Skip to main content

Advertisement

SpringerLink
Log in

MicroRNA analysis in mouse neuro-2a cells after pseudorabies virus infection

  • Published:
Journal of NeuroVirology Aims and scope Submit manuscript

Abstract

Pseudorabies virus (PRV), an alpha herpesvirus can enter the mammalian nervous system, causing Aujezsky’s disease. Previous studies have reported an alteration of microRNA (miRNA) expression levels during PRV infections. However, knowledge regarding miRNA response in nervous cells to PRV infection is still unknown. To address this issue, small RNA libraries from infected and uninfected mouse neuroblastoma cells were assessed after Illumina deep sequencing. A total of eight viral miRNA were identified, and ten host miRNAs showed significantly different expression upon PRV infection. Among these, five were analyzed by stem-loop RT-qPCR, which confirmed the above data. Interestingly, these viral miRNAs were mainly found in the large latency transcript region of PRV, and predicted to target a variety of genes, forming a complicated regulatory network. Moreover, ten cellular miRNAs were expressed differently upon PRV infection, including nine upregulated and one downregulated miRNAs. Host targets of these miRNAs obtained by bioinformatics analysis belonged to large signaling networks, mainly encompassing calcium signaling pathway, cAMP signaling pathway, MAPK signaling pathway, and other nervous-associated pathways. These findings further highlighted miRNA features in nervous cells after PRV infection and contributed to unveil the underlying mechanisms of neurotropism as well as the neuropathogenesis of PRV.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • An TQ, Peng JM, Tian ZJ, Zhao HY, Li N, Liu YM, Tong GZ (2013) Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012. Emerg Infect Dis 19:1749–1755

    Article  PubMed  PubMed Central  Google Scholar 

  • Anselmo A, Flori L, Jaffrezic F, Rutigliano T, Cecere M, Cortes-Perez N, Giuffra E (2011) Co-expression of host and viral microRNAs in porcine dendritic cells infected by the pseudorabies virus. PLoS One 6:e17374

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297

    Article  CAS  PubMed  Google Scholar 

  • Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B 57:289–300

    Google Scholar 

  • Besecker MI, Harden ME, Li G, Wang XJ, Griffiths A (2009) Discovery of herpes B virus-encoded microRNAs. J Virol 83:3413–3416

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Boss IW, Plaisance KB, Renne R (2009) Role of virus-encoded microRNAs in herpesvirus biology. Trends Microbiol 17(12):544–553

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cai X, Lu S, Zhang Z, Gonzalez CM, Damania B, Cullen BR (2005) Kaposi’s sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells. Proc Natl Acad Sci U S A 102:5570–5575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cai Y, Zhu L, Zhou Y, Liu X, Liu X, Li X, Lang Q, Qiao X, Xu Z (2015) Identification and analysis of differentially-expressed microRNAs in Japanese encephalitis virus-infected PK-15 cells with deep sequencing. Int J Mol Sci 16:2204–2219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cameron JE, Yin Q, Fewell C, Lacey M, McBride J, Wang X, Flemington EK (2008) Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways. J Virol 82:1946–1958

    Article  CAS  PubMed  Google Scholar 

  • Fuchs W, Granzow H, Klupp BG, Kopp M, Mettenleiter TC (2002) The UL48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions. J Virol 76:6729–6742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grey F (2015) Role of microRNAs in herpesvirus latency and persistence. J Gen Virol 96:739–751

    Article  CAS  PubMed  Google Scholar 

  • Grey F, Antoniewicz A, Allen E, Saugstad J, McShea A, Carrington JC, Nelson J (2005) Identification and characterization of human cytomegalovirus-encoded microRNAs. J Virol 79:12095–12099

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grey F, Meyers H, White EA, Spector DH, Nelson J (2007) A human cytomegalovirus-encoded microRNA regulates expression of multiple viral genes involved in replication. PLoS Pathog 3:e163

    Article  PubMed  PubMed Central  Google Scholar 

  • Grey F, Hook L, Nelson J (2008) The functions of herpesvirus-encoded microRNAs. Med Microbiol Immunol 197:261–267

    Article  CAS  PubMed  Google Scholar 

  • Hicks J, Liu HC (2013) Involvement of eukaryotic small RNA pathways in host defense and viral pathogenesis. Viruses 5:2659–2678

    Article  PubMed  PubMed Central  Google Scholar 

  • Hill JM, Zhao Y, Clement C, Neumann DM, Lukiw WJ (2009) HSV-1 infection of human brain cells induces miRNA-146a and Alzheimer-type inflammatory signaling. Neuroreport 20:1500–1505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hou J, Wang P, Lin L, Liu X, Ma F, An H, Cao X (2009) MicroRNA-146a feedback inhibits RIG-I-dependent type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2. J Immunol 183:2150–2158

    Article  CAS  PubMed  Google Scholar 

  • Hou ZH, Han QJ, Zhang C, Tian ZG, Zhang J (2014) miR146a impairs the IFN-induced anti-HBV immune response by downregulating STAT1 in hepatocytes. Liver Int 34:58–68

    Article  CAS  PubMed  Google Scholar 

  • Huang J, Ma G, Fu L, Jia H, Zhu M, Li X, Zhao S (2014) Pseudorabies viral replication is inhibited by a novel target of miR-21. Virology 456:319–328

    Article  PubMed  Google Scholar 

  • Kincaid RP, Sullivan CS (2012) Virus-encoded microRNAs: an overview and a look to the future. PLoS Pathog 8:e1003018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klupp BG, Hengartner CJ, Mettenleiter TC, Enquist LW (2004) Complete, annotated sequence of the pseudorabies virus genome. J Virol 78:424–440

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Labbaye C, Testa U (2012) The emerging role of MIR-146A in the control of hematopoiesis, immune function and cancer. J Hematol Onco l5:13

    Article  Google Scholar 

  • Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C, Voinnet O (2005) A cellular microRNA mediates antiviral defense in human cells. Science 308:557–560

    Article  CAS  PubMed  Google Scholar 

  • Li Y, Wang Y, Yu L, Sun C, Cheng D, Yu S, An T (2013) miR-146b-5p inhibits glioma migration and invasion by targeting MMP16. Cancer Lett 339:260–269

    Article  CAS  PubMed  Google Scholar 

  • Li W, Chang J, Wang S, Liu X, Peng J, Huang D, Sun M, Chen Z, Zhang W, Guo W, Li J (2015a) miRNA-99b-5p suppresses liver metastasis of colorectal cancer by down-regulating mTOR. Oncotarget 6:24448–24462

    Article  PubMed  PubMed Central  Google Scholar 

  • Li Y, Chang H, Yang X, Zhao Y, Chen L, Wang X, Zhao J (2015b) Antiviral activity of porcine interferon regulatory factor 1 against swine viruses in cell culture. Viruses 7:5908–5918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lin X, Liang D, He Z, Deng Q, Robertson ES, Lan K (2011) miR-K12-7-5p encoded by Kaposi’s sarcoma-associated herpesvirus stabilizes the latent state by targeting viral ORF50/RTA. PLoS One 6:e16224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu J, Xu J, Li H, Sun C, Yu L, Li Y, Wen Y (2015) miR-146b-5p functions as a tumor suppressor by targeting TRAF6 and predicts the prognosis of human gliomas. Oncotarget 6:29129–29142

    PubMed  PubMed Central  Google Scholar 

  • Liu F, Zheng H, Tong W, Li GX, Tian Q, Liang C, Tong GZ (2016) Identification and analysis of novel viral and host dysregulated microRNAs in variant pseudorabies virus-infected PK15 cells. PLoS One 11:e0151546

    Article  PubMed  PubMed Central  Google Scholar 

  • Mahjoub N, Dhorne-Pollet S, Fuchs W, Ahanda MLE, Lange E, Klupp B, Giuffra E (2015) A 2.5-kilobase deletion containing a cluster of nine microRNAs in the latency-associated-transcript locus of the pseudorabies virus affects the host response of porcine trigeminal ganglia during established latency. J Virol 89:428–442

    Article  PubMed  Google Scholar 

  • Mellencamp MW, O'brien PC, Stevenson JR (1991) Pseudorabies virus-induced suppression of major histocompatibility complex class I antigen expression. J Virol 65:3365–3368

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mulder WAM, Pol JMA, Gruys E, Jacobs L, De Jong MCM, Peeters BPH, Kimman TG (1997) Pseudorabies virus infections in pigs: role of viral proteins in virulence, pathogenesis and transmission. Vet Res 28:1–17

    CAS  PubMed  Google Scholar 

  • Otsuka M, Jing Q, Georgel P, New L, Chen J, Mols J, Das SC (2007) Hypersusceptibility to vesicular stomatitis virus infection in Dicer1-deficient mice is due to impaired miR24 and miR93 expression. Immunity 27:123–134

    Article  CAS  PubMed  Google Scholar 

  • Perng GC, Jones C (2010) Towards an understanding of the herpes simplex virus type 1 latency-reactivation cycle. Interdiscip Perspect Infect Dis 2010:262415

    Article  PubMed  PubMed Central  Google Scholar 

  • Pfeffer S, Zavolan M, Grässer FA, Chien M, Russo JJ, Ju J, Tuschl T (2004) Identification of virus-encoded microRNAs. Science 304:734–736

    Article  CAS  PubMed  Google Scholar 

  • Piedade D, Azevedo-Pereira JM (2016) The role of microRNAs in the pathogenesis of herpesvirus infection. Viruses 8:156

    Article  PubMed Central  Google Scholar 

  • Pivovarova NB, Andrews SB (2010) Calcium-dependent mitochondrial function and dysfunction in neurons. FEBS J 277:3622–3636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pomeranz LE, Reynolds AE, Hengartner CJ (2005) Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev 69(3):462–500

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Punj V, Matta H, Schamus S, Tamewitz A, Anyang B, Chaudhary PM (2010) Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 suppresses CXCR4 expression by upregulating miR-146a. Oncogene 29:1835–1844

    Article  CAS  PubMed  Google Scholar 

  • Saba R, Sorensen DL, Booth SA (2014) MicroRNA-146a: a dominant, negative regulator of the innate immune response. Front Immunol 5:578

    Article  PubMed  PubMed Central  Google Scholar 

  • Singh Y, Kaul V, Mehra A, Chatterjee S, Tousif S, Dwivedi VP, Suar M, Van Kaer L, Bishai WR, Das G (2013) Mycobacterium tuberculosis controls microRNA-99b (miR-99b) expression in infected murine dendritic cells to modulate host immunity. J Biol Chem 288:5056–5061

    Article  CAS  PubMed  Google Scholar 

  • Stewart CR, Marsh GA, Jenkins KA, Gantier MP, Tizard ML, Middleton D, Deffrasnes C (2013) Promotion of Hendra virus replication by microRNA 146a. J Virol 87:3782–3791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Taganov KD, Boldin MP, Chang KJ, Baltimore D (2006) NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A 103:12481–12486

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tang S, Patel A, Krause PR (2009) Novel less-abundant viral microRNAs encoded by herpes simplex virus 2 latency-associated transcript and their roles in regulating ICP34.5 and ICP0 mRNAs. J Virol 83:1433–1442

    Article  CAS  PubMed  Google Scholar 

  • Tang S, Bertke AS, Patel A, Margolis TP, Krause PR (2011) Herpes simplex virus 2 microRNA miR-H6 is a novel latency-associated transcript-associated microRNA, but reduction of its expression does not influence the establishment of viral latency or the recurrence phenotype. J Virol 85:4501–4509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Timoneda O, Núñez-Hernández F, Balcells I, Muñoz M, Castelló A, Vera G, Rosell R (2014) The role of viral and host microRNAs in the Aujeszky’s disease virus during the infection process. PLoS One 9:e86965

    Article  PubMed  PubMed Central  Google Scholar 

  • Tong W, Liu F, Zheng H, Liang C, Zhou YJ, Jiang YF, Tong GZ (2015) Emergence of a pseudorabies virus variant with increased virulence to piglets. Vet Microbiol 181:236–240

    Article  PubMed  Google Scholar 

  • Tong W, Li G, Liang C, Liu F, Tian Q, Cao Y, Li L, Zheng X, Zheng H, Tong GZ (2016) A live, attenuated pseudorabies virus strain JS-2012 deleted for gE/gI protects against both classical and emerging strains. Antivir Res 130:110–117

    Article  CAS  PubMed  Google Scholar 

  • Umbach JL, Kramer MF, Jurak I, Karnowski HW, Coen DM, Cullen BR (2008) MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs. Nature 454:780–783

    CAS  PubMed  PubMed Central  Google Scholar 

  • Van Opdenbosch N, Van den Broeke C, De Regge N, Tabarés E, Favoreel HW (2012) The IE180 protein of pseudorabies virus suppresses phosphorylation of translation initiation factor eIF2α. J Virol 86:7235–7240

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Varkonyi-Gasic E, Hellens RP (2011) Quantitative stem-loop RT-PCR for detection of microRNAs. Methods Mol Biol 744:145–157

    Article  CAS  PubMed  Google Scholar 

  • Wang LL, Huang Y, Wang G, Chen SD (2012) The potential role of microRNA-146 in Alzheimer’s disease: biomarker or therapeutic target. Med Hypotheses 78:398–401

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Diao C, Yang X, Yang Z, Liu M, Li X, Tang H (2016) ICP4-induced miR-101 attenuates HSV-1 replication. Sci Rep 6:23205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu YQ, Chen DJ, He HB, Chen DS, Chen LL, Chen HC, Liu ZF (2012) Pseudorabies virus infected porcine epithelial cell line generates a diverse set of host microRNAs and a special cluster of viral microRNAs. PLoS One 7(1):e30988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu S, He L, Li Y, Wang T, Feng L, Jiang L, Huang X (2013) miR-146a facilitates replication of dengue virus by dampening interferon induction by targeting TRAF6. J Infect 67:329–341

    Article  PubMed  Google Scholar 

  • Wu BW, Engel EA, Enquist LW (2014) Characterization of a replication-incompetent pseudorabies virus mutant lacking the sole immediate early gene IE180. MBio 5:e01850

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xiang M, Birkbak NJ, Vafaizadeh V, Walker S, Yeh JE, Liu S, Richardson AL (2014) STAT3 induction of miR-146b forms a feedback loop to inhibit the NF-κB to IL-6 signaling axis and STAT3-driven cancer phenotypes. Sci Signal 7(310):ra11

    Article  PubMed  PubMed Central  Google Scholar 

  • Ye X, Luo H, Chen Y, Wu Q, Xiong Y, Zhu J, Diao Y, Wu Z, Miao J, Wan J (2015a) MicroRNAs 99b-5p/100-5p regulated by endoplasmic reticulum stress are involved in Abeta-induced pathologies. Front Aging Neurosci 7:210

    Article  PubMed  PubMed Central  Google Scholar 

  • Ye C, Zhang QZ, Tian ZJ, Zheng H, Zhao K, Liu F, Shi M (2015b) Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence: evidence for the existence of two major genotypes. Virology 483:32–43

    Article  CAS  PubMed  Google Scholar 

  • Zheng SQ, Li YX, Zhang Y, Li X, Tang H (2011) MiR-101 regulates HSV-1 replication by targeting ATP5B. Antivir Res 89:219–226

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The present study was funded by the National Natural Science Foundation of China (31272567), the Program for Innovative Research Team (in Science and Technology) at the University of Henan (14IRTSTHN015), and the Science and Technology Innovation Talent Support Plan of Colleges and Universities in Henan Province (14HASTIT022).

Author information

Authors and Affiliations

  1. College of Animal Husbandry and Veterinary Science, Henan Agricultural University, Zhengzhou, 450002, China

    Yongtao Li, Guanmin Zheng, Yujuan Zhang, Xia Yang, Hongying Liu, Hongtao Chang, Xinwei Wang, Jun Zhao, Chuanqing Wang & Lu Chen

Authors
  1. Yongtao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guanmin Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yujuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xia Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongying Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hongtao Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xinwei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jun Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chuanqing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lu Chen.

Ethics declarations

Conflicts of interest

The authors declare no conflict of interest.

Additional information

Yongtao Li and Guanmin Zheng contributed equally to this work.

Electronic supplementary material

Supplementary file 1

Predicted target genes of differentially expressed host miRNAs at 4 hpi. (XLS 1071 kb)

Supplementary file 2

Predicted target genes of differentially expressed host miRNAs at 28 hpi. (XLS 1194 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, Y., Zheng, G., Zhang, Y. et al. MicroRNA analysis in mouse neuro-2a cells after pseudorabies virus infection. J. Neurovirol. 23, 430–440 (2017). https://doi.org/10.1007/s13365-016-0511-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13365-016-0511-y

Keywords

Search

Navigation