Severe fever with thrombocytopenia syndrome virus (SFTSV)-host interactome screen identifies viral nucleoprotein-associated host factors as potential antiviral targets

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus that causes severe infection in humans characterized by an acute febrile illness with thrombocytopenia and hemorrhagic complications, and a mortality rate of up to 30%. Understanding on virus-host protein interactions may facilitate the identification of druggable antiviral targets. Herein, we utilized liquid chromatography-tandem mass spectrometry to characterize the SFTSV interactome in human embryonic kidney-derived permanent culture (HEK-293T) cells. We identified 445 host proteins that co-precipitated with the viral glycoprotein N, glycoprotein C, nucleoprotein, or nonstructural protein. A network of SFTSV-host protein interactions based on reduced viral fitness affected upon host factor down-regulation was then generated. Screening of the DrugBank database revealed numerous drug compounds that inhibited the prioritized host factors in this SFTSV interactome. Among these drug compounds, the clinically approved artenimol (an antimalarial) and omacetaxine mepesuccinate (a cephalotaxine) were found to exhibit anti-SFTSV activity in vitro. The higher selectivity of artenimol (71.83) than omacetaxine mepesuccinate (8.00) highlights artenimol's potential for further antiviral development. Mechanistic evaluation showed that artenimol interfered with the interaction between the SFTSV nucleoprotein and the host glucose-6-phosphate isomerase (GPI), and that omacetaxine mepesuccinate interfered with the interaction between the viral nucleoprotein with the host ribosomal protein L3 (RPL3). In summary, the novel interactomic data in this study revealed the virus-host protein interactions in SFTSV infection and facilitated the discovery of potential anti-SFTSV treatments.

Keywords: Artenimol; Bunyavirales; Interactome; Omacetaxine mepesuccinate; Pathogenesis; SFTSV; Treatment.

Graphical abstract

Fig. 1

Flowchart of the study. (A) Pulldown of viral proteins and associated proteins, followed by characterization by LC-MS/MS. (B) Functional validation of the enriched protein candidates. Corresponding siRNAs were transfected into Huh7 cells, resulting in RNAi-mediated silencing of the individual target genes. SFTSV infection was performed after gene-silencing, followed by virus titration by qRT-PCR. (C) Identification of the functional role of the selected host factors in SFTSV replication cycle. (D) Antiviral evaluation of the selected drug compounds targeting the SFTSV-host protein–protein interactions. Abbreviations: Co-IP, co-immunoprecipitation; Gc, glycoprotein C; Gn, glycoprotein N; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NP, nucleoprotein; NSs, non-structural protein.

Fig. 2

Identification of SFTSV protein-bound host factors. (A) Organization of the SFTSV genome. The tripartite segmented genome is comprised of three segments: Large (L), Medium (M), and Small (S). The L segment encodes the RNA-dependent RNA polymerase. The M segment encodes two glycoproteins Gn and Gc. The S segment encodes the viral nucleoprotein (NP) and non-structural protein (NSs), which is encoded in the opposite orientation to the viral NP. (B) Isolation and analysis of the host-bound viral protein complexes. Briefly, the SFTSV open reading frame plasmids which contains HA-tag were transfected into HEK-293T cells. After incubation for 48 h, the cell lysates were collected and pulled down using HA-tag beads, followed by protein ID by LC-MS/MS. (C) The Western blotting images of each viral protein after pull-down and detection by anti-HA tag antibody.

Fig. 3

SFTSV-host protein–protein interaction network illustrating each SFTSV protein and its associated host cellular factors. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Gene Ontology enrichment analysis was performed on the identified host cellular factors based on each SFTSV protein. Abbreviations: BP, biological process; CC, cellular components; KEGG, Kyoto encyclopedia of genes and genomes; MF: molecular function.

Fig. 5

Host small interfering RNA (siRNA) screens to identify host factors that affect SFTSV replication. (A) Upper panel: experimental design of the siRNA library screening. Briefly, Huh7 cells were transfected once daily (60 nM) for two times before SFTSV infection (MOI = 0.01). After another 48 h, cell culture supernatant was harvested for viral load determination. Lower panel: heat map showing SFTSV viral load reduction after individual siRNA knockdown. The results were normalized by the infected cells that were transfected with scrambled siRNA. (B) A list of the host factors whose knockdown reduced SFTSV replication.

Fig. 6

Primary screening of available drug compounds targeting the identified host factors. The heat map of SFTSV viral load after drug compound treatment was shown. Huh-7 cells were infected by SFTSV (MOI = 0.10) and treated by different concentrations of each drug compound as indicated. The viral load in the cell lysate was measured at 48 hpi and normalized by GAPDH.

Fig. 7

Characterization of the selected SFTSV inhibitors. (A) Dose-response analyses of artenimol and omacetaxine mepesuccinate are shown, depicting both the antiviral activity (red) and cytotoxicity (black). The grey line indicates 50% of the mock-treated control. The EC₅₀, CC₅₀, and chemical structure of each drug compound are shown. (B) Co-immunoprecipitation assay was conducted in HEK-293T cell transfected with SFTSV nucleoprotein together with either GPI or RPL3 plasmids. After pull-down, the viral nucleoprotein was detected by anti-HA antibody, while GPI and RPL3 were detected by anti-Myc antibodies. (C) Co-immunoprecipitation assay with the treatment of artenimol or omacetaxine mepesuccinate at the indicated concentrations. (D) Molecular docking analysis predicted the interface between artenimol and GPI. The protein is shown as grey ribbons and the drug compound as color sticks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)