Sigma xi presentation final1

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1. 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 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 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 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 5UTR 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 5UTR 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 5UTR 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 + + + + + _ _