Figure 5.

Soluble forms of NPC1 domain C bind directly to GP and selectively neutralize infection by viral particles containing cleaved glycoproteins. (A–C) Capacity of an rVSV displaying a domain C-VSV G chimera (rVSV-domain C) to bind to rVSV-GP and neutralize rVSV-GP infection. (A) Schematic of experiments shown in (A–C). ‘rVSV-bald’ particles lack any virus-encoded glycoproteins. (B) rVSV-GP and rVSV-GP_CL were preincubated with rVSV-domain C or rVSV-bald, and virus mixtures were stained with phosphotungstic acid and visualized by electron microscopy. Arrows indicate large clusters of viral particles. A larger version of this image is available in Supplementary Figure S5. (C) Virus mixtures were exposed to WT CHO cells, and expression of the rVSV-GP-encoded eGFP gene was quantitated at 7 h post infection (see Materials and methods for details). eGFP signal was normalized to that of uncleaved virus. (D) The capacity of rVSV-GP and rVSV-GP_CL to capture a purified, soluble form of domain C containing Flag and hexahistidine tags was determined in an ELISA, as described in Figure 3C. Results (n=3) are representative of at least three independent experiments. (E) The capacity of a purified, soluble form of GP lacking the transmembrane domain (GPΔTM) to associate with purified, soluble domain C was determined by co-IP, as described in Figure 3A. Pellets and supernatants (one gel for each) were resolved on separate gels but exposed simultaneously to the same piece of film. (F) rVSV-GP and rVSV-GP_CL were preincubated with soluble domain C, and virus–protein mixtures were exposed to the Vero African grivet monkey kidney cell line. Viral infection was enumerated by fluorescence microscopy. Results are from two independent experiments (n=4). Error bars indicate s.d. Figure source data can be found in Supplementary data.