The mechanism of translation initiation on the genomic RNA of
Cadicivirus A: a naturally occurring dicistronic picornavirus and type
member of the novel genus Dicipivirus
Mukta AsnaniDr. Tatyana Pestova
Dr. Christopher Hellen
Department of Cell Biology, SUNY Downstate Medical Center
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.
The canonical mechanism of cap-dependent translation initiation and sites of viral regulation
AUG UAG
AUG UAG
E P AAUG UAG
E P AAUG UAG
E P AAUG 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 formation48S complex
eIF4E
eIF4G
eIF4AeIF4B
eIF1
eIF1A
eIF2
eIF5
eIF3
43S complex
40S
GTP
GTP
GTP
E P AAUG 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
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)
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
PoliovirusEncephalomyocarditis (EMCV)
Hepatitis C virus (HCV)
Cricket paralysis virus (CrPV)
IRES/eIF4GIRES/eIF4G IRES/40S IRES/40S
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
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)
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.
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)
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
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)
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
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
UU
G
AUG 983
py
GNRA Tetraloop
Poliovirus IRESGNRA
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
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
Mar
ker
Cistron 1 Cistron 2Expression Expression
+
+
+
+
+
_
_
_RNA construct
IRES
Cistron 1 Cistron 2Inter-cistronic
region
IRES
IRES
5’
5’
5’
5’
3’
3’
3’
3’
IRES5’ 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
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.
- + 4AR362QM
ono-
cist
roni
c (s
tem
)
Di-c
istr
onic
Di-c
istr
onic
(s
tem
)
5’UTR CDV RNA constructs
IRES dependent 2nd cistron
5’-cap dependent 1st cistron
2535405570
15
AUG UAG
eIF4A R362Q dominant negative mutant exchanges with eIF4A in the eIF4F complex and traps it in an inactive form
AUG UAG
E P AAUG UAG
Prevents attachment of the 43S complex to mRNA
No RNA 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
Toe-printing techniqueToe-printing techniqueToe-printing techniqueToe-printing techniqueR
NA
48S/
80S
com
plex
essequence
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 system 1) Arresting 48S/80S in RRLAll 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 A48S complex
E P A
5’
80S
RTRTAUG AUG
Initiation factors: 2, 3, 4A, 4B, 4F, 1, 1A, 5, 5B
40S and 60S subunits
Met-tRNAiMet
mRNA
E P A
5’
80S
AUG
E P A48S complex
AUG5’
DHX29
5’
E P A48S complex
E P A
5’
80S
RTRTAUG AUG
Initiation factors: 2, 3, 4A, 4B, 4F, 1, 1A, 5, 5B
40S and 60S subunits
Met-tRNAiMet
mRNA
E P A
5’
80S
AUG
E P A48S 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.
Toe-printing analysis of 48S complex formation on 5’UTR IRES of CDV in RRL and in-vitro reconstitution system
C T A G
- + R
eco
mb
. itR
NA
+ E
coli
itR
NA
+ N
ativ
e it
RN
A
+ P
CB
P1
+ P
CB
P2
+40S/1/1A/2/3/4A/4B/4G
Native itRNA
AUG 983
UUG 951
UUG 974
48S on AUG
48S on UUG 974
RRL
80S on AUG 983
40S/eIF1/1A/2/3/4A/4B/4Gm
Ecoli itRNARRLCycloheximide (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 tRNAMeti (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.
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
-eIF
1
-eIF
1A
-eIF
2
-eIF
3
-eIF
4A
-eIF
4B
-eIF
4Gm
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
-eIF
1
-eIF
1A
-eIF
2
-eIF
3
-eIF
4A
-eIF
4B
-eIF
4Gm
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
NN
PABP eIF4E eIF4A eIF4A Mnk1eIF3
eIF4G12Apro
1 1599
746
992
1015
1104
eIF4G736-1115 (eIF4Gm)
eIF4G736-1008
eIF4G736-988
eIF3eIF4A
eIF4A
eIF4A
951
974 983
AUG 983 is the authentic initiation codon
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
28384962
- Lucf.
Wt.
IRES
mR
NA
Go
od
AU
G 9
50 m
RN
A
Product from AUG 950
Product from AUG 983
28384962
- Lucf.
Wt.
IRES
mR
NA
Go
od
AU
G 9
50 m
RN
A
Product from AUG 950
Product from AUG 983
40S + eIFs 1/ 1A/ 2/ 3/ 4A/ 4B/ 4GmEcoli - itRNANative total itRNAPCBP2
- + - + + +- - - + - -- + - - + +- + - - - +
good AUG950 mRNA
AUG 983
AUG 950
UUG 974
48S AUG 983
48S AUG 950
48S UUG 974
C T A G
Wt.
40S + eIFs 1/ 1A/ 2/ 3/ 4A/ 4B/ 4GmEcoli - itRNANative total itRNAPCBP2
- + - + + +- - - + - -- + - - + +- + - - - +
good AUG950 mRNA
AUG 983
AUG 950
UUG 974
48S AUG 983
48S AUG 950
48S UUG 974
C T A G
Wt.
40S + eIFs 1/ 1A/ 2/ 3/ 4A/ 4B/ 4GmEcoli - itRNANative total itRNAPCBP2
- + - + + +- - - + - -- + - - + +- + - - - +
good AUG950 mRNA
AUG 983
AUG 950
UUG 974
48S AUG 983
48S AUG 950
48S UUG 974
C T A G
Wt.
N
951
974 983
In-vitro Translation
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 ReactionFenton
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
1) Locating binding site of Initiation factors eIF4G and eIF4A
- wt.
C-le
ss
D92
8DC
C830
Wt.
C-le
ss
D92
8DC
C830
+ eIF4A + eIF4G
C T A G S33
C
S42
C
Cys
-le
ss
S33
C
S42
C
Cys
-le
ss
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 GCysteine mutants
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)
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
Locating PCBP2’s binding site on the CDV-A IRES using directed hydroxyl radical cleavage
- C-le
ss
C54
C34
C118
C141
C308
C330
KH 1
KH 2
KH 3
C T A G
A748-U753
G734-U737G734-U737
A718-U724A718-U724
U710-A717
U700-U705A703-U707
C666-A674U673-G679
C664-U678C657-G663U650-U655
A610-C613A600-A603G881-C599U573-C578
A450-G452C436-C441U418-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 2KH 3
GNRA loop
ConclusionsSimilarities 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
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
UU
G
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.
Acknowledgements
• Mentor –
Dr. Tatyana Pestova
Dr. Christopher Hellen
• SUNY Downstate Medical Center
• And SigmaXi for hosting this showcase.
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