1. Speaker: Professor Yang Qian Nanjing Agricultural University
The Epidermal Growth Factor Receptor Regulates Cofilin Activity and Promotes Transmissible Gastroenteritis Virus Entry into Intestinal Epithelial Cells
Speaker: Professor Yang Qian
College of Veterinary
Nanjing Agricultural University
China

2. Nanjing. China.
Porcine transmissible gastroenteritis virus (TGEV) is a major pathogen that replicates in the cytoplasm of villous epithelial cells in the small intestine, leading to severe villous atrophy and malabsorptive diarrhea and resulting in significant economic losses in the swine industry, the mortality rates may reach 100% in the newborn piglets.

3. Porcine intestinal epithelium is the target cells of TGEV infection.
Nanjing. China.
TGEV is an enveloped coronavirus, contain a large positive-sense single-stranded RNA genome about 28.5 kb in length.
Porcine intestinal epithelium is the target cells of TGEV infection.

4. Nanjing. China.
Our previous study showed that TGEV could invade the host epithelium and disrupt the actin cytoskeleton in intestinal epithelium.
The mechanisms of how TGEV disrupt the actin cytoskeleton and invade the host epithelium remain largely unknown.

5. Part I. The actin cytoskeleton is required for the entry of TGEV
Nanjing. China.
Part I. The actin cytoskeleton is required for the entry of TGEV
Porcine intestinal epithelial cell line (IPEC-J2) was used. The cells were pretreated with three F-actin inhibitors: Latrunculin A (Lat A), Cytochalasin D (Cyto D), and Cucurbitacin E (Cu E) at 37 °C for 1h. All three inhibitors reduced TGEV entry in a dose dependent manner compared with mock control cells. F-actin is involved in the TGEV entry.

6. Co-location of TGEV particles and F-actin
Nanjing. China.
Co-location of TGEV particles and F-actin
The relationship between TGEV particles and F-actin in early infection was further examined by confocal fluorescence microscopy.
30 minutes after infection, TGEV had fused with the cell membrane and begun to enter the cytoplasm, cell membrane ruffles could be observed surrounding virus particles, and some of the F-actin filaments co-localized with TGEV.
By 60 mpi, TGEV had entered into the cytoplasm, TGEV particles surrounded by F-actin filaments .

7. Nanjing. China.
The results suggested that the process of TGEV infection caused F-actin accumulation around the cell membrane. F-actin is important during the TGEV entry process.
Question : Which factor regulate the F-actin in the early TGEV infection?

8. Part II. Cofilin regulates the actin cytoskeleton in TGEV infection
Nanjing. China.
Part II. Cofilin regulates the actin cytoskeleton in TGEV infection
Cofilin is a member of the actin depolymerizing-factor family and plays an important role in regulating F-actin dynamics. The active cofilin acts as severing and depolymerization of actin filaments, the inactive p-cofilin acts as polymerization and stabilization of actin filaments.
LIM-kinases (LIMKs) inhibit the activity of cofilin by phosphorylating the serine residue at position 3 (Ser-3).
SSH1/2/3 promotes the activity of cofilin by dephosphorylating the serine residue at position 3 (Ser-3).
This picture from: K.Mizuno, et Cellular Signaling 25 (2013)

9. Nanjing. China.
In infected cells, p-cofilin levels increased from 5 mpi until 30 mpi after TGEV infection, while the level of cofilin decreased from 1 mpi to 10 mpi.
The process by which TGEV causes F-actin filaments to gather around the cell membrane requires the participation of p-cofilin.

10. Cofilin is involved in TGEV infection：
Nanjing. China.
Cofilin is involved in TGEV infection：
All three Site-directed mutagenesis constructs and cofilin targeting shRNAs inhibited TGEV entry.

11. Nanjing. China.
Cofilin could regulate the actin cytoskeleton in TGEV early infection.
Question : Which factor regulate the Cofilin in the early TGEV infection? What’s the upstream signaling?

12. RhoA is responsible for the formation of stress fibers;
Nanjing. China.
RhoA, Rac1 and Cdc42
are the members of Rho GTPases family.
RhoA is responsible for the formation of stress fibers;
Rac1 induces lamellipodia or membrane ruffles;
Cdc42 regulates the formation of protrusive filopodia.

13. Part III. Rho GTPases are involved in TGEV infection
Nanjing. China.
Part III. Rho GTPases are involved in TGEV infection
The cells were transfected with lentivirus constructs that expressed wild type, constitutively-activated, and constitutively-inactivated Rho GTPases. The expression of constitutively activated Rho GTPases (L63RhoA, L61Rac1, and L61Cdc42), as well as constitutively inactivated Rho GTPases (N17Rac1 and N17Cdc42)reduced TGEV entry.

14. Nanjing. China.
Rac1 and Cdc42 GTPase are involved in cofilin phosphorylation in TGEV infection
The level of p-cofilin was significantly inhibited by constitutively activated and inactived RhoGTPases (Rac1, Cdc42).
PAKs inhibitor (IPA-3) inhibited cofilin phosphorylation, ROCK inhibitor (Y27632)had no inhibitory effects.

15. Nanjing. China.
PAKs inhibitor IPA-3 also inhibited the entry of TGEV in a dose dependent manner.

16. PAKs are involved in TGEV infection
Nanjing. China.
PAKs are involved in TGEV infection
We pretreated cells with several Rho GTPase inhibitors prior to TGEV infection. ROCK inhibitor (Y27632) had no effect on the binding or entry of TGEV. 
PAKs (IPA-3) inhibitor inhibited the entry of TGEV in a dose dependent manner.

17. PAKs are involved in cofilin phosphorylation in TGEV infection
Nanjing. China.
PAKs are involved in cofilin phosphorylation in TGEV infection
The phosphorylation of cofilin was inhibited when cells were treated with PI3K inhibitor LY and MEK1/2 inhibitor U1026
Confocal fluorescence microscopy demonstrated that LY and U0126 treatment protected the actin cytoskeleton.

18. Nanjing. China.
RHO-family-GTPases play a key role in the entry of TGEV.
Rac1 and Cdc42 GTPase are involved in cofilin phosphorylation in TGEV early infection.
Question: Which factor regulate Rac1 and Cdc42 GTPase and PAKs in the upstream signaling？

19. Part IV. PI3K-Akt pathway is involved in TGEV infection
Nanjing. China.
Part IV. PI3K-Akt pathway is involved in TGEV infection
p-Akt levels in cells were investigated after infection. From 10 mpi to 60 mpi, p-Akt levels increased in TGEV infected cells.

20. PI3K-Akt pathway is involved in TGEV infection
Nanjing. China.
PI3K-Akt pathway is involved in TGEV infection
To investigate the role of the PI3K/Akt pathway in TGEV entry, cells pre-treated with LY294002, a highly specific inhibitor of PI3K, virus binding was unaffected, but the entry of TGEV was reduced 40% compared with untreated cells.
U0126, a specific inhibitor of MEK1/2, inhibited the entry of TGEV in a dose dependent manner but did not affect the ability of the virus to bind to cells.

21. Nanjing. China.
PI3K-Akt pathway is involved in cofilin phosphorylation in TGEV infection
The phosphorylation of cofilin was inhibited when cells were treated with PI3K inhibitor LY and MEK1/2 inhibitor U1026
Confocal fluorescence microscopy demonstrated that LY and U0126 treatment protected the actin cytoskeleton.

22. Nanjing. China.
PI3K/Akt pathway is involved in the regulation of Rac1 and Cdc42 GTPase
Question : Who will be responsible for PI3K-Akt pathway ? What’s the factor control the PI3K-Akt-Rac1/Cdc42-PAK-LIMK signaling pathway to regulate the activity of cofilin ?

23. The regulation of epidermal growth factor receptor (EGFR) signaling
Nanjing. China.
The regulation of epidermal growth factor receptor (EGFR) signaling
EGFR is a member of the RTK family and is directly upstream of PI3K. Is EGFR involved in TGEV infection?

24. Part IV. EGFR is involved in TGEV early infection
Nanjing. China.
Part IV. EGFR is involved in TGEV early infection
The role of EGFR during TGEV early infection was investigated.
Fluorescence microscopy showed that at 10 mpi, both TGEV and EGF increased the levels of p-EGFR, but significantly reduced by EGFR inhibitor (AG1478).
Western blot results showed that the level of p-EGFR increased from 5 mpi to 30 mpi, reaching a peak at 10 mpi.

25. EGFR activates downstream signaling pathways in TGEV early infection
Nanjing. China.
To study the relationship between EGFR and downstream signaling pathways, we conducted experiments using different inhibitors and EGFR shRNAs. As expected, EGFR-targeting shRNAs significantly inhibited EGFR expression. The activation of Akt, LIMK, and cofilin phosphorylation was inhibited by EGFR shRNAs ,as well as with RTK inhibitor Gen, and EGFR inhibitor AG1478.

26. EGFR is involved in TGEV early infection
Nanjing. China.
EGFR is involved in TGEV early infection
The RTK specific inhibitor genistein (Gen) had no effect on the adhesion of TGEV, but inhibited the entry of TGEV in a dose dependent manner.
EGFR targeting shRNAs significantly inhibited TGEV entry.

27. Nanjing. China.
EGFR specific inhibitor AG1478 had no effect on the adhesion of TGEV, When IPEC-J2 cells were pretreated with AG1478 for 1h at 37 °C, TGEV entry was significantly inhibited. AG1478 appears to act only at early stages of entry, shortly after the virus binds to the cell.

28. EGFR is an important receptor for numerous viruses.
Nanjing. China.
EGFR is a receptor for TGEV entry
EGFR is an important receptor for numerous viruses.
The spike protein of TGEV co-localized with p-EGFR.
The interaction between TGEV S1 and EGFR were confirmed by Co-IP.
TGEV S1 directly interacts with EGFR.  

29. Nanjing. China.
Flow cytometry  analysis showed that the overexpression EGFR and APN significantly increased the TGEV invasion.

30. Nanjing. China.
Flow cytometry analysis showed that interference for EGFR and APN significantly reduced the TGEV invasion.

31. EGFR acts as a membrane receptor for TGEV entry.
Nanjing. China.
EGFR is involved in the entry of TGEV and the activation of downstream pathways early in the infection process;
EGFR acts as a membrane receptor for TGEV entry.

32. Nanjing. China.
Membrane microdomains, also called lipid rafts, function as platforms to concentrate receptors and induce downstream signal transduction upon the binding of viral protein.
Whether the lipid rafts could play an important role in TGEV infection?

33. Part V. Lipid rafts are involved in TGEV infection.
Nanjing. China.
Part V. Lipid rafts are involved in
TGEV infection.
Lipid rafts were distributed evenly in the cell
membranes of untreated cells. In contrast, the
lipid rafts were clustered in TGEV infected
cells or EGF treated.
EGFR and the attaching TGEV particles
were located together in the lipid rafts during infection.

34. Lipid rafts are involved early in TGEV infection.
Nanjing. China.
Lipid rafts are involved early in TGEV infection.
For studying the role of lipid rafts in the entry process of TGEV, both MβCD and nystatin were used to remove the cholesterol from the cell membrane. The binding and entry of TGEV was inhibited in a dose dependent manner compared with mock control cells.

35. Nanjing. China.
Lipid rafts activate downstream signaling pathways early in TGEV infection
During the early phase of TGEV infection, the destruction of lipid rafts inhibited the activation of EGFR, Akt, and LIMK, and also inhibited the phosphorylation of coffilin.

36. Nanjing. China.
The signaling pathway of regulating the actin dynamics by cofilin and initiating TGEV entry
Lipid rafts
EGFR

37. Nanjing. China.
EGFR is a receptor for TGEV invasion, and is involved in the regulation of actin dynamics and TGEV invasion. Lipid rafts are clustered and act as signaling platforms for EGFR- mediated signaling. TGEV stimulates the phosphorylation of cofilin and the polymerization of F-actin through the EGFR- PI3K-Rac1/Cdc42-PAK-LIMK signaling pathway.
The entry mechanism of TGEV and provide a potential target for the development of new anti-TGEV therapies.

38. Hu weiwei
Acknowledgement
Zhu liqi
表面糖脂、糖链结构以及病毒的特异性受体

39. Thanks for your attention!

40. The distribution of p-cofilin in IPEC-J2 cells
Nanjing. China.
To examine the role of cofilin more closely, cells were fixed at the indicated time points, stained with anti-p-cofilin antibody, and observed by confocal microscopy. Most p-cofilin is found in the nucleus of uninfected cells. In contrast, early in TGEV infection, p-cofilin redistributes to the cytoplasm and is less abundant in the nucleus. However, by 60 mpi, p-cofilin appears to shift from the cytoplasm back to the nucleus.