doi:10.1128/JVI.02879-15. were purchased from BioLegend (San Diego, CA). Polyclonal goat anti-THY-1 was obtained from Novus (Littleton, CO). Transferrin-conjugated Alexa 488- and AlexaFluo-conjugated secondary antibodies were purchased Mouse monoclonal to CD40 from Invitrogen (Grand Island, NY). IPA-3 (EMD, Chicago, IL), dynasore monohydrate, and filipin III (Santa Cruz, Santa Cruz, CA), jasplakinolide (Calbiochem, San Diego, CA), 5-( 0.0001, 3 independent experiments). The inhibitory effect of EIPA on infectivity was dose dependent (Fig. 7B). The level of GAPDH RNA was the same in cells treated with the highest dose of EIPA and DMSO (the solvent for EIPA). In addition, cell viability, determined by CytoTox-One assay (Promega, Madison, WI) which measures cell membrane integrity, was similar in EIPA-treated cells and solvent controls (Fig. 7C and ?andD),D), indicating that EIPA was not cytotoxic under these conditions. Previously, we reported that soluble THY-1 (sTHY-1) blocks HCMV entry (29). Here we compared the inhibitory effects of EIPA and sTHY-1. Treatment of HS-578T cells with EIPA or sTHY-1 alone reduced HCMV infectivity by 90% and 60%, respectively (Fig. 7E). Less than 5% of the total infectivity was resistant to combined treatment with EIPA and sTHY-1. We previously showed that entry of HCMV into SNB-19 glioblastoma cells is THY-1 dependent (29). Pretreatment of SNB-19 cells with EIPA reduced HCMV infectivity by 80% in multiple independent experiments, and treatment with sTHY-1 reduced HCMV infectivity by 75% (Fig. 7F). Treatment with combined sTHY-1 and EIPA slightly reduced the HCMV infectivity compared to that with EIPA alone or sTHY-1 alone. These data suggest that macropinocytosis is an important pathway for internalization of HCMV. Since 80% of HCMV infectivity was THY-1 dependent and EIPA sensitive, the SEA0400 data imply that THY-1 mediates HCMV entry by macropinocytosis. Open in a separate window FIG 7 Macropinocytosis inhibition of HCMV infection by EIPA is dose dependent, and EIPA and soluble THY-1 protein block HCMV infection to similar extents. (A and B) HS-578T cells were pretreated with EIPA at 215 M (A) or at various concentrations (B), followed by HCMV infection for 4.5 to 5.5 h. RNA was extracted, and HCMV transcripts were detected using RT-qPCR and normalized against GAPDH SEA0400 amplified from the same reaction as an internal control. (C) To assess potential cytotoxicity, the level SEA0400 of GAPDH RNA was determined by RT-qPCR at the highest dose used for panel B (100 M). (D) CytoTox-One assay was used to assess cytotoxicity based on cell membrane damage at the end of the infection. (E and F) HS-578T (E) and SNB-19 (F) cells were pretreated with 50 M EIPA or DMSO solvent. HCMV was incubated with soluble THY-1 protein or control (filtrates that contained the same buffer composition) at room temperature for 10 min, and cells SEA0400 were infected for 4.5 h. RNA was extracted, and HCMV transcripts were detected using RT-qPCR and normalized against GAPDH amplified from the same reaction as an internal control. Actin remodeling is essential for macropinosome formation, and inhibitors of actin remodeling such as jasplakinolide and cytochalasin D have been used to assess the role of macropinocytosis in virus infection (38, 40, 63,C65). Treatment of HS-578T cells with jasplakinolide reduced HCMV infectivity (Fig. 8A) ( 0.001, 6 independent experiments) at a nontoxic dose (Fig. 8B). Inhibition of actin remodeling with cytochalasin D also impaired virus infection in a dose-dependent manner (Fig. 8C). Within the dose range used, no detectable cytotoxicity was observed as assessed by monitoring the GAPDH RNA level and cell viability (Fig. 8D and ?andEE). Open in a separate window FIG 8 Actin remodeling is important for HCMV-induced macropinocytosis. (A) HS-578T cells were pretreated with jasplakinolide (200 nM) for 60 min, followed by HCMV infection for 60 min. Virus internalization was then terminated by a low-pH buffer wash to inactivate any remaining extracellular virus. After an additional 5 h in culture, SEA0400 RNA was extracted and HCMV transcripts were detected using RT-qPCR and normalized against GAPDH amplified from the same reaction as described for Fig. 7. (B) The quantity of GAPDH RNA was determined by RT-qPCR to assess potential cytotoxicity. (C) HS-578T cells were pretreated with cytochalasin D (CytoD) at the indicated concentration, followed by.