1. Sensing Parainfluenza virus infection:
The role of RIG-I and MDA5
John A L Short, Rick Randall E-mail: jals4@st-andrews.ac.uk
Parainfluenza: The Essentials
What is a Parainfluenza virus?
 Parainfluenza virus (PIV) is a single stranded, RNA negative sense virus
 Infects the upper and lower respiratory tract
Who is at risk of infection?
 All children are infected at some stage
 Immunocompromised patients e.g. AIDS
What are the symptoms?
 Severe fever, coughing, sneezing, croup
There are NO antiviral drugs or vaccines
1
RIG-I and MDA5: viral sensors
RIG-I detects short double stranded viral RNA and 5’ triphosphate capped
single stranded viral RNA
MDA5 detects viral long double stranded viral RNA
RIG-I MDA5
IFNCell
Antiviral
IFN
2
Virus Infects Cell
RIG-I and MDA5 sensors
recognise viral RNA
Sensors activate pathway for
Interferon (IFN) expression
IFN is secreted & absorbed by
neighbouring uninfected cell
Cell expresses antivirals,
generating antiviral state
Inhibition of viral replication
upon viral infection
3 How do we visualise infection?
Plaque Assay
 Plaques formed from virus
spreading from infected cell to
neighbouring cells over 10 days
 Use cells that “knock down” or
decrease levels of RIG-I and MDA5
 2o Antibody conjugated to alkaline
phosphatase that reacts with
substrate to stain plaques black
 A549 NPro cells is a +ve control:
NPro inhibits IFN expression
Immunofluorescence
 Cells infected for 2 days
 Marker for IFN signalling to
neighbouring cells: cellular antiviral
MxA stained with Cy5
 Cells express Green Fluorescent
Protein (GFP) when IFN is induced
 2o Antibody conjugated to
fluorophore Texas Red
Plaque
2
MergeAnti-MxAAnti-PIV5GFP
Plaque
1
4
Mock
GFP +IFN-IFNAnti-PIV5
Immunofluorescence
- AIM: To determine the IFN response to PIV5 infection
Key: Green = GFP; Red = Viral NP;
Blue = Antiviral MxA
 Adding IFN to cells
induces expression of
antiviral MxA
 PIV5 infects cells,
forming a plaque
 No IFN induction =
no MxA expression
 IFN expressing cells
leads to induction of
MxA in neighbouring
uninfected cells
 Heterogeneous
cellular response:
Only some cells
express IFN, the
majority respond to IFN
signaling
 Infect monolayer of A549
alveolar epithelial cells with PIV5
NPro +ve
Control
Naïve MDA5 KD
RIG-I KD RIG-I/
MDA5 KD
0
0.5
1
1.5
2
2.5
3
Naïve
MDA5KD
RIG-IKD
RIG-
I/MDA5
KD
Npro+ve
Control
Relative sizes of largest plaques following
PIV5 infection
PlaquesizerelativetoNaive
5 Plaque Assay
- AIM: To visualise virus infection in cells lacking RIG-I and MDA5
 Naïve and MDA5 knock down (KD) cell lines had similar
plaque sizes after viral infection
 RIG-I and the RIG-I/MDA5 double KD cells had larger plaque
sizes than Naïve and MDA5 KD cells
 In RIG-I KD and RIG-I/MDA5 KD cells, plaques were
different sizes, indicating asynchronous viral IFN evasion
6 What does the data mean?
 IFN is expressed in some cells following virus infection
 Plaque assay data suggests that RIG-I is more important than MDA5 in
sensing PIV infection
The Future:
 Why is there a heterogeneous cellular response to virus infection?
 Are there differences in the number IFN expressing cells between RIG-I KD
and MDA5 KD cell lines?
7 Reference
Randall, R. E. and S. Goodbourn (2008). "Interferons and viruses: an interplay
between induction, signalling, antiviral responses and virus countermeasures."
J Gen Virol 89(Pt 1): 1-47.
 Stain cells with 1o antibody
for virus nucleoprotein