Interplay between coronavirus and nonsense mediated mRNA decay (NMD)

1. A
PRESENTATION ON
ADVANCED MICROBIOLOGY
Presented by
MD ROBEL AHMED
STUDENT ID:
L20192E020101
1ST YEAR 1ST SEMESTER
(Master’s)
FACULTY OF LIFE SCIENCE
AND MECICINE
Presented to
XIUFANG HU
PROFESSOR ,
FACULTY OF LIFE SCIENCE AND
MEDICINE
ZHEJIANG SCI-TECH UNIVERSITY
DATE OF PRESENTATION: 12th MAY 2020

3. RNA Decay
► Post transcriptional quality control pathway.
► Intrinsic conserved mechanism in Eukaryotic organisms like plants and animals
► Detect and disrupt mRNA having negative activity.
► mRNA have to harvour PTC, uORF, long 3’ UTR sensitive to decay.

4. Nonsense Mediated Decay
 Detects mRNAs harboring aberrant features.
 Due to alternative splicing or mutation in nonsense bases of DNA.
 Degrades target via various exo-nuclease pathway.
 NMD inducing features
 uORF with PTC
 Long 3’UTR
 Viral RNA with (iTC).

5. What is nonsense?
A T G C A G C A G C A G C A G C A G C A G C A G C A G
Start Gln Gln Gln Gln Gln Gln Gln Gln
A T G C A G C A G C A G C A G C A G T A G C A G C A G
Start Gln Gln Gln Gln Gln ? Gln Gln
A U G C A G C A G C A G C A G C A G U A G C A G C A G
Start Gln Gln Gln Gln Gln STOP Codon Gln Gln
mRNA
Truncated protein
Anti-sense
Sense

6. +
Translation
RdRP
+
-
ds RNA intermediate
- Strand synthesis
+
+
+
+
+
+
+ sense RNA act as viral mRNA
PTC
+
UPF2
UPF3
EJC
UPF1 P +

7. Genomic Information of Coronavirus
 Plus sense (+) single stranded RNA (ssRNA) genome
 Genome length is about
 The virus is cytoplasmic origin
 Lifecycle is common like other + sense viruses.

8. Coronavirus and NMD
 Murine Hepatitis Virus (MHV) as a Model CoV.
 Hypothesis is that CoV genomic and sub-genomic RNA like rLucRNA3 is
susceptible to NMD.

9. RESULT
MHV genomic RNA is a NMD target
Weather depletion of host NMD factor enhances viral replication ?
Or not?
If so happened, virus may be NMD target.
17Cl-1 cell transfected with
 NMD reporter plasmid NS39 having PTC
 Or Wild type reporter plasmid not have PTC.
qRT-PCR result shows that NS39 is a NMD target.
Conc. Of NS39 transcript raised high in cells depleting NMD factors.

10. NMD negative cell with MHV
genomic RNAs (M protein)
 Number of M protein positive
cell increased
 Number of virus titer also
increased
RESULT

11. Degradation kinetics of MHV genomic RNA
NMD deficient cell:
• Level of genomic RNA is Higher
RESULT
NMD competent cell:
• qRT-PCR result showed no
substantial increase in the levels of
genomic RNA.

12. Cytoplasmically Synthesized MHV Sub genomic like RNA (rLucRNA3) Is Susceptible to NMD:
RESULT

13. MHV N Protein Inhibits the
NMD Pathway
Viral protection of incoming
MHV genomic RNA from
NMD.
Cells transfected with
 NS39 plasmid
 Wild Type plasmid
Along with CAT or TGEV-nsp1
RESULT
Level of NS39 higher in TGEV-nsp1 expressing cell than CAT
expressing cell, indicate TGEV-nsp1 inhibit NMD

14. RESULT
To further confirm, stable cells
transfected with NS39 or Wild type
plasmid in the presence of N
protein or TGEV-nsp1
Levels of NS39 reporter transcripts were higher in cells
expressing MHV N or TGEV nsp1, but not CAT,

15. RESULT
MHV genomic RNA targeted by host NMD in early infections

16. UPF2
UPF3
EJC
PTC
3’UTR
P
3’UTR
UPF1
Decay
Resistanc
e
UPF1
UPF1
Viral protein synthesis
Down-regulation NMD factor
Viral mRNA synthesis
Via ds intermediate
3’UTR
infection
Translation termination
Due to PTC and NMD
initiation

17. Discussion
Out study shows a common interplay between CoV and NMD
In Host cell Depletion of NMD done by
 Silencing essential NMD factors as UPF1, UPF2,
SMG proteins and others
 Chemically cellular NMD silenced by CHX and
wortmannin.
Result shows that CoV genomic and sub-genomic
RNA are attacked by host cellular NMD machinery
subsequently try to be resistant for multiplication.
MHV N protein is an important protein helps virus to
be resistant against Nonsense mediated decay.

18. Copyright: All design and figure made by Ahmed Md Robed. It is illegal to copy of them.