Intrinsic features of Zika Virus non-structural proteins NS2A and NS4A in the regulation of viral replication
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.