Virus Genes

pp 1–10 | Cite as

Suppression of µ1 subunit of the adaptor protein complex 2 reduces dengue virus release

  • Nopprarat Tongmuang
  • Umpa Yasamut
  • Sansanee Noisakran
  • Gopinathan Pillai Sreekanth
  • Pa-thai Yenchitsomanus
  • Thawornchai LimjindapornEmail author
Original Paper


Dengue virus (DENV) requires clathrin-mediated endocytosis for its entry into the cells where the adaptor protein complex (AP) is vital for the clathrin-coated vesicle formation. The role of AP-2 was previously examined in the early stages of DENV infection; however, the role of AP-2 in the late stage of DENV infection was not determined. The µ1 subunit of AP-2 (AP2M1) is one of the most important cytoplasmic carrier domains in clathrin-mediated endocytosis and the phosphorylation of this subunit by the kinase enzyme, AP-2 associated protein kinase 1 (AAK1), stimulates clathrin and supports the cell surface receptor incorporation. In the present study, we primarily aimed to investigate the role of AP2M1 by gene silencing approach as well as using naked DENV RNA transfection into AP2M1 knockdown cells. Secondarily, an inhibitor of AAK1, sunitinib was used to investigate whether AAK1 could influence the virus production in DENV-infected Huh7 cells. The knockdown of AP2M1 in the DENV-infected Huh7 cells displayed a reduction in the viral titer at 24 h post-infection. Furthermore, experiments were conducted to bypass the DENV internalization using a naked DENV RNA transfection into the AP2M1 knockdown cells. Higher intracellular DENV RNA, DENV E protein, and intracellular virion were observed, whereas the extracellular virion production was comparably less than that of control. Treatment with sunitinib in DENV-infected Huh7 cells was able to reduce extracellular virion production and was consistent with all four serotypes of DENV. Therefore, our findings demonstrate the role of AP2M1 in the exocytosis step of DENV replication leading to infectious DENV production and the efficacy of sunitinib in suppressing virus production during the infection with different serotypes of DENV.


Dengue virus Adaptor protein complex 2 AP-2-associated protein kinase 1 Sunitinib 



This work was supported by a Siriraj Research and Developmental Grant to TL (grant no. R016134005), Royal Golden Jubilee Ph.D. scholarships to NT (PHD/0140/2554), Mahidol University Post-Doctoral Fellowship Grant (grant no. R016120002) to GPS, and a grant from the Thailand Research Fund (IRG5980006).

Author Contributions

The study was designed by NT, UY, and TL. NT and UY carried out the experiments and analyzed data. NT, UY, GPS, and TL wrote the paper. TL, SN, and PY reviewed and finalized the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human and animal participants

This article does not contain any studies with human or animal subjects performed by any of the authors.


  1. 1.
    Chatel-Chaix L, Bartenschlager R (2014) Dengue virus- and hepatitis C virus-induced replication and assembly compartments: the enemy inside–caught in the web. J Virol 88:5907–5911PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    van der Schaar HM, Rust MJ, Chen C, van der Ende-Metselaar H, Wilschut J, Zhuang X, Smit JM (2008) Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells. PLoS Pathog 4:e1000244PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Ang F, Wong AP, Ng MM, Chu JJ (2010) Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus. Virol J 7:24PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Mercer J, Schelhaas M, Helenius A (2010) Virus entry by endocytosis. Annu Rev Biochem 79:803–833PubMedCrossRefGoogle Scholar
  5. 5.
    Smit JM, Moesker B, Rodenhuis-Zybert I, Wilschut J (2011) Flavivirus cell entry and membrane fusion. Viruses 3:160–171PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Park SY, Guo X (2014) Adaptor protein complexes and intracellular transport. Biosci Rep 34(4):e00123PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Yasamut U, Tongmuang N, Yenchitsomanus PT, Junking M, Noisakran S, Puttikhunt C, Chu JJ, Limjindaporn T (2015) Adaptor protein 1A facilitates dengue virus replication. PLoS ONE 10:e0130065PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Canagarajah BJ, Ren X, Bonifacino JS, Hurley JH (2013) The clathrin adaptor complexes as a paradigm for membrane-associated allostery. Protein Sci 22:517–529PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Christensen JG (2007) A preclinical review of sunitinib, a multitargeted receptor tyrosine kinase inhibitor with anti-angiogenic and antitumour activities. Ann Oncol 18(Suppl 10):x3–10PubMedCrossRefGoogle Scholar
  10. 10.
    Kollmannsberger C, Soulieres D, Wong R, Scalera A, Gaspo R, Bjarnason G (2007) Sunitinib therapy for metastatic renal cell carcinoma: recommendations for management of side effects. Can Urol Assoc J 1:S41–54PubMedPubMedCentralGoogle Scholar
  11. 11.
    Dorosky D, Prugar LI, Pu S, O’Brien C, Bakken R, De Jonghe S, Herdewijn P, Brannan J, Dye JM, Einav S (2018) AAK1 and GAK inhibitors demonstrate activity against Filoviruses. J Immunol 200:50–57Google Scholar
  12. 12.
    Neveu G, Ziv-Av A, Barouch-Bentov R, Berkerman E, Mulholland J, Einav S (2015) AP-2-associated protein kinase 1 and cyclin G-associated kinase regulate hepatitis C virus entry and are potential drug targets. J Virol 89:4387–4404PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Ohno H, Aguilar RC, Fournier MC, Hennecke S, Cosson P, Bonifacino JS (1997) Interaction of endocytic signals from the HIV-1 envelope glycoprotein complex with members of the adaptor medium chain family. Virology 238:305–315PubMedCrossRefGoogle Scholar
  14. 14.
    Bhattacharyya S, Hope TJ, Young JA (2011) Differential requirements for clathrin endocytic pathway components in cellular entry by Ebola and Marburg glycoprotein pseudovirions. Virology 419:1–9PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Helbig I, Lopez-Hernandez T, Shor O, Galer P, Ganesan S, Pendziwiat M, Rademacher A, Ellis CA, Humpfer N, Schwarz N, Seiffert S, Peeden J, Shen J, Sterbova K, Hammer TB, Moller RS, Shinde DN, Tang S, Smith L, Poduri A, Krause R, Benninger F, Helbig KL, Haucke V, Weber YG (2019) A recurrent missense variant in AP2M1 impairs clathrin-mediated endocytosis and causes developmental and epileptic encephalopathy. Am J Hum Genet 104:1060–1072PubMedCrossRefGoogle Scholar
  16. 16.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Brunelle JL, Green R (2014) One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE). Methods Enzymol 541:151–159PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Agrawal T, Schu P, Medigeshi GR (2013) Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle. Sci Rep 3:1813PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Camus G, Segura-Morales C, Molle D, Lopez-Verges S, Begon-Pescia C, Cazevieille C, Schu P, Bertrand E, Berlioz-Torrent C, Basyuk E (2007) The clathrin adaptor complex AP-1 binds HIV-1 and MLV Gag and facilitates their budding. Mol Biol Cell 18:3193–3203PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Wang PG, Kudelko M, Lo J, Siu LY, Kwok KT, Sachse M, Nicholls JM, Bruzzone R, Altmeyer RM, Nal B (2009) Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins. PLoS ONE 4:e8325PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Yu IM, Holdaway HA, Chipman PR, Kuhn RJ, Rossmann MG, Chen J (2009) Association of the pr peptides with dengue virus at acidic pH blocks membrane fusion. J Virol 83:12101–12107PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Yu IM, Zhang W, Holdaway HA, Li L, Kostyuchenko VA, Chipman PR, Kuhn RJ, Rossmann MG, Chen J (2008) Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319:1834–1837PubMedCrossRefGoogle Scholar
  23. 23.
    Pan XB, Han JC, Cong X, Wei L (2012) BST2/tetherin inhibits dengue virus release from human hepatoma cells. PLoS ONE 7:e51033PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Krauss M, Kukhtina V, Pechstein A, Haucke V (2006) Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes. Proc Natl Acad Sci USA 103:11934–11939PubMedCrossRefGoogle Scholar
  25. 25.
    He B, Xi F, Zhang X, Zhang J, Guo W (2007) Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane. Embo J 26:4053–4065PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Martin TF (2015) PI(4,5)P(2)-binding effector proteins for vesicle exocytosis. Biochim Biophys Acta 1851:785–793PubMedCrossRefGoogle Scholar
  27. 27.
    Chen Z, Lin X, Zhang Z, Huang J, Fu S, Huang R (2011) EXO70 protein influences dengue virus secretion. Microbes Infect 13:143–150PubMedCrossRefGoogle Scholar
  28. 28.
    Ricotta D, Conner SD, Schmid SL, von Figura K, Honing S (2002) Phosphorylation of the AP2 mu subunit by AAK1 mediates high affinity binding to membrane protein sorting signals. J Cell Biol 156:791–795PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Conner SD, Schmid SL (2002) Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis. J Cell Biol 156:921–929PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Lee DW, Zhao X, Zhang F, Eisenberg E, Greene LE (2005) Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors. J Cell Sci 118:4311–4321PubMedCrossRefGoogle Scholar
  31. 31.
    Neveu G, Barouch-Bentov R, Ziv-Av A, Gerber D, Jacob Y, Einav S (2012) Identification and targeting of an interaction between a tyrosine motif within hepatitis C virus core protein and AP2M1 essential for viral assembly. PLoS Pathog 8:e1002845PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Zhang F, Landford WN, Ng M, McNatt MW, Bieniasz PD, Hatziioannou T (2011) SIV Nef proteins recruit the AP-2 complex to antagonize Tetherin and facilitate virion release. PLoS Pathog 7:e1002039PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT, Faraoni R, Floyd M, Hunt JP, Lockhart DJ, Milanov ZV, Morrison MJ, Pallares G, Patel HK, Pritchard S, Wodicka LM, Zarrinkar PP (2008) A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 26:127–132PubMedCrossRefGoogle Scholar
  34. 34.
    Sreekanth GP, Yenchitsomanus PT, Limjindaporn T (2018) Role of mitogen-activated protein kinase signaling in the pathogenesis of dengue virus infection. Cell Signal 48:64–68PubMedCrossRefGoogle Scholar
  35. 35.
    Sreekanth GP, Chuncharunee A, Cheunsuchon B, Noisakran S, Yenchitsomanus PT, Limjindaporn T (2017) JNK1/2 inhibitor reduces dengue virus-induced liver injury. Antiviral Res 141:7–18PubMedCrossRefGoogle Scholar
  36. 36.
    Sreekanth GP, Chuncharunee A, Sirimontaporn A, Panaampon J, Noisakran S, Yenchitsomanus PT, Limjindaporn T (2016) SB203580 modulates p38 MAPK signaling and dengue virus-induced liver injury by reducing MAPKAPK2, HSP27, and ATF2 phosphorylation. PLoS ONE 11:e0149486PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Sreekanth GP, Chuncharunee A, Sirimontaporn A, Panaampon J, Srisawat C, Morchang A, Malakar S, Thuwajit P, Kooptiwut S, Suttitheptumrong A, Songprakhon P, Noisakran S, Yenchitsomanus PT, Limjindaporn T (2014) Role of ERK1/2 signaling in dengue virus-induced liver injury. Virus Res 188:15–26PubMedCrossRefGoogle Scholar
  38. 38.
    Sreekanth GP, Panaampon J, Suttitheptumrong A, Chuncharunee A, Bootkunha J, Yenchitsomanus PT, Limjindaporn T (2019) Drug repurposing of N-acetyl cysteine as antiviral against dengue virus infection. Antiviral Res 166:42–55PubMedCrossRefGoogle Scholar
  39. 39.
    Leela SL, Srisawat C, Sreekanth GP, Noisakran S, Yenchitsomanus PT, Limjindaporn T (2016) Drug repurposing of minocycline against dengue virus infection. Biochem Biophys Res Commun 478:410–416PubMedCrossRefGoogle Scholar
  40. 40.
    Malakar S, Sreelatha L, Dechtawewat T, Noisakran S, Yenchitsomanus PT, Chu JJH, Limjindaporn T (2018) Drug repurposing of quinine as antiviral against dengue virus infection. Virus Res 255:171–178PubMedCrossRefGoogle Scholar
  41. 41.
    Bekerman E, Neveu G, Shulla A, Brannan J, Pu SY, Wang S, Xiao F, Barouch-Bentov R, Bakken RR, Mateo R, Govero J, Nagamine CM, Diamond MS, De Jonghe S, Herdewijn P, Dye JM, Randall G, Einav S (2017) Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects. J Clin Invest 127:1338–1352PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Morchang A, Lee RCH, Yenchitsomanus PT, Sreekanth GP, Noisakran S, Chu JJH, Limjindaporn T (2017) RNAi screen reveals a role of SPHK2 in dengue virus-mediated apoptosis in hepatic cell lines. PLoS ONE 12:e0188121PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand
  2. 2.Graduate Program in Molecular Medicine, Multidisciplinary Unit, Faculty of Science, Faculty of Medicine Ramathibodi Hospital, Faculty of Medicine Siriraj Hospital, Faculty of Dentistry, Faculty of Tropical MedicineMahidol UniversityBangkokThailand
  3. 3.Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical SciencesChiang Mai UniversityChiang MaiThailand
  4. 4.Medical Biotechnology Research Unit, National Center for Genetic Engineering and BiotechnologyNational Science and Technology Development AgencyBangkokThailand
  5. 5.Department of Anatomy, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand

Personalised recommendations