In Vitro Study On Molecular Mechanism Of Antibody-Dependent Enhancement Of Dengue Virus Infection

Dengue virus (DENV) is a mosquito-borne virus causing dramatic public health issues in more than 100 countries, particularly in Asia and Latin America, and an estimated more than 50 million people are infected annually. The geographic expansion of the vector, the Aedesaegypti mosquitoes and Aedes albopictus, contributes to a continuous increase in incidence and disease severity.Each of the four serotypes of DENV can lead a spectrum of outcomes from subclinical to death. Based on clinical observations, second heterotypic infection or waning immunity of infant born to mother infected by DENV significantly increase the possibility of acquiring severe disease. Antibody-dependent enhancement (ADE) has been thought to be involved in the immunopathogenesis of severe Dengue symptoms like dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). It has been hypothesized that the preexisting heterotypic antibodies form a complex with the virus and target cells, via Fc receptors in the target cells, stimulate the infectious properties of virions and facilitate the infection of target cells including monocyte, macrophages and mature DC. Current studies suggested that DENV-ADE infection could suppress IFN-I secretion and function and sequentially promote viral production. Among which IL-10 play a vital role in DENV-ADE infection through suppressing of IFN signaling. However, the suppression of type I IFN secretion in DENV-ADE infection has not heen found in other in vitro studies using human primary monocytes. Therefore, it is reasonable to postulate that a more pervasive mechanism may exist which does not rely on the suppression of IFNα/β or the increased production of IL-10/IL-6.In this study, we used an in vitro model, IFN-I deficient K562 monocytic cell line, exhibited a typical intrinsic ADE infection. Through detection of antiviral gene NOS2 levels both in DENV and DENV-ADE infected K562cells, we found that the reduced NOS2 expression and following nitric oxide production in DENV-ADE infected cells resulted in elevated viral production. Further analysis revealed that the suppressed NOS2 expression was due to suppressed RIG-I/MDA-5—NF-κB—IRF-1 signaling cascade. Over-expression of RIG-I W/WT MDA-5 synergistically suppress intracellular viral replication through promotion of NF-κB activation and NOS2 expression.For examined the function of anti-inflammatory cytokine IL-10 in DENV-ADE infection, we detected the activation of IL-10/IL-6—SOCS3 signaling in DENV-ADE infected cells. The results showed that no significant increased production of IL-10, IL-6 and SOCS3 in the DENV-ADE infected K562 cells. Four strains of K562 carrying homozygous deletions of IL-10 were generated with CRISPR-Cas9 approach, and all four strains expressed a similar enhanced fold of DENV-ADE compared with the wide-type K562 cell lines. The results indicated that the marked increase of virus production in DENV-ADE infected K562 was independent of IL-10 function.Autophagy process was shown to play an important role in DENV life cycles. Through detection of the marker protein of autophagy and autophagy related proteins, we found that DENV-ADE infection induced increased cellular autophagy process. Pharmacally activated or inhibited autophagy process would increase or suppress DENV intracellular viral RNA replication in a dose dependent manner. And knockout of ATG5 significantly suppressed viral production. These data suggested the elevated virus production in DENV-ADE infection of K562 is attribute to increased cellular autophagy process. Besides, over-expression of ATG5 impaired the NF-kB activation and following NOS2 expression, and eventually lead to increased intracellular viral RNA replication.In a word, this study demonstrated that the elevated viral production in DENV-ADE infected cells was due to decreased RIG-I/MDA-5—NF-κB—IRF-1— NOS2 signaling cascade, and such suppression was independent of IL-10 function and inhibition of IFN signaling. In addition, DENV-ADE infection induced autophagisome formation to promote DENV proliferation partially through suppression of MAVS mediated innate immune signaling. This research will enhance our comprehension of the complexity in DENV-ADE infection, and provide a theoretical support and potential targets for severe dengue therapy. Furthermore, autophagy inhibitors have potential value for clinical application as anti-DENV drug.