1. The mechani smof t ransl at i on
i ni t i at i on on t he genomi c RNA of
Cadi ci vi rus A: a nat ural l y occurri ng
di ci st roni c pi cornavi rus and t ype
member of t he novel genus
Di ci pi vi rus
Mukta Asnani
Dr. Tatyana Pestova
Dr. Christopher Hellen
Department of Cell Biology, SUNY Downstate Medical Center

2. RNA viruses: Infection and hijacking of cellular translation
apparatus
Viruses depend on the host cell's translation apparatus.
They commonly suppress translation of cellular mRNAs by inhibiting the canonical mechanism
of cap-dependent initiation of translation – to favor viral protein synthesis and to impair host
antiviral responses.
This raises the question:
How does viral translation proceed in these circumstances?
Investigation of this question may reveal unique aspects of viral translation initiation that are
potential targets for therapeutic inhibition.

3. The canonical mechanism of cap-dependent translation initiation and sites of
viral regulation
AUG UAG
AUG UAG
E P A
AUG UAG
E P A
AUG UAG
E P A
AUG
UAG
E P
A
AUG UAG
1. mRNA Activation by
eIF4F cap-binding complex
2. Recruitment of
43S complexE
P
A
3. 5’ to 3’ Scanning
4. Initiation codon recognition
and
48S complex formation
48S complex
eIF4E
eIF4G
eIF4A
eIF4B
eIF1
eIF1A
eIF2
eIF5
eIF3
43S complex
40S
G
TP
GTP
GTP
E P A
AUG UAG
5. GTP hydrolysis by eIF2,
release of factors,
60S Subunit joining
6. Hydrolysis of GTP
by eIF5B & release
of eIF5B
80S complex
eIF5B
60S
GTP
GTP
DHX29
GDP
Viral proteases (2A and
3C) synthesized during
infection cleaves host
initiation factors and
hence shuts off the
canonical translation
initiation and allow
selective translation of
viral RNA genome
2A
3C
eIF4F complex

4. The genomes of several families of RNA viruses contain internal ribosomal entry sites
(IRESs), which mediate end-independent initiation, enabling viral mRNAs to bypass
the canonical cap-dependent mechanism
Characteristics of IRES-
1. Long highly structured positioned in 5’-untranslated region of mRNA, which serves the function of interacting with many
canonical initiation factors and other cellular factors.
2. Reduced requirement of initiation factors particularly cap-binding eIF4F complex.
3. Recruits 40S directly onto the mRNA in the vicinity of initiation codon.
4. Requires certain cellular factors called ITAFs (IRES-trans acting factors) which is generally not required during
canonical cap-dependent translation. In addition to modulating IRES activity, these ITAFs also plays an important role
in various cellular functions.
This alternative mechanism of translation initiation was first observed to be used by poliovirus RNA
genome in infected cells in late 1980s.
Poliovirus genome
Poliovirus IRES (~450 nt)
eIF4Gm
PCBP2
PCBP2 – ITAF
eIF4Gm – cleaved eIF4G
Sweeney et. al. (EMBO, 2014)

5. Classification of Viral IRESs
Family Genus Example IRES
class
Key
interaction
ITAFs (IRES Trans acting
factors)
Picornaviridae Aphthovirus Foot-and-mouth disease virus (FMDV)
Type 2 eIF4G
PTB, ITAF45
Cardiovirus Encephalomayocarditis virus (EMCV) PTB
Enterovirus Polio virus
Type 1
eIF4G
PTB
Rhinovirus Human rhinovirus (HRV) PTB, PCBP2, La, hnRNP A1, unr?
Flaviviridae Hepatitis C virus (HCV))
Type 3
40S subunit
Cripaviridae Cricket paralysis virus (CrPV) Type 4 40S subunit
IRESs are classified into different types depending on their secondary structure and initiation factors
requirements.
Non-canonical interactions of IRESs with
canonical components of the translational
apparatus
Poliovirus
Encephalomyocarditis
(EMCV)
Hepatitis C virus
(HCV)
Cricket paralysis
virus (CrPV)
IRES/eIF4G
IRES/eIF4G IRES/40S IRES/40S

6. Internal Ribosomal Entry Site (IRES) links to past of the translation
initiation mechanism ??
Canonical initiation-
In 1988 first IRES was found in
Poliovirus and EMCV
In 1991 first cellular IRES was found in
IgG heavy chain binding protein (BiP)
Quick response under stress
condition such as hypoxia, DNA
damage by UV, nutrient deprivation
etc.
Highly regulated process
(Cap-dependent)
Relic of the past and
evolved in matured
eukaryotes ??
Evolved in eukaryotes to
regulate gene expression
under stress ??
IRES study will shed light on
past of the translation initiation
mechanism
Cap-Independent

7. Viral Zoonoses – Cause of Human Infectious Diseases
 Animals like bats and migratory water birds are always found to be reservoir host of zoonotic pathogens.
Cross species transmission has given rise to 70% zoonotic diseases in humans by host switching and adaption
leading to outbreaks in new hosts.
 Thus zoonotic viruses always pose a threat to human health.
 Understanding of these viruses might prevent the dreadful epidemic.
Bean et. al. (Nature, 2013)

8. Why is it important to study IRES - dependent Translation?
To understand not only the translation mechanism used by different viruses but also the
processes and regulation of cellular mRNA translation.
 To understand how does cells and viruses impart specific translation of mRNAs in sea of
competent transcripts.
 The understanding of IRES mediated translation and role of various initiation factors in
stimulating their activity can be extended to the cellular translation as well.
 Understanding of the viral IRESs can also help to understand the translation of various
cellular IRESs present in the transcript encoding proteins expressed under compromised
conditions such as apoptosis, differentiation, hypoxia and nutrient deprivation when cap-
dependent translation is inhibited.
To study various antiviral and signaling pathways activated during viral infection.
 The study of one virus IRES can be extrapolated to understand the mechanism of
translation used by novel or already known IRESs.
Thus there is always a constant hunt for the new viruses from different
species.

9. Dicistroviridae
Before genome
sequencing era
(2 families were unrelated)
Picornaviridae
?
After genome
sequencing era
(both are related)
Picornavirus –like
superfamily
Multiple steps of translocation and
IRES deletion/duplication
Found in arthropods such as shrimps,
honey bee and insect pests of
agricultural and medical importance
(eg- triatoma virus cause chagas’
disease, infected many Latin
Americans)
Found in humans and wide variety of
animals in which they can cause
respiratory, cardiac, hepatic,
neurological diseases.
Hosts different
but contain same
gene contents
Different genome organization
Search of new viruses – To understand evolutionary past
Woo et. al. (J Virol, 2012)

10. Discovery of Canine dicistronic picornavirus (Cadicivirus A, CDV-A)
•In order to study picornavirus family and distantly related members, current screening efforts have
identified growing numbers of picornaviruses with 5'UTRs that diverge from known IRES types, and that
may therefore contain novel IRESs or variants of known IRESs.
• We became interested in Canine dicistronic picornavirus (Cadicivirus A or CDV) which was recently
characterized in the course of efforts to identify novel viruses in dogs. This was undertaken because
viruses occasionally gain the ability to spread within new hosts, leading to the emergence of new
epidemic diseases. An understanding of mechanisms underlying viral emergence is necessary for the
rational design of antiviral control strategies, and cross-species transmission of viruses from dogs is
possible because of their long history of cohabitation with humans.
•Cadicivirus A has a dicistronic genome with a 982nt-long 5'UTR and a 588nt-long intergenic region
(IGR).These noncoding regions have both been shown to function as IRESs.
• 982 bases
• 42% G-C rich
• 3’ end shows strong
sequence similarity to stem
loop V of the poliovirus IRES
5’UTR IRES
844 amino acids 1406 amino acids
IGR IRES
• 588 bases
• 3’ end shows strong sequence
similarity to stem loop V of the
poliovirus IRES
My Topic of Interest

11. Prediction of 5’UTR IRES Structure of CDV-A and analyzation using
SHAPE (Selective 2’-hydroxyl acylation analyzed by primer extension)
Binding sites for primers used for probing modifications across the RNA
Reverse
transcriptase
Primer-extension analysis
of modified RNA using
radiolabeled primer
A
B
C
D
F
G
H
I
J
K
L
M
N
AUG
983
NMIA
(N-methylnitroisatoic
anhydride)
Sequence of DNA
- + NMIA
Full length
RNA
Modified
nucleotides
C T A G
Predicted Structure using sequence co-variation
analysis and MFold software
Mechanism of Action
Different primers used to probe the modification along
the IRES
1
2
3
4
5
6
7
8
9
Jennifer et. al. (JACS, 2012)

12. Correlation of SHAPE analysis with the predicted structure
SHAPE data almost perfectly fit the predicted structure of the IRES
and hence confirmed the predicted structure.
B
C
D
F
G
H
I
J
K
L
M
N
Representative gel using primer 2

13. II
III
IV
V
VI
VII
py
AUG
Comparison between the structures of Cadicivirus-A 5’UTR
and poliovirus IRESs
A
B
C
D
F
G
H
I
J
K
L
M
N
UUG
AUG
983
py
GNRA
Tetraloop
Poliovirus IRES
GNRA
Tetraloop
Highlights-
1. CDV-A domain M resembles domain V of the poliovirus IRES.
2. CDV-A domain N (∆G = -4.2 kcal/mol) containing UUG-951 is much less stable
than poliovirus domain VI (∆G = -17.1 kcal/mol).
3. The GNRA tetraloop in CDV-A Domain K is rotated 90 degree clockwise
compared to that in domain IV of poliovirus.
4. Domain L (∆G = -5.9 kcal/mol) separates domain K and M by a greater distance
than that between domains IV and V. This greater distance may confer flexibility to
domain K so that the GNRA tetraloop can be oriented in a proper conformation.
28 nts 22 nts
CDV-A 5’-UTR IRES

14. How are IRESs studied in in-vitro?
• IRES-mediated translation of Cistron 2 occurs independently of translation of the upstream Cistron 1
• It is unaffected when Cistron 1 translation is abrogated by inserting a hairpin at a cap-proximal position that prevents
ribosomal attachment.
RRL (Rabbit
Reticulocyte Lysate) RNA construct
+ S35-Methionine (radioactive
amino acid)
@37C, 60’
Protein expressed is exposed to
film after running on gel
Expected protein size
Marker
Cistron 1 Cistron 2
Expression Expression
+
+
+
+
+
_
_
_
RNA construct
IRES
Cistron 1 Cistron 2
Inter-cistronic
region
IRES
IRES
5’
5’
5’
5’
3’
3’
3’
3’
IRES
5’ 3’ +_
Dicistronic
construct
Dicistronic
construct (ΔIRES)
Dicistronic
construct (stem)
Monocistronic
construct (stem)
Different RNA constructs with IRES inserted in the intergenic region are in-vitro translated in mammalian system
such as rabbit reticulocyte lysate (RRL) and protein expressed determines the IRES activity.
In-vitro Translation in RRL

15. Translational activity of 5’UTR CDV IRES in
Rabbit Reticulocyte lysate (RRL)
Conclusions –
• The CDV-A 5’UTR IRES can promote translation in
RRL and requires eIF4A for its activity.
Next Step -
• The activity of these IRESs in RRL justifies the use
of (a) our mammalian in vitro reconstituted system
and (b) Toe-printing analysis of 48S complex
formation in RRL to investigate their mechanisms of
action.
- + 4AR362QMono-cistronic
(stem)
Di-cistronic
Di-cistronic
(stem)
5’UTR CDV RNA
constructs
IRES dependent 2nd
cistron
5’-cap dependent 1st
cistron
25
35
40
55
70
15
AUG UAG
eIF4A R362Q dominant negative
mutantexchangeswith eIF4A in
the eIF4F complex and traps it in
an inactive form
AUG UAG
E P A
AUG UAG
Prevents attachment of
the43S complex to
mRNA
NoRNA binding No
helicase activity
Inhibition of translation of an mRNA by a
dominant-negative form of eIF4A indicates
that initiation on the mRNA occurs by an
eIF4G/eIF4A-dependent mechanism.
- + -
Mechanism of Action of
eIF4AR362Q mutant

16. Toe-printing techniqueToe-printing technique RNA
48S/80S
complexes
sequence
Full-length cDNA
48S/80S complex
(15-17 nts from the P-site codon (AUG))
P-site codon
(AUG)
Analysis of 48S/80S complex formed in RRL and in in-vitro
reconstitution system using Toeprinting approach
2) In vitro reconstituted system1) Arresting 48S/80S in RRL
All the initiation factors and ITAFs required for the
activity of the CDV-A 5’UTR IRES are present in RRL.
80S complexes formed in RRL are then arrested using
cycloheximide.
Using Cycloheximide
(CHX), a protein
translation inhibitor
- Arrest translation after the first
cycle of elongation
5’
E P A
48S complex
E P A
5’
80S
RTRT
AUG AUG
Initiation factors:
2, 3, 4A, 4B, 4F, 1, 1A, 5, 5B
40S and 60S
subunits
Met-tRNAi
Met
mRNA
E P A
5’
80S
AUG
E P A
48S complex
AUG5’
DHX29
5’
E P A
48S complex
R
AUG
Initiation factors:
2, 3, 4A, 4B, 4F, 1, 1A, 5, 5B
40S and 60S
subunits
Met-tRNAi
Met
mRNA
E P A
48S complex
AUG5’
DHX29
Reverse Transcription
The required initiation factors and ITAFs are either
purified from RRL or expressed recombinantly in E.coli
and then added to the reaction in-vitro separately to
assemble 48S complex on the desired messenger RNA.

17. Toe-printing analysis of 48S complex formation on 5’UTR IRES of CDV in
RRL and in-vitro reconstitution system
C T A G
-
+Recomb.itRNA
+EcoliitRNA
+NativeitRNA
+PCBP1
+PCBP2
+40S/1/1A/2/3/
4A/4B/4G
Native
itRNA
AUG 983
UUG 951
UUG 974
48Son AUG
48Son UUG 974
RRL
80S on AUG 983
40S/eIF1/1A/2/3/4A/4B/4Gm
Ecoli itRNA
RRL
Cycloheximide (20ug)
- - - -
- - - -
- + + +
- - + +
Conclusions –
1. 48S complexes form on the authentic AUG both in
the in vitro reconstituted mammalian system and in
RRL.
2. In the absence of ITAFs, 48S complexes formed
on the authentic CDV-A initiation codon (AUG-983)
and upstream near cognate UUG 974 with E.coli
and in vitro transcribed mammalian Met-tRNAMet
i,
but not with native crude mammalian Met-tRNAMet
i,
in which case 48S complex formation additionally
required PCBP2.
3. The contaminants present in native tRNAMet
i (total)
compete with the IRES for RNA binding proteins
such as eIF4G or eIF4A and thus do not allow 48S
complexes to assemble on this IRES. PCBP2
enables the IRES to win this competition either by
increasing the binding of initiation factors or by
changing the conformation of IRES to facilitate
attachment of 43S complexes.
Next Step –
1. To test which canonical initiation factors are
necessary for assembly of 48S complexes on the
5’UTR IRES.

18. Conclusion -
•eIF2, 3, 4A and 4G are essential for 48S
assembly, while eIF4B stimulated the
activity of this IRES.
• In the absence of eIF1 or 1A, near-
cognate codons such as UUG951 and
UUG974 upstream of the authentic AUG983
were selected. Selection of the authentic
initiation codon is thus determined by
eIF1/1A.
• The 43S pre-initiation complex attaches
to the IRES upstream of domain N and
scans downstream towards the authentic
codon AUG983.
•The eIF4G-eIF3 interaction is not
obligatory for ribosome loading onto the
CDV-A IRES (in contrast to poliovirus).
•Next Step-
• Since the upstream UUG951 and UUG974
were selected, IRES mutants will be
designed to determine the earliest point
from which incoming 43S complexes can
begin inspection of the mRNA.
Initiation Factor requirements for 48S complex formation on the
CDV-A 5’UTR IRES
C T A G
-40S
-eIF1
-eIF1A
-eIF2
-eIF3
-eIF4A
-eIF4B
-eIF4Gm
40S/Native itRNA + PCBP2 +
initiation factors except
AUG 983
UUG 951
UUG 974
48S AUG 983
48S UUG 974
48S UUG 951
C T A G
-40S
-eIF1
-eIF1A
-eIF2
-eIF3
-eIF4A
-eIF4B
-eIF4Gm
40S/Native itRNA + PCBP2 +
initiation factors except
AUG 983
UUG 951
UUG 974
48S AUG 983
48S UUG 974
48S UUG 951
5’UTR MC RNA
40S/ Native itRNA/eIF1/1A/2/3/4A/4B/PCBP2
eIF4F
eIF4Gm 736-1115
eIF4G 736-1008
eIF4G 736-988
+ + + + + +
+ + + + +
+
+
+
+
48S AUG 983
5’UTR MC RNA
40S/ Native itRNA/eIF1/1A/2/3/4A/4B/PCBP2
eIF4F
eIF4Gm 736-1115
eIF4G 736-1008
eIF4G 736-988
+ + + + + +
+ + + + +
+
+
+
+
48S AUG 983
N
N
951
974 983
AUG 983 is the authentic initiation codon

19. UUG 951 – good AUG 950
Introduced AUG950 is in-frame with the authentic AUG983
Conclusion -
•An optimized AUG triplet introduced at nt. 950 (upstream of the
authentic AUG983) is active in the in vitro reconstitution system and
functions independently of PCBP2.
Mutational Analysis of the CDV-A 5’UTR IRES to locate the point from which
ribosomal inspection of the mRNA begins
28
38
49
62
-
Lucf.
Wt.IRESmRNA
GoodAUG950mRNA
Product from
AUG 950
Product from
AUG 983
28
38
49
62
-
Lucf.
Wt.IRESmRNA
GoodAUG950mRNA
Product from
AUG 950
Product from
AUG 983
N
951
974 983
In-vitro Translation

20. Fe(III) 1- (p-Bromoacetamidobenzyl)
ethylene diamine tetraacetic acid
Iron –EDTA (chelating agent)
Mechanism of Action of HRC Assay
Locating binding site and Orienting Initiation factors and PCBP2
on 5’UTR CDV IRES
Cleavages generated are
then analyzed using
Reverse transcription.
Fe-BABE
The sulfhydryl group of endogenous
cysteine or single cysteine mutants of the
protein are reacted with bromoacetyl
moiety of FeBABE. (Site specific iron
chelates)
The hydroxyl radicals are generated
using ascorbic acid and H2O2. The target
nucleic acid if known to be bound by the
chelated protein, the radicals will cleave
the nucleic acid in the vicinity of binding
site.
Reacted with target
RNA
+ Ascorbic acid, H2O2,
@37C, 10’
Fenton
Reaction
Fenton
Reaction
A480C
mutant of
protein
In order to locate the binding site of initiation factors such as eIF4G and eIF4A and ITAF (PCBP2)
which are known to bind the poliovirus IRES, I used Hydroxyl Radical cleavage assay (HRC)
Schematic
diagram

21. 1) Locating binding site of Initiation factors eIF4G and eIF4A
-
wt.
C-less
D928DC
C830
Wt.
C-less
D928DC
C830
+ eIF4A + eIF4G
C T A G
S33C
S42C
Cys-less
S33C
S42C
Cys-less
Cysteine
mutants
a) FeBABE-eIF4G wt/mutants b) FeBABE-eIF4A mutants
Conclusions –
• eIF4G interacts with domain M.
• The interaction is enhanced by eIF4A
•eIF4A does not bind directly but is
recruited by eIF4G in the
vicinity of domain M.
G905 - U911
A804 - C812
C897- C903
G819 - C829
C872 – U877
A882- U885
A835
C T A G
Cysteine
mutants

22. Comparison of eIF4G and eIF4A binding site on PV1 and CDV-A IRESs
Cys929
Wt/ Cys819/821/847/919/934/936
Cys829
Cys33
Cys42
eIF4GI736–1115
eIF4A
Poliovirus
(domain V)
Cadicivirus A
(domain M)

23. KH3
domain
KH1
domain
KH2
domain
E34C
S141C
A308C
S330C
C54
C118
119aa linker
GXXG
motif
GXXG
motif
GXXG
motif
• Common ITAF necessary for Type1 IRESs.
• It binds to Type 1 IRESs via cooperative interactions
at distinct sites.
•Three hnRNP K-homology domains – KH1 and KH2 are
arranged back to back while KH3 is mobile, being
separated by 119 amino acid from KH1-KH2 domains.
•Each KH domain accommodates 4 nucleic bases in
the binding cleft formed by
α1, α2 and invariant GXXG connecting motif on
one side
Β2 and a variable loop on the other
•Forms hetero-multimers with PCBP1
Locating PCBP2’s binding site on the CDV-A IRES using directed
hydroxyl radical cleavage
KH1 KH2 KH3
13 81 97 169 288 356
Protein
(cys-less)
Surface exposed Single cysteine
mutants
PCBP2 E34C, S54C, S118C, S141C, A308C,
S330C

24. Locating PCBP2’s binding site on the CDV-A IRES using directed
hydroxyl radical cleavage
-
C-less
C54
C34
C118
C141
C308
C330
KH
1
KH
2
KH
3
C T A G
A748-U753
G734-U737
G734-U737
A718-U724
A718-U724
U710-A717
U700-U705
A703-U707
C666-A674
U673-G679
C664-U678
C657-G663
U650-U655
A610-C613
A600-A603
G881-C599
U573-C578
A450-G452
C436-C441
U418-G420
Conclusion –
•As seen for Poliovirus, KH2 and KH3 domains are close to each
other when bound to IRES. KH1 gave strong cleavages near GNRA
loop of 5’UTR CDV IRES.
• Being flexible, Domain 3 can also bind to a distant stem of domain
H/I.
Domain K of
5’UTR CDV IRES Domain IV of
Poliovirus IRES
GNRA loop
KH 1
KH 2
KH 3
GNRA loop

25. Conclusions
Similarities and Differences between the mechanisms of 48S complex formation on the CDV-A IRES
and on the Type 1 (poliovirus) IRES
SIMILARITIES
1. Initiation depends on specific binding of eIF4G’s central domain to homologous, conserved domains of these IRESs.
2. Initiation requires eIF4A, which is recruited by eIF4G to the same site on both IRESs.
3. Both IRESs depend on the same ITAF, PCBP2, that binds to structurally similar sites on both.
4. Following attachment to the IRES, the 43S complex reaches the initiation codon by scanning.
DIFFERENCES
1. The PCBP2 binding site is differently arranged in CDV-A and Type 1 IRESs.
eIF4G
eIF4A
PCBP2
eIF4G
eIF4A
PCBP2
2. Domain VI is unwound
‘Poor context’ AUG is not inspected
eIF1 is not required.
3. eIF3 – eIF4G is required
2. Domain N is unwound
‘Near-cognate’ UUG is inspected
eIF1/1A is required.
3. eIF3 – eIF4G is not required
5’UTR CDV-A IRESPoliovirus IRES

26. 5’UTR CDV IRES –
1. To locate the exact ribosomal loading site
2. To map the 5’-terminal border of the IRES
Future Plans
∆nt. 341 – 982 (domain H – N)
∆nt. 518 – 982 (domain K – N)
∆nt. 553 – 982 (domain ΔK-N)
A
B
C
D
F
G
H
I
J
K
L
M
N
UUG
AUG
983
Placing good AUG upstream
of Domain N at 944 in the wild
type construct
a) By replacing domain N of CDV-A with
Domian VI of poliovirus IRES
b) By placing good context AUG at 944
upstream of Domain N of wild type
construct.
a) By truncating the IRES from 5’ end
of the IRES and testing its activity in
RRL.

27. Acknowledgements
• Mentor –
Dr. Tatyana Pestova
Dr. Christopher Hellen
• SUNY Downstate Medical Center
• And SigmaXi for hosting this showcase.