Sigma xi presentation final1

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  • 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

    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

  • 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)

  • Co