Session III The innate immunity represents an early innate host antiviral defense mechanism that takes place shortly after virus invasion and long before the development of adaptive immunity. Viruses must outcompete or subvert the initial antiviral innate immune response, allowing the establishment of persistent replication that leads to continuous stimulation of both the innate and adaptive immune components. Similar to other viruses, foot-and-mouth disease virus (FMDV), an etiological agent of severe and highly contagious viral disease, is reported to have evolved multiple strategies to evade and attenuate the host innate immunity to facilitate viral replication. In our lab, based on a subset of luciferase assays using promoter constructs of key components of the innate immunity coupled with IP-MS and Y2H, we decipher the mode of action of how viral proteins interact with host factors and further disrupt the host factor-mediated innate immunity, especially in the context of the type I interferon (IFN) system.

1. Virus-host interactions in foot-and-mouth disease virus infection
Prof. Dr. Haixue Zheng
State Key Laboratory of Veterinary Etiological Biology,
College of Veterinary Medicine, Lanzhou University,
Lanzhou Veterinary Research Institute (LVRI),
Chinese Academy of Agricultural Sciences (CAAS)
Innate immune evasion by foot-and-
mouth disease virus

2. Foot-and-mouth disease (FMD)
Background
Foot-and-mouth disease (FMD) is a severe, highly contagious viral disease of livestock that results in
significant economic impact.
FMD incubation period varies between 1 and 10 days (normally 2–7 days). It induces acute disease after
infection and can spread rapidly within a population, infecting whole herds of animals.
2001 UK FMD 2010 Japan FMD 2017 Jordan FMD

3. Why/How dose FMDV replicate rapidly and cause acute disease in animals after infection?
Inflammation
NF-kB
TNFa
IL-1
NF-
kB
IFNa/b
IRF3
ISGs Innate
immunity
Cell adhesion
molecules
Chemokines
Cytokines
(IL6)
Virus
clearance
Adaptive
immunity
Recovered
or Illness
Scientific questions:
(1) How dose FMDV regulate innate immune system (break/block)?
(2) How dose innate immune evasion contribute to pathogenesis of FMDV and concurrent adaptive
immunity?

4. Innate immunity is critical for induction of host antiviral response during viral infection
Phosphorylation
Ubiquitination
SUMOylation
Complex formation
……
Proteasome
kB ISRE
IFNa/b
Virus
IKKs
p
p p
TBK1
K63-linked Ub ISG15 phosphorylation
p S
SUMOylation
p
S
p
K48-linked Ub
Host/Viral
proteins
Virus infection triggers
IFN production
The mechanisms of innate immune
evasion by FMDV are complicated.

5. Our findings
Outlines
1. Regulation of pattern-recognition receptors (PRRs) by FMDV
2. Regulation of key innate immune molecules by FMDV
3. Host protein-mediated antiviral response against FMDV
4. Antiviral strategies exploited based on the properties of FMDV proteins

6. 1. Regulation of pattern-recognition receptors (PRRs) by FMDV
Our findings
(1) FMDV 2B and 3B proteins interact with
RIG-I to degrade RIG-I and promote FMDV
replication (J Virol 2016；J Immunol 2020).
(2) FMDV 2B interacts with and degrade
LGP2 (Cell Death Dis 2017).
(3) FMDV 2B and 2C degrade NOD2 to block
innate immune response (J Virol 2019).
ISRE
IFNa/b
Virus
MAVS
p p
TBK1
K63-linked Ub ISG15 phosphorylation
p S
p
K48-linked Ub
IRF3
IRF3 P

7. FMDV 3B interacts with RIG-I to block IFN-β production
Conclusion
We found that 3B protein
interacted with the viral RNA sensor RIG-I to
block RIG-I–mediated immune signaling..
A
B
C
RIG-I
FLAG-3B
RIG-I IgG RIG-I IgG
IB: FLAG
IB: RIG-I
CO-IP
IP: RIG-I
Vec FLAG-3B
FLAG-3B
RIG-I
WCL
b-actin
FLAG-3B
RIG-I
b-actin
WCL
IB: FLAG
IB: RIG-I
CO-IP
IP: FLAG
FLAG-3B
RIG-I
D PK-15
WCL
IP
IB: 3B
IP:
IB: RIG-I
IB: 3B
IB: RIG-I
IB: b-actin
E
IFN-b
FMDV
MAVS
IRF3 P
IRF3 P
IRF3
P
P
TBK1
P
3B
Interaction with and degradation of RIG-I
by FDMV 3B
Zhang et al. J Immunol 2020.

8. 2. Regulation of key innate immune molecules by FMDV
MAVS:
(1) FMDV VP3 interacts with MAVS and interferes with the
formation of MAVS complex, further blocking type I IFN
production (FASEB J 2016).
(2) FMDV VP0 interacts with PCBP2 to degrade MAVS through
autophagy-dependent pathway (Cell Death Dis 2019).
TBK1:
(1) The E3 ubiquitin ligase activity is essential for TBK1-
mediated degradation of VP3 (J Virol 2019).
IRF3:
(1) FMDV VP1 interacts with IRF3 and inhibits its
phosphorylation and nuclear translocation (J Virol 2019，
Cover Story).
(2) DDX56 interacts with IRF3, inhibiting IRF3-IPO5 complex
formation and contributing to decreased IFN production. 3A
interacts with DDX56, suppressing IRF3 phosphorylation and
promotes viral replication (J Cell Sci. 2019, Cell Signal. 2019).
ISRE
IFNa/b
Virus
MAVS
p p
TBK1
K63-linked Ub ISG15 phosphorylation
p S
p
K48-linked Ub
IRF3
IRF3 P

9. FMDV VP3 interacts with MAVS and interferes with the formation of MAVS
complex, further blocking type I IFN production
Conclusion
Transient transfection and CO-IP confirm that the
structural protein VP3 interacts with virus-
induced signaling adapter (VISA), which is
dependent on the C-terminal aa 111-220 of VP3.
Interaction between VP3 and MAVS

10. 3. Host protein-mediated antiviral response against FMDV
Findings:
(1) Picornavirus 3A protein degrades G3BP1 through
autophagic protein LRRC25 which impairs host antiviral
repsones (J Virol 2020).
(2) VP1 interacts with RPSA to maintain the activation of
MAPK pathway and promote FMDV replication (J Virol 2020).
(3) G3BP1 interacts with RIG-I, and decreases the expression
of E3 ubiquitin ligase RNF125, contributing to the
accumulation of RIG-I and increase of type I IFN production
(Cell Death Dis 2019).
(4) TPL2 regulates host innate immune response through
multiple mechanisms (J Virol, 2020； Frontiers Immunol，
2020).
(5) JMJD6 recruits RNF5 to induce the K48 ubiquitination of
IRF3 and blocks host antiviral response (Plos Pahogens, 2021).
ISRE
IFNa/b
Virus
MAVS
p p
TBK1
K63-linked Ub ISG15 phosphorylation
p
S
SUMOylation
p
K48-linked Ub
IRF3
IRF3 P
JMJD6

11. JMJD6 negatively regulates cytosolic RNA induced antiviral signaling by recruiting
RNF5 to promote activated IRF3 K48 ubiquitination
Conclusion
JMJD6 recruits RNF5 to induce the K48-linked polyubiquitination and proteasomal degradation of
activated IRF3

12. Application of our findings in vaccine development
………
………
………
………
………
………
………
………
20 118
O-type FMDV VP3:
A-type FMDV VP3:
Asia 1-type FMDV VP3:
C-type FMDV VP3:
SAT 2-type FMDV VP3:
SAT 1-type FMDV VP3:
SAT 3-type FMDV VP3:
-HA-TBK1
WB: aHA
WB: aMyc -Myc-VP3
WB: aactin -β-actin
HA-TBK1
Myc-VP3
Myc-VP3 K20R
- + - + - +
- - - - + +
- - + + + +
+ + - - - -
TBK1 C426/605A and VP3 K118R are the critical sites for
degradation of VP3.
WB: aHA -HA-TBK1
WB: aactin -β-actin
WB: aFlag -Flag-VP3
Flag-VP3
HA-TBK1 C426/605A
HA-TBK1 - + -
-
- +
+ +
+
C
TBK1 catalyzes the K48 ubiquitination of VP3.
Abs: αM αMαMαMαM
-F-TBK1
-Myc-VP3
F-TBK1:
_ _ _
+ +
Myc-VP3
Input
-VP3-K63Ub
IP: aM
Improvement of FMDV antigen by
targeting TBK1
(Patent, ZL201910401904.8，2020)
Knockout of TBK1 in BHK-
21 cells and introduction of
VP3K118R mutation in
FMDV substantially
increase the antigen
production.

13. 4. Antiviral strategies exploited based on the properties of FMDV proteins
Conclusion:
 FMDV 2C structure comprises an
ATPase domain, a ZFER, and a C-
terminal domain.
 The PBL-Pocket interaction between
FMDV 2C is essential for its functions.
 PBL-peptide impairs FMDV 2C
oligomerization and inhibits its
activities.
 PBL-peptide exhibits anti-FMDV
activity in cells with an IC50 of 5.5 μM
Cell reports. 2022

14. Summary: Schematic representation of the antagonistic mechanisms
exploited by FMDV
The multiple mechanisms of immune suppression used by FMDV
Yang et al., J Virol, 2019
Zhang et al., J Virol, 2019
Li et al., J Virol, 2019
Li et al., J Virol, 2020
Zhu et al., J Virol, 2020
Yang et al., J Virol, 2020
Zhang et al., J Immunol, 2020
Li et al., J Virol, 2021
Zhang et al., J Virol, 2021
Zhang et al., PLoS Pothog, 2021

15. ACKNOWLEDGEMENTS
Xiangtao Liu (Director of NRL-FMD, LVRI)
Zixiang Zhu (LVRI)
Dan Li (LVRI)
Yang Fan (LVRI)
Wen Dang (LVRI)
Weijun Cao (LVRI)
Jijun He (LVRI)
Jianhong Guo (LVRI)
Ye Jin (LVRI)
Yanmin Li (SMU)

16. Thanks for your attention!
Lanzhou Veterinary Research Institute

17. ABSL-3
Vaccine
Factory
NFMDRL
Office Building
Living Areas
Director General: Prof. Haixue Zheng
LVRI, CAAS
 Found in 1957
 Nation-wide top academic
research Institute
 3 OIE Reference Labs
 The largest platform of high-
level laboratory facilities for
animal biosafety in China

18. My research field
Such as:
1. Epidemiological patterns and evolution
2. Molecular basis for phenotypic variation
3. Asymptomatic/persistent infection and possible transmission by carrier
4. Molecular mechanism for immune evasion
5. Adaptive immune response
6. FMDV infection in pigs
Transmission of FMD(v)
Dr. Haixue Zheng zhenghaixue@caas.cn
Dr. Zixiang Zhu zhuzixiang@caas.cn
Response to FMDV infection or vaccination