Ebola virus VP35 has novel NTPase and helicase-like activities

Shu, Ting; Gan, Tianyu; Bai, Peng; Wang, Xiaotong; Qian, Qi; Zhou, Hui; Cheng, Qi; Qiu, Yang; Yin, Lei; Zhong, Jin; Zhou, Xi

Helicase activities are important for the replication of diverse viruses (52) , thus it would be intriguing to determine whether GuHCl can also inhibit the replication of EBOV. Because the live virus experiments of EBOV can only be conducted in the biosafety level 4 (BSL-4) facilities, EBOV minigenome (MG) replicon systems are usually used to study EBOV replication under common BSL-2 conditions (53, 54) . To this end, we utilized a stable EBOV minigenome replicon system (EBOV replicon cells) that can stably replicate and transcribe the EBOV minigenomic RNA [the Gaussia luciferase (GLuc)-encoding ORF was flanked by the minimal cis-elements located at the two ends of EBOV genome] in Huh7 cells stably expressing NP, VP35, VP30, and L (41). The EBOV replicon cells were treated with GuHCl at the indicated concentrations for 96 h. After that, we examined the cell viability at the doses we applied, and found no obvious cytotoxicity until the concentration of GuHCl up to 2 mM ( Figure 8D ). And 1 mM GuHCl did not affect the expression levels of NP, VP30, VP35 and ␤-actin (Supplementary Figure S11A) . Then, the replication and transcription of the EBOV minigenome replicon were examined by measuring the levels of GLuc activity, minigenomic viral RNA (vRNA), cRNA (complementary to vRNA; replication intermediate), and viral mRNA. Our data show that both the replication and transcription of the EBOV minigenome were inhibited by GuHCl treatment in a dose-dependent manner ( Figure 8E and F).