Porcine FcγRIIb mediated PRRSV ADE infection through inhibiting IFN-β by cytoplasmic inhibitory signal transduction

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Abstract

Antibody-dependent enhancement (ADE) in porcine reproductive and respiratory syndrome virus (PRRSV) infection is a significant obstacle to the development of effective vaccines for controlling PRRS. Our previous results have demonstrated that porcine FcγRIIb (poFcγRIIb) play an important role in mediating ADE of PRRSV infection in vitro. However, the underlying mechanisms involved in poFcγRIIb mediated-ADE are still not clear. In this study, MARC-145 cel1 lines stably expressing mutated poFcγRIIb (MARC-poFcγRIIb-T and MARC-poFcγRIIb-CT) in cytoplasm were established and the capacity of poFcγRIIb mutants in mediating ADE of PRRSV was investigated. Our results showed that removal of cytoplasmic domain or disruption the tyrosine residue within ITIM (immunoreceptor tyrosine-based inhibition motif) of the poFcγRIIb abolished the ability of poFcγRIIb to mediate ADE of PRRSV. Furthermore, we found that SHIP1 and TBK1 were involved in poFcγRIIb-mediated ADE of PRRSV infection. Taken together, our findings indicated that poFcγRIIb mediated the ADE pathway of PRRSV infection through recruiting SHIP-1, which further inhibited of TBK-1-IRF3-IFN-β signaling pathway to enhance PRRSV infection. These findings will contribute to the molecular mechanism of ADE infection and provide some implications for vaccine development.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) is a porcine viral disease which is an economically important in the world [1]. Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen that causes PRRS. PRRS is a positive envelope RNA virus, belonging to the family of arteria viruses [2]. It has been reported that swine are the only hosts of PRRSV. Swine macrophages and dendritic cells are the main permitted cells of the virus [3]. Previous studies have shown that infectious PRRSV immune complexes mediated by Fc gamma receptor (Fc gamma R) enter macrophages and play a key role in the pathogenesis of PRRSV [[4], [5], [6], [7]]. The presence of low neutralizing anti-PRRSV serum significantly enhanced the infection of PRRSV. The duration of viremia of swine injected with low neutralizing antibodies was significantly longer than that of pigs injected with normal IgG [5,6]. These observations suggest that PRRSV-induced low titer antibodies exposed to wild-type or vaccine strains may increase the severity of the disease and promote susceptibility to PRRSV infection, i.e. antibody dependence enhancement (ADE) [[6], [7], [8]]. ADE infection of PRRSV is an important obstacle to the development of effective vaccine to control PRRSV.

Our previous research demonstrated that porcine FcγRIIb (poFcγRIIb) plays a crucial role in mediating the ADE of PRRSV infection [9]. Fc receptors are important mediators of immune responses. FcγRIIb is the only inhibitory receptor that interacts with immune complexes. It prevents immune cells from over-activation by introducing SH2-containing tyrosine inositol phosphate 1 (SHIP1) into its cytoplasmic immunoreceptor tyrosine inhibitory motif (ITIM) [9,10]. Cytoplasmic structures of FcγR are involved in interacting with intermediate signal transduction elements and are indispensable for its function [11]. However, the intracellular molecular mechanism of poFcγRIIb in mediating the ADE of PRRSV infection has not been thoroughly elucidated to date. In this study, we investigated the effect of cytoplasmic and ITIM regions on poFcγRIIb mediated PRRSV infection in the presence of antibodies. Our results indicated that infection with PRRSV-antibody complexes could activate ITIM-associated signal transduction and inhibit IFN-β production, resulting in the enhancement of PRRSV infection. Our findings help to elucidate the molecular mechanisms underlying the ADE of virus infection.

Section snippets

Cell lines

MARC-145 cells, derived from the fetal green monkey fibroblast line MA-104 [12], 10% fetal bovine serum (FBS, Hyclone) and penicillin streptomycin (100 U/ml penicillin and 100 mg/ml streptomycin) were added to Dulbecco's modified Eagle medium (DMEM, Gibco). The porcine alveolar macrophages (PAMs) were collected from 4- to 6-week-old piglets free of PRRSV by lung lavage, as previously described [9]. PAM was dispensed into 24-well plates at a rate of 1 × 105 cells/well and stored in RPMI-1640

Preparation of stable MARC-145 cell lines expressing poFcγRIIb receptor containing mutations in the signaling domains

It has been reported that poFcγRIIb mediates ADE in PRRSV infection when a stable MARC-145 cell line expressing poFcγRIIb (MARC-poFcγRIIb) is used [8]. Previous studies demonstrated that the cytoplasmic regions and ITIM are essential for the FcγRIIb mediated inhibitory function [[15], [16], [17], [18]]. Thus, to investigate cytoplasmic signal transduction in poFcγRIIb mediated ADE, we introduced a series of point and deletion mutations in the cytoplasmic domain of poFcγRIIb and constructed a

Discussion

Our previous results have demonstrated that poFcγRIIb plays a crucial role in mediating the ADE of PRRSV infection [8,20]. However, the intracellular molecular mechanism of the ADE of PRRSV infection mediated by poFcγRIIb has not been thoroughly elucidated to date. In the present study, we demonstrated that cytoplasmic signaling transduction of poFcγRIIb is necessary for ADE of PRRSV infection. Considering our previous research [8,20], we proposed a model of the signaling pathway in ADE of

Declaration of Competing Interest

There have no conflicts of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31502041; 31490601), the National Key Technology Support Program of China (Grant No. 2015BAD12B02) and the Key Science and Technology Fund of Henan Province of China (NO. 182102310120).

References (33)

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    Citation Excerpt :

    PoFcγRIIb mediated ADE pathway of PRRSV infection through recruitment of ship-1. The TBK-1-IRF-3-IFN-β signal transduction pathway was further inhibited to enhance PRRSV infection (Wan et al., 2019). In addition, the ability of poFcγRII to mediate PRRSV ADE can be eliminated by interrupting the tyrosine residues in ITIM or removing the cytoplasmic domain of Fc.

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1

These authors contributed equally to this work.

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