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. 2020 Jan 22;9:480. doi: 10.3389/fcimb.2019.00480

Figure 4.

Figure 4

HCAR2 knockout enhanced the replication of ZIKV in A549 cells. (A) Confirmation of HCAR2 knockout efficiency by DNA sequencing. (B) Confirmation of HCAR2 knockout efficiency by real-time RT-PCR assay. (A,B) The CRISPR/Cas9 mediated A549 HCAR2-KO1 or A549 HCAR2-KO2 cell clone was isolated. Then, the genomic DNAs or total RNAs were extracted for DNA sequencing (A) or real-time RT-PCR assay (B) with HCAR2-specific probes. (C–E) The effect of HCAR2 depletion on ZIKV RNA level (C), E protein level (D), and titer (E). A549 HCAR2-deficient cells were infected with ZIKV (MOI 3) for 1 h and collected at 2, 6, 12, 18, and 24 h p.i. The viral RNA level was measured by real-time RT-PCR using primers specific to ZIKV NS1 mRNA. Human GAPDH mRNA level was measured as an internal control (C). Western blotting was performed to detect ZIKV E protein expression at 24 h p.i. (D). GAPDH served as an internal control. The supernatants of infected cells were harvested at 24 h p.i. and plaque assay was performed to measure viral titer (E). All the data were shown as means ± S.D. (error bars) from at least three independent experiments. Statistically significant, **p ≤ 0.01 (t-test); ***p ≤ 0.001 (t-test); NS, not significant.