xrRNA structure (Akiyama et al., 2016) DENV 3‘UTR (Slonchak et al., 2015)
Subgenomic flaviviral RNA
Functions are very little known:

• Inhibiting mRNA degradation pathways (e.g., DENV, Moon et al., 2012)

• Influencing the host immune system and the pathogenicity of the virus

• Supressing the type I interferon response (WNV, Schuessler et al., 2012; ZIKV, Donald et al., 2016)

• Supressing RNAi antiviral response (DENV, Moon et al., 2015; WNV, Schnettler et al., 2012)

• DENV sfRNA has been observed to bind and inactivate RNA-binding proteins (G3BP1, G3BP2, and CAPRIN1;
Bidet et al., 2014)
Tick-borne encephalitis virus sfRNA

i
hp
i
hp

48
i
24

hp
i
hp

r fl
Hy erfl

24

oe
24
do

fjin

ud
pr
u

Ne
So
Ne
size
• Crystal structure has not yet been described, only predicted. gRNA [bases]
6000
• TBFs halt sites do not contain observed conserved sequences of MBFs. 4000
3000
2000
• The presence of sfRNA was demonstrated in our lab occuring during 1500
the infection of TBEV. 1000
500
sfRNA 200

18S RNA

Neudoerfl

DENV (Ng et al., 2017)

Tick-borne encephalitis virus
Translational shut-off
• TBEV decreases de novo host synthesis of
proteins, 28S and 18S rRNA and 45-47S
pre-rRNA transcripts
45-47S pre-rRNA

(Selinger et al., 2019)

 Optimisation of cell transfection with in vitro transcribed sfRNA

• In vitro transcription – pDEST40 constructs- CMV and T7 promotor, 3‘ UTR (DENV, TBEV, ZIKV) and
HDVr 6.00
5.53

log10 fold-change of sfRNA levels

4.87 4.78
• Transfection of DAOY HTB-186 cells 5.00
4.54

4.00
• Polyjet transfection reagent 3.47

3.00 2.80
2.58
• Cells lysed with RNA Blue reagent (Top-Bio) 2.21
2.00

• RT-qPCR (2-ΔΔct) 1.00

0.00 24hpt 48hpt 24hpt 48hpt 24hpt 48hpt 24hpt 48hpt

DENV ZIKV TBEV N TBEV H
Determination of the role of sfRNA on de novo
host protein and rRNA synthesis

HPG-
Determination of the role of sfRNA on de novo host protein
synthesis

• Newly synthesised proteins metabolicaly labelled with HPG

DENV ZIKV N H MBP PK NK NK + M

• Click-on-membrane assay

M – marker MBP – maltose—binding protein

DENV – dengue virus PK – positive control
ZIKV – zika virus NK – negative control
N – Neudoerfl strain NK + M – NK + new medium
H – Hypr strain • Developed using WesternBright Quantum HRP substrate
Determination of the role of sfRNA on de novo host rRNA
transcription.

• Newly synthesised RNA metabolicaly labelled with 5-EU

28S • Click-on-membrane assay

18S

DENV – dengue virus MBP – maltose—binding protein

ZIKV – zika virus PK – positive control
N – Neudoerfl strain NK – negative control
H – Hypr strain • Developed using WesternBright Quantum HRP substrate
Localisation of sfRNA in transfected and infected cells using
fluorescence in situ hybridisation
• Optimisation of transfection (Lipofectamine® RNAiMAX Transfection Reagent vs PolyjetTM In
vitro DNA Transfection Reagent)

• Optimisation of probe labelling method (nick translation vs random decamers)

• Optimisation of detection (direct vs indirect fluorescence)

• Finding optimal indirect label (DIG-AntiDIG vs Biotin-Streptavidin)

• Optimisation of fixation method (PFA vs methanol vs acetone vs DSP cross-linker)

• + blocking of endogenously expressed biotin

Localisation of sfRNA in transfected and infected cells using
fluorescence in situ hybridisation

DAPI 3‘UTR (sfRNA) C protein gene merge

Scale bar represents 200 µm.

• Cells infected with TBEV strain Hypr for 24 hours and fixated with DSP.

• DNA probe targetting TBEV 3‘UTR labelled with biotin and DNA probe for C protein gene labelled with DIG using
nick translation.

• Biotin detected with Streptavidin DyLight® 549 and DIG detected with Anti-DIG DyLight® 488.
Localisation of sfRNA in transfected and infected cells using
fluorescence in situ hybridisation
3‘UTR (sfRNA) C protein gene merge

Scale bar represents 200 µm.

Localisation of sfRNA in transfected and infected cells using
fluorescence in situ hybridisation

TBEV Hypr merge mock merge

 Ongoing plans

• Characterization of the interactiome of sfRNA identification of host and viral sfRNA-binding proteins using RNA pull
down assay (via S1a RNA aptamer-streptavidin system) combined with LC/MS identification

4x S1a aptamers
S1a RNA aptamer (Leppek and Stoecklin, 2013)

5‘ 3‘

S1a aptamer with Hypr sfRNA

4x S1a_Hypr 3‘UTR construct in a phMGFP plasmid

• Preparation of sfRNA-aptamer construct and optimisation of transfection and RNA pull-down

• In situ detection of sfRNA using FISH - OPTIMISATION

• Detection of newly synthesised RNA in sfRNA transfected cells using Click-on-membrane chemistry - OPTIMISATION
 Future plans

• Determination of sfRNA-binding characteristics in selected identified proteins

• Analysis of the functional relationship of selected proteins to sfRNA-mediated pathogenesis

• Further optimisation of the detection of sfRNA in situ using FISH by producing specific anti-sfRNA antibodies via
immunisation of laboratory guinea pigs or mice using purified sfRNA molecules

• + identification which RNA species binds flaviviral capsid protein in host cells and determining the key amino acid
residues responsible for such bindings
Thank you for your attention. 