Role of miRNA in host-virus interaction: current insights and future perspectives

1. Major Credit Seminar
Adil Anamul Haq
PhD Scholar
Division of Virology, IVRI Mukteswar
Welcome
Role of miRNA in host-virus interaction: current
insights and future perspectives

2.  Introduction
 Biogenesis
 Viral miRNAs
 Cellular miRNAs
 Applications
 Summary
 Future Perspectives
Contents

3. What are miRNAs?
 Non-coding RNAs ~22nts in length
 Post-transcriptionally regulate gene expression
 Regulate ~60% protein coding genes
(Friedman et al., 2009)
 Encoded both by hosts and viruses
 Important effects in
 Signaling pathway
 Metabolism
 Apoptosis
 Cell proliferation
 Regeneration
 Stem cell division
 Oncogenesis
 Nervous system control
 Developmental timing
 Hematopoietic cell fate
 Regulation of Immune system
(Libri et al., 2008)
miRNA
mRNA

4. Victor R. Ambros
Historical Background
 1993: lin-4 in C. elegans (Lee et al., 1993)
 Thought to be an oddity of nematodes
 No other miRNAs found for next 7 years !!!
 Second miRNA – let-7 in C. elegans (Reinhart et al.,2000)
 Let-7 found 100% conserved in genomes of mice and
humans (Pasquinelli et al., 2000)
 Three landmark papers in Science
1. Lagos-Quintana et al., 2001;
2. Lau et al., 2001
3. Lee and Ambros, 2001
C. elegans

5. Historical Background
Sébastien Pfeffer
 First reported by Thomas Tuschl
and colleagues (Pfeffer et al., 2004)
 First v-miRNA seen in EBV
 Most not conserved between
viruses
 Presently > 250 viral miRNAs are
known
(Cullen et al., 2013)

6. Canonical miRNA biogenesis pathways
(Sonkoly and Pivarcsi, 2009)

7. Non-canonical miRNA biogenesis pathways
Two pathways:
 Drosha-
independent
 MHV68
 BLV
 Dicer-
independent
 miR-451
(Libri et al., 2013)

8. miRNAs
mRNA Cleavage
Translational
Repression
Fate of mRNA
Boss et al., 2011

9. ‘Seed’ Sequence
(Zhao and Srivastava, 2007)
 6-7 nt signature region
at 5’ end (Bartel, 2009)
 Hexamer = 2-7 nt
 Heptamer = 2-8 nt
 Directs miRISC to
target 3’ UTR of mRNA
Plant miRNAs differ in that they are entirely complementary to their target genes

10. miRNA in host-viral Interaction

11. Which viruses encode miRNA ?
Herpesviruses code for ~ 90% of known miRNAs (Cullen, 2013)
 Herpesviridae
 Polyomaviridae
 Ascoviridae
 Baculoviridae
 Iridoviridae
 Adenoviridae
 Most recently
Retroviridae
Based on the requirements of
nuclear machinery and RNA
cleavage for miRNA
processing, it is no surprise
that cytoplasmic replicating
DNA viruses and RNA viruses
have not been found to
express miRNAs
(Boss et al., 2011)

12. Family/Sub-family Species Host Pre-miR
Hairpins
Mature
miRs
α-Herpesviruses Herpes Simplex Virus-1 Human 16 25
Herpes Simplex Virus-2 Human 18 24
Herpes B virus Human 3 3
Herpesvirus of turkey Turkey 17 28
Bovine herpesvirus 1 Bovine 10 12
Pseudorabies virus Swine 13 13
Marek’s disease virus -1 Chicken 14 26
Marek’s disease virus -2 Chicken 18 36
Β-Herpesviruses Human cytomegalovirus Human 11 17
Mouse Cytomegalovirus Murine 18 28
Human herpesvirus 6B Human 4 8
γ-Herpesviruses Kaposi’s sarcoma associated
herpes virus
Human 12 25
Epstein-Barr Virus Human 25 44
Rhesus Lymphocryptovirus Simian 36 50
Rhesus Monkey Rhadinovirus Simian 15 25
Mouse Gamma Herpesvirus 68 Murine 15 28
Herpesvirus Saimiri strain A11 Simian 3 6
List of Viral encoded miRNAs

13. Family/Sub-
family
Species Host Pre-miR
Hairpins
Mature
miRs
Polyomaviruses Simian Virus 40 Simian 1 2
BK Polyomavirus Human 1 2
JC Polyomavirus Human 1 2
Mouse polyomavirus Mouse 1 2
Merkel cell polyomavirus Human 1 2
SA12 Simian 1 2
RETROVIRUS Bovine Leukemia virus Bovine 5 8
Iridoviridae Singapore Grouper Iridovirus Fish 14 15
Ascoviridae Heliothis virescens ascovirus Insect 1 1
Baculoviridae Bombyx mori nucleopolyhedrosis virus Insect 4 4
Adenoviridae Human adenoviruses types 2 and 5
(others likely)
Human 2 3
Unclassified Bandicoot papillomatosis
carcinomatosis virus type 1
Bandicoots
(marsupial)
1 1
Bandicoot papillomatosis
carcinomatosis virus type 2
Bandicoots
(marsupial)
1 1
(Kinkaid & Sullivan, 2012)

14. Location of miRNA genes within herpesvirus
genomes
Boss et al., 2009

15. Strategies for determining viral-encoded
miRNA function
 Bottom-up
Approach
 Top-down
Approach
Grundhoff and Sullivan, 2011

16. Functions of viral encoded miRNAs
Grouped into two
classes:
 Analogs of host
miRNAs
 Specific to
viruses
 Regulation of latency/persistency
 Prevention of apoptosis
 Alteration of cell cycle
 Viral immune evasion
 Transformation
 Autoregulation of virus gene expression
(Grundhoff & Sullivan, 2011)
Gottwein & Collen, 2008

17. Regulation of latency/persistency
The role of virus-encoded miRNAs in promoting KSHV latency
(Grundhoff & Sullivan, 2011)
KSHV miR-K12-5p Rbl2
Increase in DNA
methylation
First reported evidence that viral miRNAs can directly impact the
epigenetic status of herpesvirus genomes during latency (Lu et al., 2010)

18. Evading the Immune Response
Direct Regulation Autoregulation
KSHV miR—K12-3
KSHV miR—K12-7
LIP
Secretion
IL-6 & IL-10
(Quin et al., 2010)
(Boss & Renne, 2011)

19. Autoregulation
miR—S1-
5p
miR-S1-3p
T-
Antigen
CTL
Response
(Sullivan et al., 2005)
JCV
BKV
muPyV
This study provided the first evidence that viral miRNAs
can inhibit CTL recognition by directly targeting viral antigens
(Boss et al., 2011)
T-
Antigen
SV40:
miRNAs encoded antisense to the T-antigen that
mediate its transcript cleavage during infection
(Sullivan et al., 2005)

20. Evading the Immune Response
HCMV miR-
Ul112-1
KSHV miR-K12-7
MICB
NK Cell &
CTL
Response
EBV miR-BART2-
5p NKG2D-R
(Stern-Ginossar et al., 2007; Thomas et al., 2008)
HCMV-, EBV-, and KSHV-encoded miRNAs target the MICB gene by completely different
sequences raises a very interesting question about the co-evolution of viral miRNAs and
their corresponding cellular targets (Boss et al., 2011)
(Boss & Renne, 2011)

21. Preventing Apoptosis
EBV miR-BART5
rLCV miR-BART5
PUMA
Block
Apoptosis
(Choy et al., 2008)
Repress
RNAhybrid
miRANDA
Predicted target of EBV miR-
BART5
It represents the conservation of miRNAs from
different viruses (Choy et al., 2008)

22. Alteration of cell cycle
HCMV miR-US25-1:
 ~ 15 targets
identified
 Most targets in cell
cycle
 Cyclin E2 is one of
the targets
(Grey et al., 2010)
This is the first report of a virus miRNA
targeting the 5′UTR of an mRNA
(Grey et al., 2010)
(Grey et al., 2010)

23. Transformation
12 KSHV miRNAs Thrombospondin Angiogenesis
anti-angiogenic factor frequently mutated in various cancers
KSHV
(Samols et al., 2007)

24. Viral miRNA mimicking a host miRNA
 Known as
‘analogs’
 Three Viruses
known to
encode:
 KSHV
 MDV1
 BLV
(Kincaid & Sullivan, 2012)
Mimicking host miRNAs provides obvious benefits to a
virus by allowing it to access a pre-existing target network
of numerous host transcripts that may have been selected
for a particular functional outcome (e.g., prevention of
apoptosis or evasion of immune signaling)
(Kincaid & Sullivan, 2012)

25. miR-155 mimics—viral analogues of a human
oncomir?
Kincaid & Sullivan, 2012
 miR-155 originally identified as product of bic gene
(Clurman and Hayward, 1989)
 Expressed in
 many lymphomas
 acute myeloid leukemia (AML)
 solid tumors of the breast, colon and lung
 Represents an “oncomiR”
miR-155
KSHV miR-K11 MDV1 miR-M4
Strikingly, although the lymphotropic EBV does not encode a miR-155
mimic of its own, the viral LMP-1 protein strongly induces expression of
the cellular miR-155 in latently infected B-cells (Gatto et al., 2008)

26. Potential effects of cellular miRNAs on
Viral replication
(Gottwein & Collen., 2008)

27. Antiviral
miRNAs
HIV-1
PFV-1
Influenza
VSV
- 5-human encoded miRNAs
- target nef, vpr, vif, vpu
(Hariharan et al. , 2005)
- miR-24 & miR-93
- Restrict replication
(Otsuka et al. , 2007)
- miR-32
- Restrict replication
(Lecellier et al. , 2005)
- hsa-miR-507&136
- target PB2 & HA.
- (Scaria et al., 2007)
Cellular miRNAs as Antivirals

28. miR-122: Cellular miRNA enhancing Viral
Replication
(Gupta et al., 2012)

29. Attenuated vaccines
Oncolytic Virotherapy
Determination of Cell
Tropism
Antiviral Drug Development
Applications

30. MIRA (MicroRNA Attenuation) Technology
miRNA-controlled viruses
showed no evidence of
pathogenesis, but their limited
replication made them ideal
vaccine candidates
(Barnes et al., 2008)
Poliovirus

31. Live attenuated IAV vaccine
 Incorporated MRE
 miR-93 (a
ubiquitous
miRNA)
 ORF of influenza A
nucleoprotein
coding regions
 H1N1
(Perez et al., 2009)
(Perez et al., 2009)

32. Engineering microRNA responsiveness for
Oncolytic Virotherapy
Colorectal Carcinoma
 Measles Virus
(Leber et al., 2011)
 VSV
(Edge et al., 2008)

33. Engineering microRNA to determine cell
specificity for viruses
Determine role of
haematopoietic cell
in viral replication?
 DENV strain engineered to encode four HPC- specific miR-142-
targeting sites in the 3ʹ UTR of the virus
 miR-142 completely blocked spread of the infection in vivo,
suggesting that replication in haematopoietic cells is required
for viral dissemination

34. Janssen et al., 2013
Miravirsen is a LNA modified phosphorothiolate antisense oligonucleotide
targeting and blocking miR-122

35. Miravirsen- the 1st miRNA targeted drug
 First drug to exploit miRNA for therapeutic use
 As a host targeting agent miravirsen poses a high barrier to
resistance
 Can work in all HCV genotypes because miR-122 binding
sites are conserved
 Has successfully completed Phase II clinical trial
(Janssen et al., 2013)

36. Summary
 MicroRNAs are short 22 nt ncRNAs
 MicroRNAs are involved in post transcriptional gene
regulation
 Encoded both by viruses and hosts
 Degree of complementarity between a miRNA & its target
determine the regulatory mechanism
 With notable exceptions, there is a striking lack of
evolutionary conservation of most viral miRNAs
 Have been used for therapeutic purpose
 As new discoveries in viral miRNA function are made new
questions emerge, making the complexities governing
viral-host interactions, at the least, a little more
transparent (Boss et al., 2011)

37. Future Perspectives
 Understand why some members of the same virus subfamilies do and
do not encode miRNAs (e.g., HTLV in the Delta Retroviridae or VZV in
the alpha herpesviruses
 How do viral miRNAs work synergistically with viral proteins to
regulate the viral replication cycle?
 Strategies to develop new therapeutic interventions
 Viral miRNA target
identification
 Relevant in vivo model
systems for viral miRNAs
that recapitulate all
modes of infection