Published on March 11, 2014
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 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
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
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’UTRCDV 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 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.
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.
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 PABP eIF4E eIF4A eIF4A Mnk1eIF3 eIF4G1 2Apro 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 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
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-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
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-less C54 C34 C118 C141 C308 C330 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 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 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.
Acknowledgements • Mentor – Dr. Tatyana Pestova Dr. Christopher Hellen • SUNY Downstate Medical Center • And SigmaXi for hosting this showcase.
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