miR-204 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication via inhibiting LC3B-mediated autophagy

Yao Yao; Sihan Li; Yingqi Zhu; Yangyang Xu; Siyuan Hao; Shuyuan Guo; Wen-Hai Feng

doi:10.1016/j.virs.2023.07.004

October 2023

Yao Yao, Sihan Li, Yingqi Zhu, Yangyang Xu, Siyuan Hao, Shuyuan Guo and Wen-Hai Feng. miR-204 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication via inhibiting LC3B-mediated autophagy[J]. Virologica Sinica, 2023, 38(5): 690-698. doi: 10.1016/j.virs.2023.07.004

Citation: Yao Yao, Sihan Li, Yingqi Zhu, Yangyang Xu, Siyuan Hao, Shuyuan Guo, Wen-Hai Feng. miR-204 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication via inhibiting LC3B-mediated autophagy .VIROLOGICA SINICA, 2023, 38(5) : 690-698. http://dx.doi.org/10.1016/j.virs.2023.07.004

miR-204通过靶向LC3B影响自噬抑制猪繁殖与呼吸综合征病毒复制

中国农业大学生物学院, 中国北京

通讯作者： 封文海, whfeng@cau.edu.cn
收稿日期： 2023-04-07
录用日期： 2023-07-07

摘要

猪繁殖与呼吸综合征（PRRS）是由猪繁殖与呼吸综合征病毒（PRRSV）感染引起，可导致猪的繁殖障碍和呼吸系统疾病。PRRS使我国乃至全世界的养猪产业受到了严重冲击。microRNAs（miRNAs）是一类小的不参与编码蛋白的单链RNA，通过降解mRNA或抑制蛋白翻译在转录水平后调节基因的表达，在多种生命活动中发挥重要作用。近年来，越来越多的证据表明，miRNAs参与调控PRRSV的感染和复制。然而，大多数miRNAs在PRRSV感染和复制过程中的功能和作用机制尚不清楚。在本文中，我们证明miR-204具有负调控PRRSV复制的功能。研究发现，过表达miR-204可以显著地抑制PRRSV复制。利用生物信息学分析软件发现, 自噬微管相关蛋白1轻链3B（LC3B）mRNA的3’UTR存在miR-204的潜在靶位点。通过双荧光素酶报告基因实验进一步证明，miR-204可以直接靶向LC3B mRNA，抑制其蛋白翻译，调控细胞自噬。同时，研究还揭示了细胞自噬与PRRSV复制的关系，即PRRSV感染可以诱导细胞自噬进而促进病毒自身的复制。最后，研究证明了miR-204通过抑制LC3B介导的自噬负调控PRRSV复制。本文揭示了宿主miR-204调控PRRSV复制的分子机制，进一步阐明了miRNAs在PRRSV-宿主相互作用中的发挥的功能，为PRRS的有效防控和新型疫苗的研发提供了新的靶点，为PRRSV治疗药物的开发提供了新的方向和理论依据。
- 猪繁殖与呼吸综合征病毒（PRRSV）
- , miR-204
- , 自噬
- , 微管相关蛋白1轻链3B（LC3B）

miR-204 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication via inhibiting LC3B-mediated autophagy

State Key Laboratory of Animal Biotech Breeding, Frontiers Science Center for Molecular Design Breeding, Ministry of Agriculture Key Laboratory of Soil Microbiology, and Department of Microbiology and Immunology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China

Corresponding author: Wen-Hai Feng, whfeng@cau.edu.cn
Received Date: 07 April 2023
Accepted Date: 07 July 2023

Abstract

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been regarded as a persistent challenge for the swine farms worldwide. microRNAs (miRNAs) play key roles in regulating almost every important biological process, including virus-host interaction. In this study, we found that miR-204 was highly expressed in cells that were not permissive to PRRSV infection compared with cells susceptible to PRRSV infection. Subsequently, we demonstrated that overexpression of miR-204 significantly inhibited PRRSV replication in porcine alveolar macrophages (PAMs). Through bioinformatic analysis, we found that there existed a potential binding site of miR-204 on the 3′UTR of microtubule associated protein 1 light chain 3B (MAP1LC3B, LC3B), a hallmark of autophagy. Applying experiments including luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay, we demonstrated that miR-204 directly targeted LC3B, thereby downregulating autophagy. Meanwhile, we investigated the interplay between autophagy and PRRSV replication in PAMs, confirming that PRRSV infection induces autophagy, which in turn facilitates viral replication. Overall, we verify that miR-204 suppresses PRRSV replication via inhibiting LC3B-mediated autophagy in PAMs. These findings will provide a novel potential approach for us to develop antiviral therapeutic agents and controlling measures for future PRRSV outbreaks.
- Porcine reproductive and respiratory syndrome virus (PRRSV)
- , miR-204
- , Autophagy
- , LC3B

References
1. Castillo, J.A., Castrillon, J.C., Diosa-Toro, M., Betancur, J.G., St Laurent, G., Smit, J.M., and Urcuqui-Inchima, S. (2016). Complex interaction between dengue virus replication and expression of miRNA-133a. BMC Infect Dis 16, 29.
2. Chen, P., Tan, X., Lao, M., Wu, X., Zhao, X., Zhou, S., Yu, J., Zhu, J., Yu, L., Tong, W., et al. (2021). The Novel PRRSV Strain HBap4-2018 with a Unique Recombinant Pattern Is Highly Pathogenic to Piglets. Virol Sin 36, 1611-1625.
3. Chen, Q., Fang, L., Wang, D., Wang, S., Li, P., Li, M., Luo, R., Chen, H., and Xiao, S. (2012). Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication. Virus Res 163, 650-655.
4. Derstuganova, T.M., Velichkovski, I.B., Varaksin, A.N., Gurvich, V.B., Malykh, O.L., Kochneva, N.I., and Iarushin, S.V. (2013).[Assessment of the impact of socio-economic factors on the health state of the population of the Sverdlovsk region in the system of social-hygienic monitoring]. Gig Sanit, 87-89.
5. Dhakal, S., and Renukaradhya, G.J. (2019). Nanoparticle-based vaccine development and evaluation against viral infections in pigs. Vet Res 50, 90.
6. Diao, F., Jiang, C., Sun, Y., Gao, Y., Bai, J., Nauwynck, H., Wang, X., Yang, Y., Jiang, P., and Liu, X. (2023). Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication. PLoS Pathog 19, e1011295.
7. Dikic, I., and Elazar, Z. (2018). Mechanism and medical implications of mammalian autophagy. Nat Rev Mol Cell Biol 19, 349-364.
8. Duan, X., Nauwynck, H.J., and Pensaert, M.B. (1997). Effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV). Archives of Virology 142, 2483-2497.
9. Fu, L., Fu, X.Y., Mo, J., Li, X.M., Li, R.H., and Peng, S.F. (2019). miR-146a-5p enhances hepatitis B virus replication through autophagy to promote aggravation of chronic hepatitis B. Iubmb Life 71, 1336-1346.
10. Guo, X.K., Zhang, Q., Gao, L., Li, N., Chen, X.X., and Feng, W.H. (2013). Increasing expression of microRNA 181 inhibits porcine reproductive and respiratory syndrome virus replication and has implications for controlling virus infection. J Virol 87, 1159-1171.
11. Guo, Y., Xu, X., Tang, T., Sun, L., Zhang, X., Shen, X., Li, D., Wang, L., Zhao, L., and Xie, P. (2022). miR-505 inhibits replication of Borna disease virus 1 via inhibition of HMGB1-mediated autophagy. J Gen Virol 103, doi:10.1099/jgv.0.001868.
12. Kappes, M.A., and Faaberg, K.S. (2015). PRRSV structure, replication and recombination:Origin of phenotype and genotype diversity. Virology 479-480, 475-486.
13. Keshavarz, M., Dianat-Moghadam, H., Sofiani, V.H., Karimzadeh, M., Zargar, M., Moghoofei, M., Biglari, H., Ghorbani, S., Nahand, J.S., and Mirzaei, H. (2018). miRNA-based strategy for modulation of influenza A virus infection. Epigenomics 10, 829-844.
14. Klionsky, D.J. (2008). Autophagy revisited:a conversation with Christian de Duve. Autophagy 4, 740-743.
15. Klionsky, D.J., Abdel-Aziz, A.K., Abdelfatah, S., Abdellatif, M., Abdoli, A., Abel, S., Abeliovich, H., Abildgaard, M.H., Abudu, Y.P., Acevedo-Arozena, A., et al. (2021). Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy 17, 1-382.
16. Levine, B., and Kroemer, G. (2019). Biological Functions of Autophagy Genes:A Disease Perspective. Cell 176, 11-42.
17. Li, N., Du, T., Yan, Y., Zhang, A., Gao, J., Hou, G., Xiao, S., and Zhou, E.M. (2016a). MicroRNA let-7f-5p Inhibits Porcine Reproductive and Respiratory Syndrome Virus by Targeting MYH9. Sci Rep 6, 34332.
18. Li, S., Zhang, X., Yao, Y., Zhu, Y., Zheng, X., Liu, F., and Feng, W. (2022). Inducible miR-150 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by Targeting Viral Genome and Suppressor of Cytokine Signaling 1. Viruses 14, 1485.
19. Li, S., Zhou, A., Wang, J., and Zhang, S. (2016b). Interplay of autophagy and apoptosis during PRRSV infection of Marc145 cell. Infect Genet Evol 39, 51-54.
20. Liu, F., Du, Y., and Feng, W.H. (2017). New perspective of host microRNAs in the control of PRRSV infection. Vet Microbiol 209, 48-56.
21. Liu, F., Wang, H., Du, L., Wei, Z., Zhang, Q., and Feng, W.H. (2018). MicroRNA-30c targets the interferon-alpha/beta receptor beta chain to promote type 2 PRRSV infection. J Gen Virol 99, 1671-1680.
22. Meulenberg, J.J. (2000). PRRSV, the virus. Vet Res 31, 11-21.
23. Montaner-Tarbes, S., Del Portillo, H.A., Montoya, M., and Fraile, L. (2019). Key Gaps in the Knowledge of the Porcine Respiratory Reproductive Syndrome Virus (PRRSV). Front Vet Sci 6, 38.
24. Neumann, E.J., Kliebenstein, J.B., Johnson, C.D., Mabry, J.W., Bush, E.J., Seitzinger, A.H., Green, A.L., and Zimmerman, J.J. (2005). Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J Am Vet Med Assoc 227, 385-392.
25. Orvedahl, A., MacPherson, S., Sumpter, R., Jr., Talloczy, Z., Zou, Z., and Levine, B. (2010). Autophagy protects against Sindbis virus infection of the central nervous system. Cell Host Microbe 7, 115-127.
26. Pileri, E., and Mateu, E. (2016). Review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination. Vet Res 47, 108.
27. Renken, C., Nathues, C., Swam, H., Fiebig, K., Weiss, C., Eddicks, M., Ritzmann, M., and Nathues, H. (2021). Application of an economic calculator to determine the cost of porcine reproductive and respiratory syndrome at farm-level in 21 pig herds in Germany. Porcine Health Manag 7, 3.
28. Rhman, M.A., and Pmo, O. (2022). Potential therapeutic applications of microRNAs in cancer diagnosis and treatment:Sharpening a double-edged sword? Eur J Pharmacol, 175210.
29. Ribeiro, C.M., Sarrami-Forooshani, R., Setiawan, L.C., Zijlstra-Willems, E.M., van Hamme, J.L., Tigchelaar, W., van der Wel, N.N., Kootstra, N.A., Gringhuis, S.I., and Geijtenbeek, T.B. (2016). Receptor usage dictates HIV-1 restriction by human TRIM5alpha in dendritic cell subsets. Nature 540, 448-452.
30. Schaaf, M.B., Keulers, T.G., Vooijs, M.A., and Rouschop, K.M. (2016). LC3/GABARAP family proteins:autophagy-(un)related functions. FASEB J 30, 3961-3978.
31. Sun, M.X., Huang, L., Wang, R., Yu, Y.L., Li, C., Li, P.P., Hu, X.C., Hao, H.P., Ishag, H.A., and Mao, X. (2012). Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication. Autophagy 8, 1434-1447.
32. Wang, H., Xu, Y., and Feng, W. (2021). Porcine Reproductive and Respiratory Syndrome Virus:Immune Escape and Application of Reverse Genetics in Attenuated Live Vaccine Development. Vaccines (Basel) 9, 480.
33. Wang, J., Liu, J.Y., Shao, K.Y., Han, Y.Q., Li, G.L., Ming, S.L., Su, B.Q., Du, Y.K., Liu, Z.H., Zhang, G.P., et al. (2019). Porcine Reproductive and Respiratory Syndrome Virus Activates Lipophagy To Facilitate Viral Replication through Downregulation of NDRG1 Expression. J Virol 93, e00526-19.
34. Wang, L., Tian, Y., and Ou, J.H. (2015). HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. PLoS Pathog 11, e1004764.
35. Wu, J., Peng, X., Zhou, A., Qiao, M., Wu, H., Xiao, H., Liu, G., Zheng, X., Zhang, S., and Mei, S. (2014). MiR-506 inhibits PRRSV replication in MARC-145 cells via CD151. Mol Cell Biochem 394, 275-281.
36. Xu, C., Wang, M., Song, Z., Wang, Z., Liu, Q., Jiang, P., Bai, J., Li, Y., and Wang, X. (2018). Pseudorabies virus induces autophagy to enhance viral replication in mouse neuro-2a cells in vitro. Virus Res 248, 44-52.
37. Yang, Y., and Klionsky, D.J. (2020). Autophagy and disease:unanswered questions. Cell Death Differ 27, 858-871.
38. Yao, Y., Zhang, X., Li, S., Zhu, Y., Zheng, X., Liu, F., and Feng, W.H. (2022). miR-142-3p suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by directly targeting Rac1. Vet Microbiol 269, 109434.
39. You, X., Lei, Y., Zhang, P., Xu, D., Ahmed, Z., and Yang, Y. (2022). Role of transcription factors in porcine reproductive and respiratory syndrome virus infection:A review. Front Microbiol 13, 924004.
40. Zhang, H., Sha, H., Qin, L., Wang, N., Kong, W., Huang, L., and Zhao, M. (2022). Research Progress in Porcine Reproductive and Respiratory Syndrome Virus-Host Protein Interactions. Animals (Basel) 12, 1381.
41. Zhang, Q., Guo, X.K., Gao, L., Huang, C., Li, N., Jia, X., Liu, W., and Feng, W.H. (2014). MicroRNA-23 inhibits PRRSV replication by directly targeting PRRSV RNA and possibly by upregulating type I interferons. Virology 450-451, 182-195.
42. Zhang, Q., Huang, C., Yang, Q., Gao, L., Liu, H.C., Tang, J., and Feng, W.H. (2016). MicroRNA-30c Modulates Type I IFN Responses To Facilitate Porcine Reproductive and Respiratory Syndrome Virus Infection by Targeting JAK1. J Immunol 196, 2272-2282.
43. Zhang, W., Yang, H., Kong, X., Mohapatra, S., San Juan-Vergara, H., Hellermann, G., Behera, S., Singam, R., Lockey, R.F., and Mohapatra, S.S. (2005). Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat Med 11, 56-62.
44. Zhang, X., and Feng, W.H. (2021). Porcine Reproductive and Respiratory Syndrome Virus Evades Antiviral Innate Immunity via MicroRNAs Regulation. Front Microbiol 12, 804264.
45. Zhou, A., Li, S., Khan, F.A., and Zhang, S. (2016). Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1 interaction. Virulence 7, 98-109.
46. Zhu, M., Zhang, Y., Pan, J., Tong, X., Zhang, X., Hu, X., and Gong, C. (2022). Grass Carp Reovirus triggers autophagy enhancing virus replication via the Akt/mTOR pathway. Fish Shellfish Immunol 128, 148-156.
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