Table 1.
Sequences of primers for qRT-PCR used in the study.
Fig 1.
NS4A protein occurred later than NS2B, NS3 and NS5 during ZIKV infection.
(a-b) Replication of ZIKV genome in microglial cells (HMC3) and astrocytic cells (U251). (c-e) Expression of ZIKV proteins in HMC3, U251 and Vero cells. (f-h) Gray scale analysis of Fig 1C (f), Fig 1D (g) and Fig 1E (h). After ZIKV/SZ01 (0.1 MOI) infection of HMC3, U251 or Vero cells for 0, 6, 18, 30, 42, and 54 h, the viral RNA or viral proteins in cell lysates was determined by qRT-PCR or western blot analysis. Data are means ± SEM of triplicate experiments. E, envelope protein; NS2B, non-structural 2B protein; NS3, non-structural 3 protein; NS4A, non-structural 4A protein; NS5, non-structural 5 protein.
Fig 2.
NS2A and NS4A were reversely associated with cell death induced by ZIKV infection.
(a-b) Cell death after ZIKV/SZ01 infection of HMC3 or U251 cells expressing ZIKV NS proteins. (c-d) Cytopathic effect after ZIKV/SZ01 infection of HMC3 (72 h p.i.) or U251 (48 h p.i.) cells expressing ZIKV NS proteins. Scale bar, 50 μm. HMC3 or U251 cells (expressing ZIKV NS1, NS2A, NS2B, NS3, NS4A, or NS4B) were infected by 0.1 MOI of ZIKV/SZ01. Cell death was determined by flow cytometry and Annexin V/PI apoptosis kit at 48 (U251) or 72 (HMC3) h p.i. The cytopathic effect was observed under a light microscope. The differences between groups of control and NS proteins were evaluated by two-tailed Student’s t test. Data are means ± SEM of triplicate experiments; *, P< 0.05; **, P< 0.01; ***, p < 0.001.
Fig 3.
NS2A and NS4A inhibited ZIKV infection.
(a-b & e) NS2A and NS4A reduced the production of infectious viral particles in HMC3 or U251 cells (48 h p.i.). (c) NS2A and NS4A inhibited the expression of ZIKV E protein in HMC3 cells. (d) NS2A and NS4A inhibited viral RNA replication in HMC3 cells. (f) NS2A and NS4A inhibited the replication of viral genome in U251 cells. HMC3 or U251 cells (expressing ZIKV NS1, NS2A, NS2B, NS3, NS4A, or NS4B) were infected by 0.1 MOI of ZIKV/SZ01 for 48 or 72 h. ZIKV titers in the culture supernatants were determined by viral plaque formation unit assay (Fig 3A, 3B and 3E) and the expression of ZIKV envelope (E) at 48 h p.i. (Fig 3C) was examined by western blot analysis. Fig 3B was the quantitative analysis of results shown in Fig 3A. ZIKV E was stained by an anti-E rabbit antibody. HMC3 or U251 cells (expressing ZIKV NS2A or NS4A) were infected by 0.1 MOI of ZIKV/SZ01 and total RNA was prepared at various time points p.i. for measuring viral RNA copies in the culture supernatants (HMC3, Fig 3D) or viral genome in cell lysates (U251, Fig 3F), determined by TaqMan or SYBR Green qRT-PCR. The differences between groups of control and NS proteins were evaluated by two-tailed Student’s t test. Data are means ± SEM of triplicate experiments; *, P< 0.05; **, P< 0.01; ***, p < 0.001.
Fig 4.
NS2A and NS4A inhibited ZIKV RNA replication.
(a) Inhibition of ZIKV attachment to cells by NS2A and NS4A. HMC3 cells expressing NS2A or NS4A were incubated with 2 MOI of ZIKV at 4°C for 1 h, followed by washes extensively with PBS. Viral attachment was assessed by qRT-PCR. (b) Inhibition of ZIKV entry into cells by NS2A and NS4A. HMC3 cells expressing NS2A or NS4A were incubated with 2 MOI of ZIKV at 4°C for 1 h, followed by incubation at 37°C for another 10 min. Viral entry into cells was determined by qRT-PCR. (c & f) Inhibition of ZIKV dsRNA production by NS2A and NS4A. DsRNA synthesis was analyzed by IFA in HMC3 cells (expressing ZIKV NS2A or NS4A) infected with 0.1 MOI of ZIKV for 24 or 48 h (Fig 4F). DsRNA was probed by the J2 mouse monoclonal anti-dsRNA antibody (green) with cell nuclei stained by 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 50 μm. Four fields of each group were randomly selected for statistical analysis by Image J (Fig 4C). (d) Inhibition of ZIKV RNA replication by NS2A and NS4A. (e) Inhibition of ZIKV NS2B expression by NS2A and NS4A. HMC3 cells expressing NS2A or NS4A were transfected with RNA from ZIKV replicons. At 48 or 96 h post transfection, total RNA was prepared from the cells and ZIKV RNA copies were measured by qRT-PCR (Fig 4D). Lysates were collected to determine the expression of NS2B by western blot assay at 24, 48, 72 or 96 h.p.i. (Fig 4E). The differences between groups of controls and NS proteins were evaluated by two-tailed Student’s t test. Data are means ± SEM of at least triplicate experiments; **, P< 0.01; ***, p < 0.001.
Fig 5.
NS2A and NS4A broadly inhibited viral infection.
(a-c & e-h) The antiviral activity of NS2A and NS4A in HMC3 or U251 cells. HMC3 or U251 cells expressing NS2A or NS4A were infected by 0.1 MOI of ZIKV/SZ01, ZIKV/MR766, SFTSV or EV-A71 for 24 and 48 h. Viral RNA copies in infected cells were determined by qRT-PCR. (d & i-l) Antiviral activity of NS2A and NS4A in HEK293 or HeLa cells. HEK293 or HeLa cells were transfected with plasmids expressing NS2A or NS4A for 24 h, followed by infection with ZIKV/SZ01, ZIKV/MR766, SFTSV or EV-A71 for 24 (Fig 5D) or 48 h. Viral RNA copies in infected cells were determined by qRT-PCR. The differences between groups of control and NS2A or NS4A were evaluated by two-tailed Student’s t test. Data are means ± SEM of triplicate experiments; *, P < 0.05; **, P< 0.01; ***, p < 0.001.
Fig 6.
NS4A enhanced ISGs expression by activating ISGF3 pathway in HMC3 cells.
(a) Venn Diagram of differentially expressed genes. After HMC3 cells with or without NS4A expression were infected with 0.1 MOI of ZIKV/SZ01 for 24 h, total RNA from the mock and virus infected cells were collected for RNA-Seq, followed by analyses of the differentially expressed genes. Vec-M or Vec-S, mock or ZIKV/SZ01 infected- HMC3 cells without NS4A expression; NS4A-M or NS4A-S, mock or ZIKV/SZ01 infected-group of HMC3 cells with NS4A expression. (b) Expression of ISGs in HMC3 cells expressing NS4A determined by RNA-Seq. (c) Expression of ISGs in HMC3 cells expressing NS4A verified by qRT-PCR. The differences between groups of control and NS4A were evaluated by two-tailed Student’s t test. Data are means ± SEM of triplicate experiments; *, P < 0.05; **, P< 0.01; ***, p < 0.001. (d) The top 20 enriched pathways of Vec-M vs NS4A-M. The pathway enrichment analysis was used to group the differentially expressed genes in HMC3 cells expressing NS4A and vector. (e) Detection of STAT1, p-STAT1 (Tyr701), STAT2, p-STAT2 (Tyr690) and IRF9 protein expression in the ISGF3 signaling pathway in HMC3 cells expressing NS4A by western blot analyses.
Fig 7.
The ISGF3 signaling pathway was partially responsible for the antiviral effect of NS4A.
(a) Effect of Abrocitinib on the expression of ZIKV E protein in HMC3 cells. (b) Effect of Abrocitinib on the reproduction of ZIKV in HMC3 cells. HMC3 cells expressing NS4A were infected with 0.1 MOI of ZIKV/SZ01, with (1 μM or 5 μM) or without the treatment of Abrocitinib. Protein expression of STAT1, p-STAT1 (Tyr701), STAT2, p-STAT2 (Tyr690), IRF9 and ZIKV E was detected by western blot analyses (Fig 7A) and viral RNA copies in the culture supernatants were determined by TaqMan qRT-PCR at 48 h p.i. (c) Effect of knockdown of STAT2 on ZIKV E protein expression in U251 cells. (d) Effect of knockdown of STAT2 on ZIKV E RNA replication in U251 cells. The STAT2 in U251 cells with or without NS4A expression was knocked down by shSTAT2. Subsequently, the above cells and the control cells were infected with 0.1 MOI of ZIKV /SZ01. Protein expression of STAT1, p-STAT1 (Tyr701), STAT2, p-STAT2 (Tyr690), IRF9 and ZIKV E was detected by western blot analyses (Fig 7C) and viral RNA in the cell lysates was determined by TB Green qRT-PCR (Fig 7D). The differences of ZIKV viral RNA copies between groups of control and Abrocitinib or shSTAT2 were evaluated by two-tailed Student’s t test. Data are means ± SEM of triplicate experiments; *, P< 0.05; ***, p < 0.001.
Fig 8.
The ISGs expression induced by NS4A in U251 or HeLa cells.
(a) U251 cells with or without NS4A expression were cultured by DMEM with 2% FBS for 24 h. Total RNA was prepared from the cells for ISG transcription by qRT-PCR. (b) HeLa cells were transfected with plasmids expressing NS4A or empty vector for 24 h. After the cells were cultured in DMEM with 2% FBS for 24 h, total RNA was prepared from the cells for ISG transcription by qRT-PCR. The differences between groups of control and NS4A were evaluated by two-tailed Student’s t test. Data are means ± SEM triplicate experiments; *, P< 0.05; **, P< 0.01; ***, p < 0.001.
Fig 9.
Aborting NS4A protein expression rescued ZIKV RNA replication and protein expression.
(a & c) RNA replication of ZIKV E and HA-NS4A in HMC3 (Fig 9A) or U251 (Fig 9C) cells expressing HA-NS4A or NS4A-X. (b & d) Protein expression of ZIKV E and HA-NS4A in HMC3 (Fig 9B) or U251 (Fig 9D) cells expressingHA-NS4A or NS4A-X. Cells expressing HA-NS4A or NS4A-X were infected with 0.1 MOI of ZIKV/SZ01 for 24 or 48 h. Viral RNA copies and HA-NS4A in the cell lysates were determined by TB Green qRT-PCR. Protein expression of ZIKV E and HA- NS4A were detected by western blot analyses. NS4A-X: The cells were transfected with plasmids expressing HA-NS4A, which has a stop codon introduced to abort the translation of NS4A. The differences between groups of control and HA- NS4A or NS4A-X were evaluated by two-tailed Student’s t test. Data are means ± SEM triplicate experiments; *, P< 0.05; **, P< 0.01; ***, p < 0.001.