As with GP, differences in MAb binding to unheated GPTHL did not correlate with the thermostability of a given epitope and could not explain the stability of the ADI-16061 stalk epitope (data not shown). Open in a separate window FIG 2 Proteolytic cleavage with thermolysin (THL) and cathepsin L (CatL) destabilizes EBOV GP. epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, EACC acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals. IMPORTANCE The development of Ebola virus EACC countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability. ~ 59C) were obtained for GP and GPMuc, suggesting that Muc does not contribute to the stability of the GP prefusion conformation. Therefore, we largely used viral particles bearing GPMuc in the following experiments. Open in a separate window FIG 1 Thermal denaturation curves for prefusion epitopes in uncleaved EBOV GP. (A) rVSV-GP and rVSV-GPMuc were incubated at the indicated temperatures for 10?min, after which the samples were cooled to 4C and KZ52 binding was assessed by ELISA. Averages standard deviations (SD) are shown; values of 59C (Fig. 1C). In contrast, the ADI-16061 epitope was largely resistant to elevated temperatures, possibly because this epitope in the GP2 HR2 domain is stabilized by its proximity to the GP membrane anchor. Alternatively, it is possible that the ADI-16061 epitope partially renatures during the cooling step or subsequent steps in the ELISA (Fig. 1C). The increased thermostability of the ADI-16061 stalk epitope was not explained by its increased binding to GP; ADI-16061 (and the other MAbs tested) were found to have related relative binding to unheated GP (data not shown). In contrast to the GP foundation and glycan cap subdomains probed above, Muc was shown to be mainly disordered, with several Muc-specific MAbs realizing linear epitopes (10, 11, 39,C41). To investigate the thermostability of Muc, we used MAb 14G7, which recognizes a linear Muc epitope (39). On the heat range at which the base and glycan cap epitopes were lost, we observed no appreciable reduction in the 14G7 epitope (Fig. 1D). Instead, 14G7 acknowledgement was enhanced at very high temps (Fig. 1D), probably due to the improved exposure of its linear epitope (39). Taken together, these experiments demonstrate the highly organized regions of the GP trimer, including the foundation and glycan cap subdomains, suffer a EACC concerted, irreversible loss of their prefusion conformation at elevated temps. EBOV GP is definitely destabilized by proteolytic cleavage. Proteolytic cleavage of GP by endosomal cysteine cathepsins CatB and CatL exposes the binding site for its crucial endo/lysosomal receptor, NPC1, and primes it to undergo subsequent entry-related conformational changes (35, 42, 43). Earlier work also suggests that cleaved GP conformers (GPCL) generated are more conformationally labile than their uncleaved counterparts (35, 43). To investigate the consequences of proteolytic processing on GP thermostability, rVSV-GP was incubated with thermolysin (THL) as explained previously and tested in the epitope-loss ELISA (22, 43). THL is definitely proposed to PPARG mimic the cleavage of GP by CatB during viral access (22). GP cleavage was verified by Western blot analysis (Fig. 2A). Even though thermal denaturation curve for THL-cleaved GPCL (GPTHL) resembled those of GP and GPMuc in sigmoidal shape, it was remaining shifted by 6C relative to the second option, indicating decreased stability (Fig. 2B and ?andC).C). We acquired similar findings with the GP foundation epitope of MAb ADI-15878 and the RBS epitope of MR72 (8, 44). ADI-16061s GP stalk epitope was even more resistant to denaturation in.