Host serine protease ACOT2 assists DENV proliferation by hydrolyzing viral polyproteins

Abstract

Dengue fever is a mosquito-borne tropical disease caused by the dengue virus (DENV). The replication of DENV relies on the processing of its genome-encoded polyprotein by both viral protease NS3 (NS3^pro) and host proteases. However, the impact of host proteases on DENV proliferation is not well understood. In this study, we utilized fluorophosphonate-based probes (FPs) to investigate the up-regulation of host serine proteases during DENV infection in detail. Among the identified proteases, acyl-CoA thioesterase 2 (ACOT2), an enzyme that hydrolyzes acyl-CoA molecules to generate fatty acids and free CoA, exhibited cleavage activity against DENV polypeptide substrates. Enzymatic assays and virological experiments confirmed that ACOT2 contributes to DENV propagation during the replication stage by cleaving the viral polyprotein. Docking models provided insights into the binding pocket of viral polypeptides and the catalytic mechanism of ACOT2. Notably, this study is the first to demonstrate that ACOT2 functions as a serine protease to hydrolyze protein substrates. These findings offer novel insights into DENV infection, host response, as well as the potential development of innovative antiviral strategies.IMPORTANCEDENV, one of the major pathogens of Dengue fever, remains a significant public health concern in tropical and subtropical regions worldwide. How DENV efficiently hijacks the host and accesses its life cycle with delicate interaction remains to be elucidated. Here, we deconvoluted that the host protease ACOT2 assists the DENV replication and characterized the ACOT2 as a serine protease involved in the hydrolysis of the DENV polypeptide substrate. Our results not only further the understanding of the DENV life cycle but also provide a possibility for the usage of activity-based proteomics to reveal host-virus interactions.

Keywords: ABPP; ACOT2; DENV; serine protease; virus replication.

Fig 1

Selection of activity-based serine ABP probe. (A) Scheme of comparative ABPP to identify proteins. (B) Structure of DENV viral protein NS2B/NS3^pro and its catalytic triad His51-Asp75-Ser135. (C) Inhibitors of NS2B/NS3^pro and fluorophosphonate probe.

Fig 2

Target identification by comparative ABPP. (A) Targets identified in native A549 cells (A549-1^st, A549-2^nd, and A549-3^rd represent the A549 cell lysates assay from three biologically independent experiments). (B) Targets identified in DENV-infected A549 cells (DENV-1^st, DENV-2^nd, and DENV-3^rd represent DENV-infected A549 cell lysates assay from three biologically independent experiments). (C) Overlap of the proteins identified in A549 cells and DENV-infected A549 cells. (D) Representative ratio plot for up-regulated proteins identified by FP from the active proteome infected versus mock. Red points indicate the up-regulated host proteases with serine residues. (E) Biological processes analysis of up-regulated proteins identified by FP. (F) Cellular component analysis of up-regulated proteins. (G) GO analysis of molecular functions. (H) KEGG analysis of target proteins. ER, endoplasmic reticulum.

Fig 3

The hydrolytic activities of target proteins on the substrate of DENV NS2B/NS3^pro. (A) The structure of fluorophore peptide substrate for NS2B/NS3^pro. (B) SDS-PAGE analysis of the expression and purification for up-regulate host protease. (C) The fluorophore substrate cleavage activities of host protease. Different proteases were diluted into an assay buffer containing the peptide substrate. Only substrate reaction without protease served as a negative control. Assays were conducted in black 96-well plates.

Fig 4

ACOT2 functions at the DENV replication stage. (A) The luciferase activity of DENV on different host protease knockdown cell lines, Y-axis represents the percentage of luciferase activity of different knockdown cell lines relative to the A549 cell line (sh-NC) after infection with DENV. (B) Western blot detected the expression of endogenous ACOT2 and production of progeny virus in sh-NC and sh-ACOT2 A549 cell line (sh-ACOT2) with infected DENV. Total cell lysates were immunoblotted with anti-ACOT2, anti-DENV Envelope, and anti-GADPH antibodies (loading control). (C) The growth curve of DENV in sh-NC and sh-ACOT2. Detected the luciferase activity at 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 60 h after DENV-infected, and infection was expressed as a percentage relative to that sh-NC. Graphpad Prim was utilized to plot the growth curves and calculate the statistical significance. (D) Detected the expression of ACOT2 in sh-NC, sh-ACOT2, and sh-ACOT2 which overexpressed ACOT2^WT, ACOT2^res, and ACOT2^S294A-res, respectively. (E) qRT-PCR examination of the expressions of ACOT2^WT, ACOT2^res, and ACOT2^S294A-res. DENV replicon (DENV) and DENV mutant replicon (DENV-NS3^mut)-infected sh-NC, sh-ACOT2, and sh-ACOT2 which overexpressed ACOT2^WT, ACOT2^res, and ACOT2^S294A-res, respectively. qRT-PCR then examined ACOT2 in each experimental group, and the expression was shown as a percentage relative to that sh-NC. (F) ACOT2 rescued DENV replication. RNA levels of DENV in different experimental groups were detected by qRT-PCR, and DENV infection was then expressed by a percentage relative to that sh-NC. Graphs show mean ± SEM (n = 3 biologically independent experiments). *P < 0.05; ***P < 0.005; ****P < 0.001.

Fig 5

ACOT2 hydrolyzed DENV viral polyproteins. (A) ACOT2 cleavaged the polypeptide substrates of NS2B/NS3^pro. ACOT2 was diluted into a test buffer containing the different polypeptide substrates. Assays were tested in black 96-well plates. (B) The structure of recombinant model proteins (GST: aqua; EGFP: green; Polypeptide substrate sequence of NS2B/NS3^pro (SIT): rose color; His-tag: blue). (C) The hydrolytic activity of ACOT2 on model polyproteins. The reaction products were detected by His-tag which assay western blot. The names and positions of detected SIT and proteins are shown at the up and left, respectively.

Fig 6

Binding mode between peptides and ACOT2. (A) The structure of ACOT2 is shown as a b-factor to indicate that the catalytic center is stable in the structure. (B) HPEPDOCK server prediction for five peptide docking results where the receptor is represented in cartoon structure. (C) The electrostatic surface potential of the binding pocket. (D) The catalytic centers of ACOT2 are shown in the center of the image. The ACOT2 catalytic triad is displayed as sticks and the protease is displayed as cartoon. (E through I) The binding poses of the five peptides in the binding pocket, the peptides are shown as sticks of different colors. (J) The cleavage activity of ACOT2^WT; ACOT2^Y235A and ACOT2^H425A on DENV polypeptides.

Fig 7

Structural basis of substrate catalysis. (A-C) Substrate active site and binding site. (D-E) ESP-mapped molecular van der Waals surface of the catalytic triad. (F) ESP before and after S294 deprotonation. (G) Schematic diagram of the mechanism of ACOT2-catalyzed peptide hydrolysis.