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