Mukta Asnani Dr. Tatyana Pestova
Dr. Christopher Hellen
Department of Cell Biology, SUNY Downstate Medical Center
RNA viruses: Infection and hijacking of cellular translation
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
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
E P A AUG UAG
E P A AUG UAG
E P A AUG
E P A
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
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
Viral proteases (2A and
3C) synthesized during
infection cleaves host
initiation factors and
hence shuts off the
initiation and allow
selective translation of
viral RNA genome
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
This alternative mechanism of translation initiation was first observed to be used by poliovirus RNA
genome in infected cells in late 1980s.
Poliovirus IRES (~450 nt)
eIF4Gm cleaved eIF4G
Sweeney et. al. (EMBO, 2014)
Classification of Viral IRESs
Family Genus Example IRES
ITAFs (IRES Trans acting
Picornaviridae Aphthovirus Foot-and-mouth disease virus (FMDV)
Type 2 eIF4G
Cardiovirus Encephalomayocarditis virus (EMCV) PTB
Enterovirus Polio virus
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
Non-canonical interactions of IRESs with
canonical components of the translational
Hepatitis C virus
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
Highly regulated process
Relic of the past and
evolved in matured
Evolved in eukaryotes to
regulate gene expression
under stress ??
IRES study will shed light on past
of the translation initiation
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
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.
(2 families were unrelated)
(both are related)
Picornavirus like superfamily
Multiple steps of translocation and
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,
but contain same
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
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
of modified RNA using
Sequence of DNA
- + NMIA
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
Jennifer et. al. (JACS, 2012)