Fig 1.
Bat cells cleave cathepsin-dependant Hendra virus F and furin-dependant PIV5 F.
(A) Cells were transfected with pCAGGS-Hendra virus F or pCAGGS-PIV5 F and 18–24 hours post transfection, metabolically labeled with Tran 35S for 3 hours at 37°C. Following labeling, cells were lysed and immunoprecipitated. Proteins were analyzed by 10%SDS-PAGE and visualized by autoradiography. (B) Cells were transfected with Hendra virus F or PIV5 F alone or in combination with Hendra virus G or PIV5 HN. 24 to 48 hours post transfection, cells were washed and images were taken using a Nikon digital camera mounted atop a Nikon TS100 microscope with 10x objective. Arrows indicate syncytia.
Fig 2.
Kinetics of PIV5 Fusion protein cleavage is faster in bat cells compared to Vero cells.
(A) Cells transiently transfected with pCAGGS-Hendra virus F or pCAGGS-PIV5 F were metabolically labeled with Tran 35S for 30 minutes and chased for the indicated times. Cells were immediately lysed and cell lysates were immunoprecipitated. Proteins were migrated on 10% SDS-PAGE and analyzed by autoradiography. (B) Quantification of F1 densitometry was done using ImageQuant, TL software (GE Healthcare, Piscataway, NJ) and results are represented as percent cleavage defined as F1/(F1+F0). Error bars represent the mean ± standard deviation for three independent experiments. Two-way ANOVA, **p<0.01, ***p<0.001.
Fig 3.
Inhibition of cathepsin L and vesicular trafficking prevent cleavage of Hendra virus F.
(A) Vero cells or bat cells were transfected with pCAGGS-Hendra virus F and 24 hours post transfection, cells were metabolically labeled with Tran 35S in the absence or presence of the indicated inhibitor. E64-d and cathepsin L inhibitor I were added at 20μM. Cells were lysed, immunoprecipitated and anaylzed on 10% SDS-PAGE. Arrow indicates the position of a novel band. (B, C) Cells transiently transfected with Hendra virus F (B) or PIV5 F (C) were labeled with Tran 35S for 45 minutes and then chased for 3 hours at either 20°C or 37°C. Following lysis and immunoprecipitation, proteins were run on 10% SDS-PAGE and visualized by autoradiography.
Fig 4.
Effect of dec-RVKR-cmk on PIV5 F cleavage and furin enzyme activity in fruit bat cells.
(A) Cells transfected with pCAGGS-PIV5 F were labeled with Tran 35S for 3 hours in the absence or presence of cathepsinL I (20μM) or dec-RVKR-cmk (50 μM). Prior to SDS-PAGE analysis, cells were lysed and subject to immunoprecipitation. Images were visualized by autoradiography. (B) 2×106 cells of each cell type were lysed for 10 minutes on ice followed by shearing with a 10-gauge needle. Cell lysates were incubated with increasing concentrations of dec-RVKR-cmk for 3 hours at 37°C prior to addition of Pyr-Arg-Thr-Lys-Arg-AMC furin substrate. End-point fluorescence was measured after 4 hours using an XPS plate reader. Error bars represent the mean ± standard deviation for three independent experiments.
Fig 5.
Different mammalian cell types show differences in furin-like enzyme activity.
2×106 cells of each cell type were lysed for 10 minutes on ice followed by shearing with a 10-gauge needle. Clarified lysates were then incubated with 10 μM of Pyr-Arg-Thr-Lys-Arg-AMC furin substrate for 4 hours at 37°C with fluorescence measured every 3 minutes. Each cell type was assayed in duplicate and the progress curves are representative of 3 separate experiments.
Fig 6.
Sequence alignment of Pteropus alecto cathepsin L1 and cathepsin L1 of other mammalian species show Pteropus-specific amino acid changes.
Sequence alignment was generated using ClustalW. (GenBank accession numbers are given in parentheses): human (P07711.2), rhesus macaque (EHH24212.1), horse (XP_001494409.1), dolphin (XP_004320974.1), dog (Q9GL24.1), cow (P25975.3), mouse (NP_034114.1). Yellow indicates amino acid changes that are specific to P. alecto cathepsin L. The asterisk “*” indicates identical residues, “:” indicates conserved substitutions and “.” semi-conserved substitutions.
Fig 7.
The C-terminus region of P. alecto furin has the highest degree of amino acid variation compared to furin proteases from other mammalian species.
Sequence alignment was generated using ClustalW. (GenBank accession numbers are given in parentheses): human (NP_002560.1), rhesus macaque (EHH27600.1), horse (XP_005602832.1), dolphin (XP_004322334.1), dog (XP_850069.2), cow (NP_776561.1), mouse (NP_001074923.1). Highlighted in gray is the catalytic site of furin and in green the changes in amino acid residues in P. alecto compared to other mammalian furins. The asterisk “*” indicates identical residues, “:” indicates conserved substitutions and “.” semi-conserved substitutions.