Mukta Asnani Dr. 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 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 complex

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

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 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

Poliovirus Encephalomyocarditis

(EMCV)

Hepatitis C virus

(HCV)

Cricket paralysis

virus (CrPV)

IRES/eIF4G

IRES/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

AU

G

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 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

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

Mark

er

Cistron 1 Cistron 2

Expression Expression

+

+

+

+

+

_

_

_

RNA construct

IRES

Cistron 1 Cistron 2Inter-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

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.

- + 4AR362Q M

on

o-c

istr

on

ic

(ste

m)

Di-

cist

ron

ic

Di-

cist

ron

ic

(ste

m)

5’UTR CDV RNA constructs

IRES dependent 2nd cistron

5’-cap dependent 1st cistron

25

35 40

55

70

15

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 technique R

NA

48

S/8

0S

co

mp

lexe

s 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 system 1) 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 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

. it

RN

A

+ E

co

liit

RN

A

+ N

ati

ve itR

NA

+ 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 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-tRNAMeti,

but not with native crude mammalian Met-tRNAMeti,

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 -4

0S

-e

IF1

-e

IF1

A

-e

IF2

-e

IF3

-e

IF4

A

-e

IF4

B

-e

IF4

Gm

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 -4

0S

-e

IF1

-e

IF1

A

-e

IF2

-e

IF3

-e

IF4

A

-e

IF4

B

-e

IF4

Gm

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

eIF4G12A

pro

1 1599

74

6

99

21

01

5

11

04

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

28

3849

62

- Lucf

.

Wt.

IRES

mRN

A

Goo

d A

UG

950

mRN

A

Product from

AUG 950

Product from

AUG 983

28

3849

62

- Lucf

.

Wt.

IRES

mRN

A

Goo

d A

UG

950

mRN

A

Product from

AUG 950

Product from

AUG 983

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

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

1) Locating binding site of Initiation factors eIF4G and eIF4A

-

wt.

C-l

ess

D

92

8D

C

C8

30

Wt.

C-l

ess

D9

28

DC

C8

30

+ eIF4A + eIF4G

C T A G S3

3C

S4

2C

Cys

-le

ss

S3

3C

S4

2C

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 G Cysteine

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-l

ess

C5

4

C3

4

C1

18

C1

41

C3

08

C3

30

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

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 IRES Poliovirus 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.