Fig 1: Knockdown of tetraspanins increases Zika virus infectivity and secretion of infectious particles/vesicles. (A) Immunoblot of tetraspanin CD9, CD63, and CD81 EV immunoprecipitated proteins probed for EV markers and Zika virus proteins. (B) RNA was extracted from cells 48 h after Zika virus infection, and RT-qPCR was performed to obtain transcript levels of CD9, CD63, and CD81 (error bars represent standard errors of the means [SEM] from 3 independent experiments; samples normalized to control and control set to 100); ****, P < 0.0001. (C) Immunoblots of cell and EV 100K lysate harvested at 72 h from uninfected or Zika virus-infected control (Ctrl) and inducible tetraspanin knockdown cells that were probed for EV markers and Zika virus proteins. Quantitation of 100K EV Zika virus capsid levels of 3 independent experiments (error bars displayed as SEM; *, P < 0.05). (D) Tetraspanin inducible shRNA knockdowns and control cells were monitored for CPE via live cell imaging every 24 h for 4 days after infection with Zika virus or mock. (E) Quantitation of the percent change in Zika virus 100K EV RNA levels of 3 independent experiments (error bars display SEM from 3 independent experiments; samples normalized to control and control set to 100). (F) Immunoblot of cell lysate from cells exposed to EVs from tetraspanin knockdown cells.