DDX21 translocates from nucleus to cytoplasm and stimulates the innate immune response due to dengue virus infection

https://doi.org/10.1016/j.bbrc.2016.03.120Get rights and content

Highlights

  • DDX21 translocates from nucleus to cytoplasm in response to DENV infection.

  • DDX21 is involved in activation of the innate immune response for the inhibition of DENV infection.

  • The DENV NS2B/3 complex degraded DDX21 in cytoplasm to facilitate DENV replication.

Abstract

Successful DENV infection relies on its ability to evade the host innate immune system. By using iTRAQ labeling followed by LC-MS/MS analysis, DDX21 was identified as a new host RNA helicase involved in the DENV life cycle. In DENV infected cells, DDX21 translocates from nucleus to cytoplasm to active the innate immune response and thus inhibits DENV replication in the early stages of infection. DDX21 is then degraded by the viral NS2B-NS3 protease complex and the innate immunity is thus subverted to facilitate DENV replication. The results reveal a new mechanism in which DENV subverts the host innate immune system to facilitate its replication in host cells.

Introduction

Dengue virus (DENV), as well as West Nile virus (WNV), Zika virus (ZIKV), and Yellow fever virus (YFV) belongs to the genus of Flavivirus and is transmitted by the bite of mosquitoes. The flaviviruses have a 10–11 kb single positive-stranded RNA genome with short untranslation regions (UTRs) on the 5′ and 3′ ends, and firstly served as mRNA encoding a single polyprotein which is cleaved into three structural proteins and seven nonstructural proteins [1]. The DENV NS2B and NS3 proteins form a viral protease complex together with host proteases to process the polyprotein [2].

Host factors are absolutely essential for successful DENV infection and viral replication [3]. Currently, a limited number of host proteins have been reported to be involved in DENV replication. Yocupicio-Monroy [4], [5] reported that the calreticulin and La bind to the 3′ UTR of DENV-4, and La is redistributed in response to DENV infection. In a separate study De Nova-Ocampo [6] showed that the La, PTB, and EF-1alpha interact with DENV RNA. Recently, Ward [7] reported that the DDX6 bound to DENV RNA during infection and is required for DENV replication.

The DExD/H box protein family includes a large number of proteins that have functional roles in kinds of RNA metabolic processes, including modulation of complex RNA structures, association/dissociation of some ribonucleoprotein complexes by the RNA helicase activity, regulation of some virus life cycles [8]. DDX20 was shown to be associated with the nuclear proteins of Epstein-Barr virus and may play a role in the transcriptional regulation of latent viral genes [9]. The DDX21 was first isolated as a nucleolar protein that was recognized by an autoantibody [10]. DDX21 was then identified as an interacting partner for the transcription factor c-Jun and its helicase activity was involved in relocalization of DDX21 from the nucleolus to the nucleoplasm [8]. It is reported that DDX21 interacted with the 5′ UTR of borna disease virus (BDV) mRNA and controlled its translation [11]. Recently, DDX1, DDX5, DDX17 and DDX21 were identified as HIV Rev interacting proteins that enhance the HIV-1 Rev-dependent RNA export in the nucleolus or nucleus [12], [13]. DDX21 was also reported to be a host regulator involved in influenza A virus replication [14].

By combining isobaric tags for relative and absolute quantitation (iTRAQ) labeling with liquid chromatography/tandem mass spectrometry (LC-MS/MS), we quantified the differential proteomes of 293T cells with DENV infection or not and identified DDX21 as a DENV-downregulated host protein. Further study presents the role of DDX21 in DENV life cycle for the first time that DDX21 translocates from nucleus to cytoplasm to active the innate immune response for the inhibition of early infection, and later during infection, DDX21 is degraded by the DENV NS2B/3 protease complex and the innate immunity is thus subverted to facilitate DENV replication.

Section snippets

Cell culture and virus infection

The 293T cells and A549 cells were cultured in DMEM containing 10% FBS, 10 U/mL penicillin and 10 μg/mL streptomycin at 37 °C and 5% CO2. C6/36 (Aedes albopictus) cells were cultured at 28 °C in RPMI 1640 supplemented with 10% FBS.

The DENV-2 infection was conducted by incubating the cells with virus (M.O.I = 1) for 2 h at 37 °C with occasional rocking. Afterward, the cells were rinsed with PBS, overlaid with complete medium, and incubated at indicated time points.

iTRAQ-2DLC-MS/MS analysis

The iTRAQ-2DLC-MS/MS analysis

DENV infection down regulates DDX21

To implicate the molecular pathways impacted by DENV-2 infection, proteins of DENV-infected and mock-infected 293T cells were extracted and prepared for iTRAQ analysis. DDX21 was identified in the down-expression group in infected cells for the first time (Table 1). We confirmed the change in DDX21 expression in A549 cells infected with DENV-2 and harvested at 12 h intervals. DDX21 expression was significantly reduced over the first day and rebounded after 36 h post-infection (Fig. 1).

DENV infection causes DDX21 to translocate from nucleus to cytoplasm

As DDX21

Discussion

The DDX21 is a helicase that is reported to have functional roles in viruses replication. For example, the interaction between DDX21 and influenza A virus PB1 protein results in inhibition of viral polymerase assembly and reduction of viral RNA and protein synthesis. To overcome this restriction, the viral NS1 protein binds to DDX21 and displacing PB1 during late infection. Thus the role of DDX21 in influenza A virus replication transforms from the host restriction factor into a host regulator

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NSFC 31370193).

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    These authors contributed equally to this manuscript.

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