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