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. 2021 Mar 5;13(3):409.
doi: 10.3390/v13030409.

Caprine MAVS Is a RIG-I Interacting Type I Interferon Inducer Downregulated by Peste des Petits Ruminants Virus Infection

Qiuhong Miao  1   2 , Ruibing Qi  1 , Chunchun Meng  1 , Jie Zhu  1 , Aoxing Tang  1 , Dandan Dong  1 , Hongyuan Guo  1 , Monique M van Oers  2 , Gorben P Pijlman  2 , Guangqing Liu  1
Affiliations

Caprine MAVS Is a RIG-I Interacting Type I Interferon Inducer Downregulated by Peste des Petits Ruminants Virus Infection

Qiuhong Miao et al. Viruses. .

Abstract

The mitochondrial antiviral-signaling protein (MAVS, also known as VISA, IPS-1, or CARDIF) plays an essential role in the type I interferon (IFN) response and in retinoic acid-inducible gene I (RIG-I) mediated antiviral innate immunity in mammals. In this study, the caprine MAVS gene (caMAVS, 1566 bp) was identified and cloned. The caMAVS shares the highest amino acid similarity (98.1%) with the predicted sheep MAVS. Confocal microscopy analysis of partial deletion mutants of caMAVS revealed that the transmembrane and the so-called Non-Characterized domains are indispensable for intracellular localization to mitochondria. Overexpression of caMAVS in caprine endometrial epithelial cells up-regulated the mRNA levels of caprine interferon-stimulated genes. We concluded that caprine MAVS mediates the activation of the type I IFN pathway. We further demonstrated that both the CARD-like domain and the transmembrane domain of caMAVS were essential for the activation of the IFN-β promotor. The interaction between caMAVS and caprine RIG-I and the vital role of the CARD and NC domain in this interaction was demonstrated by co-immunoprecipitation. Upon infection with the Peste des Petits Ruminants Virus (PPRV, genus Morbillivirus), the level of MAVS was greatly reduced. This reduction was prevented by the addition of the proteasome inhibitor MG132. Moreover, we found that viral protein V could interact and colocalize with MAVS. Together, we identified caMAVS as a RIG-I interactive protein involved in the activation of type I IFN pathways in caprine cells and as a target for PPRV immune evasion.

Keywords: Peste des Petits Ruminants Virus (PPRV); caprine; innate immunity; mitochondrial antiviral signaling protein (MAVS).

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Conflict of interest statement

The authors declare that there are no conflict of interest.

Figures

Figure 1
Figure 1
Identification of caprine MAVS. (A) Multiple sequences of MAVS from different species were selected to construct the phylogenetic tree. The tree was constructed by using the neighbour-joining method by MEGA5.0 and the scale bar is 2. The amino acid sequences of MAVS used in this program were listed in Table S2. The asterisk indicates caprince MAVS (B) Amino acid alignments of caprine, sheep, human, mouse and rabbit MAVS. Sequence alignments were performed and edited with the Lasergene MegAlign program. Structural caspase activation and recruitment domain (CARD), proline-rich region (PRR), non-characterized (NC) and C-terminal transmembrane (TM) domains are indicated in blue, yellow, purple, and green, respectively.
Figure 2
Figure 2
Schematic diagram of caprine MAVS mutants and their expression. (A) Schematic representation of caprine MAVS was presented based on the conserved points according to human MAVS. The CARD, PRR, NC and TM domains are represented in blue, yellow, purple, and green, respectively. The schematic representation is also used for constructing truncated mutants. The indicated numbers represent amino acid positions. (B) Western blot analysis of the expression of caMAVS and its mutants by transfection with caMAVS and mutant plasmids in HEK-293T cells.
Figure 3
Figure 3
Localization study of caMAVS and its mutants. (A) Expression plasmids encoding caMAVS and its different deletion mutants were transfected into Vero-SLAM cells. At 36 h post transfection, cells were fixed and stained with Myc primary antibodies and secondary Alexa-488 labeled antibodies. (B) To verify the colocalization of caMAVS (green) and Mitochondria (red), expression plasmids encoding Myc tagged caMAVS and its mutants were transfected together with pDsRed2-Mit. At 36 hpt, cells were fixed and stained as in panel A.
Figure 4
Figure 4
Overexpression of caMAVS induced IFN-β via the NF-κB and IRF-3-mediated pathways. (A) HEK-293T cells were transfected with increasing amount of caMAVS, IFN-β-Luc together with endogenous control pRL-TK Plasmid (40 ng/well). (B–D) HEK-293T cells were transfected with caMAVS or its mutants (500 ng) along with PRDII-Luc (NK-κB-luc), PRDI/III-luc (IRF3-luc)) or IFNβ-Luc together endogenous control pRL-TK Plasmid (40 ng/well). Human MAVS was used as a positive control. At 36 hpt, the HEK-293T cells were lysed, and Rluc and Fluc activities were evaluated using the Promega Dual-Luciferase Reporter Assay System. Furthermore, all the experiments were performed at least three times to ensure the results consistency (The data represent the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis; * p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 5
Figure 5
Evaluation of ISGs by overexpression of caMAVS and its mutants in EECs. (A) EECs were transfected with either empty vector or caMAVS. The relative mRNA levels of selected caprine ISGs (IFITM3, OASL, RASD2, MX1) were analyzed by qRT-PCR. (B–E) EECs were transfected with either empty vector or caMAVS and its mutants. The relative mRNA levels of selected ISGs were analyzed by qRT-PCR. Data presented were from at least three independent experiments. Significance was analyzed by GraphPad prism 5.0 software with a one-way ANOVA test (One-way ANOVA was used for statistical analysis; * p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 6
Figure 6
Identification of the interaction between caMAVS and caRIG-I by Co-IP. (A) HEK-293T cells seeded in 10 cm dishes and were transfected with HA-tagged MAVS or HA-GFP together with (without) Myc-RIG-I when cells were at around 60% confluency, respectively. Cells were harvested after 24 h post transfection. Cell lysates were precipitated with anti-Myc mAb resin overnight at 4 °C and precipitated proteins detected with anti-HA and anti-Myc mAbs. β-actin was used as protein loading control. (B) HEK-293T cells were seeded in 10 cm dishes and were transfected with HA-tagged MAVS and its mutants together with Myc-RIG-I when cells were around 60% confluency. Cells were harvested after 24 hpt and cell lysates were precipitated with anti-HA mAb resin overnight at 4 °C and precipitated proteins were detected with anti-HA and anti-Myc mAb. β-actin was used as protein loading control.
Figure 7
Figure 7
caMAVS is degraded through the proteasome pathway during PPRV infection. (A) EECs were infected with the PPRV vaccine strain, Nigeria/75/1, at MOI 10. The relative protein level of several important molecules during viral infection was checked by WB. (B) Several inhibitors were used after infection of PPRV on EECs to demonstrate which pathway is involved in MAVS degradation. The relative level of individual proteins was quantified with respect to β-actin.
Figure 8
Figure 8
caMAVS interacts and colocalizes with PPRV viral protein V. (A) Increasing amounts of plasmids encoding Flag-tagged PPEV protein V (Flag-V) (0.5 ug, 1 ug, 1.5 ug, 2 ug) were co-transfected with plasmid-encoding Myc-tagged caMAVS (2 ug) in 6-well plates. PPRV V and caMAVS were detected by Western blot using β-actin as loading control. (B) Co-IP was performed to identify interaction between PPRV V and caMAVS. Flag-V and Myc-caMAVS plasmids were transfected into HEK-293T cells independently or together. Cell lysates were subjected to immunoprecipitation with anti-Myc or anti-Flag mAb overnight at 4 °C. The expression of the transfected proteins was determined by Western blotting by anti-Flag and anti-Myc mAbs, respectively. (C) The colocalization study was performed by transfection of Myc-caMAVS (red), Flag-V (green) together with pDsRed2-Mito (pink) and subjected for IFA with anti-Flag and anti-Myc mAbs as primary antibodies.
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