USP38 Inhibits Zika Virus Infection by Removing Envelope Protein Ubiquitination

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus, and its infection may cause severe neurodegenerative diseases. The outbreak of ZIKV in 2015 in South America has caused severe human congenital and neurologic disorders. Thus, it is vitally important to determine the inner mechanism of ZIKV infection. Here, our data suggested that the ubiquitin-specific peptidase 38 (USP38) played an important role in host resistance to ZIKV infection, during which ZIKV infection did not affect USP38 expression. Mechanistically, USP38 bound to the ZIKV envelope (E) protein through its C-terminal domain and attenuated its K48-linked and K63-linked polyubiquitination, thereby repressed the infection of ZIKV. In addition, we found that the deubiquitinase activity of USP38 was essential to inhibit ZIKV infection, and the mutant that lacked the deubiquitinase activity of USP38 lost the ability to inhibit infection. In conclusion, we found a novel host protein USP38 against ZIKV infection, and this may represent a potential therapeutic target for the treatment and prevention of ZIKV infection.

Keywords: USP38; Zika virus; deubiquitinase; envelope protein; virus infection.

Figure 1

USP38 inhibits ZIKV infection. (A–D) Hela cells were transfected with HA-USP38 for 24 h then infected with ZIKV (MOI = 1) for 48 h. The levels of ZIKV proteins were detected by immunoblotting (A). E/GAPDH and NS5/GAPDH ratio was measured (B). The viral RNA content was quantified by qPCR (C) and confocal microscopy (D), bar = 10 µm. (E) Hela cells stably expressing sh-USP38 or control sh-RNA, and the USP38/GAPDH ratio was measured. (F–H) Hela cells stably expressing sh-USP38 or control sh-RNA were infected with ZIKV (MOI = 1) for 48 h. The expression level of ZIKV proteins were detected by immunoblotting (F). The ratios of E/GAPDH (G, left) and NS5/GAPDH (G, right) were measured. The viral RNA content was quantified by qPCR (H). (I) A549 cells stably expressing sh-USP38 or control sh-RNA, and the viral titers was measured by TCID₅₀. (J) Cells were measured for cell viability by CCK-8 assay, and the unit of the y axis is the readout optical density (OD) value. (K) Hela cells stably expressing sh-USP38 or control sh-RNA were incubated with ZIKV for 2 h, then continue to be cultured with serum-free medium for 48 h. The viral RNA content was quantified by qPCR. *: p < 0.05, **: p < 0.01, and ***: p < 0.001.

Figure 2

USP38 binds to E protein through its C-terminal domain. (A) HEK293T cells were co-transfected with HA-USP38 and Flag-E, Flag-C, Flag-prM, cell lysates were subjected to IP using anti-flag antibody and analyzed by immunoblotting. (B) HEK293T cells were co-transfected with HA-USP38 and Flag-E. Cell lysates were subjected to IP using control IgG, anti-HA, or anti-Flag antibody. (C) A549 cells was infected with ZIKV for 48 h. Cell lysates were subjected to IP using control IgG or anti-USP38. (D) Hela cells were transfected with HA-USP38 or Flag-E, or co-transfected with HA-USP38 and Flag-E. The sub-cellular localizations of HA-USP38 (green), Flag-E (red), and nucleus marker DAPI (blue) were analyzed with confocal microscopy. Bar = 5 µm. (E) HEK293T cells were co-transfected with HA-E and Flag-USP38, Flag-N-terminal-USP38 or Flag-C-terminal-USP38. Cell lysates were subjected to IP using anti-Flag antibody and analyzed by immunoblotting. (F) Schematic diagram of the full-length E protein and truncated E proteins: E (1–193aa), E (1–296aa), E (1–406aa), E (52–505aa), E (132–505aa) E (280–505aa). (G,H) HEK293T cells were co-transfected with HA-USP38 and Flag-E, Flag-E (1–193aa), Flag-E (1–296aa), and Flag-E (1–406aa) (G) or Flag-E, Flag-E (52–505aa), Flag-E (132–505aa), and Flag-E (280–505aa) (H). Cell lysates were subjected to IP using anti-Flag antibody and analyzed by immunoblotting (G,H). (I) HEK293T cells were co-transfected with HA-USP38 and Flag-E (1–193aa), Flag-E (1–132aa), or Flag-E (53–193aa). Cell lysates were subjected to IP using anti-Flag antibody and analyzed by immunoblotting.

Figure 3

USP38 removes both k48-linked and K63-linked polyubiquitination of E protein. (A) HEK293T cells were co-transfected with Flag-E, HA-USP38, and Myc-UB. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-Myc. (B) A549 cells were transfected with HA-UB, Flag-Vector or Flag-USP38 for 24 h then infected with ZIKV (1 MOI). Cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-E. (C) HEK293T cells were co-transfected with Flag-E, Myc-UB, Myc-k48, Myc-k63 together with HA-USP38. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-Myc. (D) HEK293T cells were co-transfected with Flag-E, Myc-UB, MycK48R, Myc-k63R together with HA-USP38. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-Myc. (E) A549 cells stably expressing sh-USP38 or control sh-RNA were transfected with HA-UB for 24h then infected with ZIKV 48h. Cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-E.

Figure 4

Deubiquitinase activity of USP38 is required for restriction of ZIKV infection. (A) HEK293T cells were co-transfected with Flag-E and Myc-UB, together with HA-USP38 or HA-USP38 (C454A/H857A/D918N). Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-Myc. (B–E) Hela cells were transfected with HA-USP38, HA-Vector or HA-USP38 (C454A/H857A/D918N) for 24 h then infected with ZIKV (MOI = 1) for 48 h. The expression level of ZIKV proteins were detected by immunoblotting (B). The ratios of E/GAPDH and NS5/GAPDH ratio were measured (C). The viral RNA content was quantified by qPCR (D) and confocal microscopy (E), bar = 10 µm.