Endocytosis plays a critical role in proteolytic processing of the Hendra virus fusion protein

Abstract

The Hendra virus fusion (F) protein is synthesized as a precursor protein, F(0), which is proteolytically processed to the mature form, F(1) + F(2). Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca(2+), and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXPhi. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins.

FIG. 1.

Schematic representation of Hendra virus F protein. The magnified region shows the sequence of the cytoplasmic tail, identifying the relevant tyrosine residue. Mutants Y525A and Y525F were obtained by site-directed mutagenesis.

FIG. 2.

Expression of wt and mutant Hendra virus F proteins. Vero cells were transfected with pCAGGS-wt Hendra F, pCAGGS-Hendra F Y525A, or pCAGGS-Hendra F Y525F. Cells were metabolically labeled for 45 min with Tran[³⁵S], chased for the indicated times, and then lysed. Lysates were immunoprecipitated with a polyclonal antibody specific to Hendra virus F, resolved via 15% polyacrylamide gel electrophoresis under reducing conditions, and visualized using storage phosphorimage autoradiography.

FIG. 3.

Biotinylation of cell surface proteins. Vero cells were transfected with pCAGGS-wt Hendra F, pCAGGS-Hendra F Y525A, or pCAGGS-Hendra F Y525F. Cells were metabolically labeled for 2 h with Tran[³⁵S] and chased for the indicated times prior to the biotinylation of surface proteins. Cells were put on ice, biotinylated, and then immediately lysed, and the lysates were immunoprecipitated. Fifteen percent of the immunoprecipitated lysate was reserved as a representative of the total protein. The remaining 85% of the lysate was subjected to a streptavidin pull-down assay to isolate biotinylated proteins. Both total protein and biotinylated surface proteins were resolved via 15% polyacrylamide gel electrophoresis under reducing conditions and visualized using storage phosphorimage autoradiography.

FIG. 4.

Endocytosis assay. (A) Vero cells were transfected in duplicate with pCAGGS expressing wild-type or mutant Hendra virus F. Cells were metabolically labeled for 2 h with Tran[³⁵S] and chased for 1 h. Plates were moved to 4°C to inhibit endocytosis, biotinylated with a cleavable form of biotin, flooded with prewarmed DMEM, and returned to 37°C for various lengths of time to permit endocytosis. Following the warm-up, cells were returned to 4°C on ice, treated or not treated with MESNa, a membrane-impermeant reducing agent, to cleave any accessible biotin, and then lysed. Lysates were then immunoprecipitated, biotinylated proteins were pulled down with streptavidin beads, and samples were resolved via 15% polyacrylamide gel electrophoresis under reducing conditions and visualized using storage phosphorimage autoradiography. (B) Percent endocytosis of Hendra virus F and mutant F proteins averaged over three experiments.

FIG. 5.

Surface expression of wt and mutant Hendra virus F. Vero cells transfected with pCAGGS-wt or mutant Hendra virus F were metabolically labeled overnight with Tran[³⁵S], biotinylated, and lysed. Lysates were immunoprecipitated, and surface proteins were separated using streptavidin beads. Biotinylated surface proteins were resolved via 15% polyacrylamide gel electrophoresis under reducing conditions and quantitated using storage phosphorimage autoradiography. The results shown are the averages of three separate experiments.

FIG. 6.

Fusion assays. (A) Syncytium assay. Vero cells were cotransfected with empty pCAGGS vector, pCAGGS-wild type F, pCAGGS-Hendra FY525A, or pCAGGS-Hendra F Y525F and pCAGGS-Hendra G. Pictures were taken at 43 h posttransfection using a Nikon Diaphot inverted phase-contrast microscope and a Kodak DCS digital camera. (B) Reporter gene assay. pCAGGS expressing Hendra virus F wt or the two mutants, along with pCAGGS-Hendra G and a plasmid containing the luciferase gene under control of the T7 promoter, was transfected into Vero cells. BSR cells, which stably express the T7 polymerase, were overlaid onto the F- and G-expressing cells, and the mixed cell populations were incubated at 37°C for 3 h. Cells were lysed and analyzed for luciferase activity on a luminometer. The results are averages of duplicates or triplicates, with the vector alone set as background, and are representative of three separate experiments.