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. 2015 Oct;89(20):10399-406.
doi: 10.1128/JVI.01736-15. Epub 2015 Aug 5.

Suppressor of Cytokine Signaling 3 Is an Inducible Host Factor That Regulates Virus Egress during Ebola Virus Infection

Atsushi Okumura  1 , Angela L Rasmussen  2 , Peter Halfmann  3 , Friederike Feldmann  4 , Akihiko Yoshimura  5 , Heinz Feldmann  4 , Yoshihiro Kawaoka  6 , Ronald N Harty  7 , Michael G Katze  8
Affiliations

Suppressor of Cytokine Signaling 3 Is an Inducible Host Factor That Regulates Virus Egress during Ebola Virus Infection

Atsushi Okumura et al. J Virol. 2015 Oct.

Abstract

Ebola virus (EBOV) initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal EBOV infections. The VP40 matrix protein is a key viral structural protein that is critical for virion egress. Physical and functional interactions between VP40 and host proteins such as Tsg101 and Nedd4 facilitate efficient release of VP40-driven virus-like particles (VLPs) and infectious virus. Here, we show that host suppressor of cytokine signaling 3 (SOCS3) can also bind to EBOV VP40, leading to enhanced ubiquitinylation and egress of VP40. Indeed, titers of infectious EBOV derived from SOCS3 knockout mouse embryonic fibroblasts (MEFs) were significantly reduced compared to those from wild-type (WT) MEFs at 24 and 48 h postinfection. Importantly, this reduced virus yield could be rescued back to WT levels by exogenously expressing SOCS3. Lastly, we show that SOCS3 expression is induced by EBOV glycoprotein (GP) expression and that VLPs containing EBOV VP40 and GP induced production of proinflammatory cytokines, which induced SOCS3 for negative-feedback regulation. These data indicate that host innate immune protein SOCS3 may play an important role in budding and pathogenesis of EBOV.

Importance: The VP40 matrix protein is a key structural protein critical for Ebola virus budding. Physical and functional interactions between VP40 and host proteins such as Tsg101 and Nedd4 facilitate efficient release of VLPs and infectious virus. We reported that host TLR4 is a sensor for Ebola GP on VLPs and that the resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines. Host SOCS3 regulates the innate immune response by controlling and limiting the proinflammatory response through negative-feedback inhibition of cytokine receptors. We present evidence that Ebola virus VLPs stimulate induction of SOCS3 as well as proinflammatory cytokines, and that expression of human SOCS3 enhances budding of Ebola VLPs and infectious virus via a mechanism linked to the host ubiquitinylation machinery.

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Figures

FIG 1
FIG 1
Induction of SOCS3 mRNA by VLPs requires EBOV GP. (A) THP-1 cells were either untreated (control), or treated with LPS (1.0 μg/ml), VP40 VLPs (10 μg/ml), VP40+GP VLPs (10 μg/ml), or VP40+GPΔmucin VLPs for 6 or 12 h. Total RNA was isolated, and RT-PCR was used to detect SOCS3 or actin (control) mRNAs. (B) THP-1 monocytic cells were infected with EBOV at an MOI of 3. At 0, 1, 3, 6, and 12 h postinfection, RT-PCR was used to detect SOCS3, TNF-α, and actin mRNAs. The data are representative of three independent experiments.
FIG 2
FIG 2
EBOV VP40+GP VLPs induce SOCS3 mRNA mediated by TLR4-MD2. HEK293-TLR4-MD2 and HEK293 cells were either untreated (cont.) or treated with LPS (1.0 μg/ml), VP40 VLPs (10 μg/ml), or VP40+GP VLPs (10 μg/ml) for 6 or 12 h as indicated. Total RNA was isolated, and RT-PCR was used to detect SOCS3, TLR4, and actin. The data are representative of three independent experiments.
FIG 3
FIG 3
EBOV VP40 VLP budding is enhanced by SOCS3 in a dose-dependent manner. Human HEK293T cells were mock transfected or transfected with the indicated plasmids. The total amount of plasmid DNA transfected was held equivalent in all samples by using empty pCAGGS vector. VLPs and cell extracts were harvested at 24 h posttransfection, and proteins were analyzed by Western blotting with the indicated antisera. The data are representative of five independent experiments.
FIG 4
FIG 4
Expression of SOCS3 enhances ubiquitinylation of VP40. Schematic diagram of SOCS3-WT and mutant ΔC41. Both SOCS3-WT and mutant ΔC41 are tagged at their C termini with a c-myc epitope tag. The SOCS box is located at the C terminus between amino acids 184 and 225. Human HEK293T cells were transfected with the indicated plasmids. Cell extracts were analyzed by IP/Western blotting or Western blotting as indicated. This is a single gel from which irrelevant lanes were removed. The data are representative of five independent experiments.
FIG 5
FIG 5
SOCS3 is required for efficient production of VLPs and live virus. (A) C57BL/6J MEFs (B6 WT MEFs) or SOCS3 KO MEFs were mock transfected or transfected with EBOV VP40, and cells and VLPs were harvested at 24 h posttransfection. Cell lysates and VLPs were analyzed by Western blotting with anti-VP40 antiserum. RNA was isolated from C57BL/6J MEFs or SOCS3 KO MEFs and analyzed by RT-PCR. The data are representative of three independent experiments. (B) WT MEFs or SOCS3 KO MEFs were infected with EBOV at an MOI of 0.1. Supernatants were collected on days 1, 2, 3, and 4 postinfection, and infectious virus was titrated by focus-forming assay. Two independent experiments were performed in triplicate. (C) WT or SOCS3 KO MEFs were infected with EBOV at an MOI of 0.1, and total cellular RNA was harvested at 24 and 48 h. postinfection and subjected to qrRT-PCR to quantify virus infection. The bars represent the averages of two independent experiments, both performed in triplicate.
FIG 6
FIG 6
SOCS3 overexpression rescues EBOV production in SOCS3 KO MEF cells. (A) SOCS3 KO MEFs stably overexpressing a WT SOCS3 in trans (SOCS-3 WT trans) were established by blasticidin selection. SOCS3 expression was verified by RT-PCR. The data are representative of two independent experiments. (B) SOCS-3 KO MEFs stably overexpressing WT SOCS3 (SOCS-3 WT trans), SOCS3 KO MEFs, and WT MEFs were infected with EBOV at an MOI of 0.1. Supernatants were collected at 24, 48, 76, and 92 h postinfection, and virus was titrated by a focus-forming assay. The data are representative of three independent experiments.
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