The requirement of environmental acidification for Ibaraki virus infection to host cells

TSURUTA, Yuya; SHIBUTANI, Shusaku T.; WATANABE, Rie; IWATA, Hiroyuki

The utilization of endosomal pathway by IBAV for infection was confirmed using endosome inhibitors. HmLu-1 (hamster lung) cells were infected with IBAV in the presence of three different endosome inhibitors, bafilomycin A1 (Baf A1, Sigma, St. Louis, MO, U.S.A.), chlorpromazine (CPZ, Abcam, Cambridge, U.K.) and dynasore (Wako, Osaka, Japan). Those drugs inhibit clathrin dependent endocytosis in different manners. Baf A1 is an antibiotic derived from Streptomyces griseus and specifically inhibits vacuolar type H+ ATPase [23] . CPZ dislocates clathrin, and its adaptor protein from plasma membrane to cytosol [22] and dynasore inhibits GTPase activity of dynamin specifically [14] . All inhibitors work reversibly, and therefore, endosome pathway can restart once the drug was removed from the culture. HmLu-1 cells prepared in 6 well plate were treated with Dulbecco's modified eagle's medium (DMEM, Wako) containing various concentrations of inhibitors for 30 min at 37°C. After cells were chilled on ice for 5 min, the media were removed, and cells were infected with IBAV at MOI=3 for 1 hr at 4°C. After a wash with PBS (−), cells were further incubated with DMEM plus inhibitors for 30 min at 37°C, and the media were replaced with DMEM containing 10% fetal bovine serum (10FDMEM). Cells were incubated for further 24 hr, and culture supernatant was collected. Collected supernatant was subjected to plaque assay, and after staining with crystal violet solution (0.1% crystal violet in 10% buffered formalin and 20% methanol), the number of plaque was counted. To analyze the statistical significance of each inhibitor concentration group against mock treated group, statistical software R [16] was used to run Dunnet's test [7] . Figure 1a shows the number of infectious IBAV in the supernatant of the cells treated with Baf A1, CPZ or dynasore. All inhibitors were shown to decrease virus titer when their concentration was elevated. The most significant decrease was observed when Baf A1 concentration was higher than 6.25 nM (P=0.0013 for 6.25 nM against 0 nM). At the same time, the effect of those inhibitors to HmLu-1 viability was tested. HmLu-1 cells in 96 well multiwell plate were treated with media containing various concentrations of inhibitors for 1 hr without IBAV infection. After 24 hr incubation, the number of viable cells was quantified using CellTiter ® Aqueous One Solution Reagent (Promega, Madison, WI, U.S.A.). As shown in Fig. 1b , no inhibitors showed significant effect on viable cell numbers. These results indicated that IBAV utilizes clathrin-dependent endosomal pathway for infection and coincided with the previous research on bluetongue virus entry [8] .