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. 2005 Aug 4;102(33):11876–11881. doi: 10.1073/pnas.0505577102

Fig. 4.

Fig. 4.

S protein-mediated intervirion fusion. (A) Intervirion fusion requires ACE2 and S protein. Bald or ACE2 particles encoding luciferase (x axis) were incubated with particles encoding GFP (SARS S and ASLV-A envelope, gray bars; SARS S alone, black bars; or ASLV-A envelope alone, white bars). Virions were mixed and used to infect HeLa/Tva cells that had been pretreated with medium in the presence and absence of leupeptin (Leu) (20 μg/ml). Intervirion fusion was measured as luciferase activity 48 h postinfection. Results represent the means of samples run in triplicate (±SD). (B) Trypsin cleavage promotes fusion mediated by S protein. Intervirion fusion between HIV-luc(ACE2) and HIV-gfp(SARS S/ASLV-A) treated with TPCK-trypsin (10 μg/ml) for 10 min at 25°C or pulsed at pH 5.0 was quantified by luciferase activity 48 h postinfection of HeLa/Tva cells pretreated with leupeptin. The results represent the means of samples run in triplicate (±SD). Mixtures of HIV-gfp(SARS S), HIV-gfp(ASLV-A), and HIV-luc(ACE2) could not be activated by trypsin cleavage, suggesting that S and ASLV-A envelope are required to be incorporated into the same particle in order for transduction of target cells by fused particles. (C) Receptor interactions at elevated temperature are required before trypsin cleavage. HIV-luc(ACE2) and HIV-GFP(SARS S/ASLV-A) particles were mixed and incubated at 4°C to allow binding. Samples were then incubated at the noted temperatures. TPCK-trypsin digestion was carried out at 4°C for 15 min. The results represent the means of samples run in quadruplicate (±SD). Similar results were observed in two additional experiments. Temp., temperature. (D) CTSL enhances intervirion fusion. HIV-luc(ACE2) and HIV-GFP(SARS S/ASLV-A) particles were mixed and incubated for 10 min at 25°C with preactivated CTSB (at pH 5.0), CTSL (at pH 6.0), CTSL buffer alone (at pH 6.0), or TPCK-trypsin (at pH 7.0). The mixed virus was used to infect HeLa/Tva cells pretreated with leupeptin. The results represent the means of samples run in quadruplicate (±SD). Similar results were observed in two subsequent experiments. (E) Acidic conditions are required for CTSL-mediated S protein activation. HIV-luc(ACE2) and HIV-GFP(SARS S/ASLV-A) particles were mixed and adjusted to various pHs and CTSL was added. After neutralization of acid conditions, the mixed virus was used to infect HeLa/Tva cells pretreated with leupeptin. The results represent the means of samples run in quadruplicate (±SD). Tryp, trypsin. Similar results were observed in an additional experiment.