Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein

Abstract

Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F(0)) to the active F(1) + F(2) disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F(0) into F(1) and F(2) fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.

FIG. 1.

General protease inhibitors do not retard proteolytic processing of Hendra virus F. Vero cells transfected with pCAGGS-Hendra F were metabolically labeled and either lysed immediately (0 h) or chased for 2 h in DMEM in the absence (DMSO) or presence of a Complete protease tablet (without EDTA), 1 kU aprotinin, 1 mM PMSF, 100 μM leupeptin, 100 μM antipain, 100 μM chymostatin, 1 μM pepstatin A, 5 μM GM6001, or 0.1 mM 1,10-phenanthroline (o-Ph). Samples were analyzed by immunoprecipitation, 15% SDS-PAGE, and the Typhoon imaging system. Positions of uncleaved F₀ and cleaved F₁ and F₂ subunits are indicated.

FIG. 2.

Nonspecific and specific cathepsin inhibitors block Hendra virus F cleavage. Vero cells expressing pCAGGS-Hendra F (A) or SV5 F (B) were starved, labeled with Tran³⁵S label, and chased for 2 h in the absence (DMSO) or presence of 10 μM E-64, E-64d, calpeptin, PD150606, caspase 3 inhibitor III (Casp 3 III) or lactacystin, 1 μM MG132, 100 μM caspase inhibitor III (Casp III), or 50 μM cathepsin inhibitor I (Cath I), cathepsin L inhibitor III (Cath LIII), or CA-074Me. Samples were prepared and analyzed as described in the text. The positions of the F₀, F₁, and F₂ proteins are indicated. All cysteine protease inhibitors, with the exception of E-64*, are membrane permeant.

FIG. 3.

Effect of cathepsin L shRNA on protein and enzymatic activity in Vero cells. (A) Cathepsin L protein expression in untransfected and shRNA-expressing Vero cells. Five micrograms of total cell extract was subjected to SDS-PAGE and analyzed by Western blotting with anti-cathepsin L antibodies (top panel) and anti-β-actin monoclonal antibodies (bottom panel). The positions of procathepsin L (ProL), single-chain cathepsin L (SCL), and double-chain cathepsin L (DCL) are indicated. (B) Cathepsin L enzyme activity levels in untransfected and shRNA-expressing Vero cells. (C) Cathepsin B enzyme activity levels in untransfected and pSuper-Scramble-, -Cath L98-, and Cath L662-expressing Vero cells.

FIG. 4.

Proteolytic processing of Hendra virus F and membrane fusion in shRNA-expressing Vero cells. (A) Expression and proteolytic processing of Hendra virus F and SV5 F in shRNA-expressing Vero cells were examined by metabolic labeling and immunoprecipitation as described previously. (B) Luciferase reporter gene assay to examine the ability of Hendra virus F and G to promote membrane fusion in shRNA-expressing Vero cells. The results presented are representative of three separate experiments.

FIG. 5.

Cathepsin L is able to cleave Hendra virus F₀ to F₁-plus-F₂ heterodimers. Immunopurified uncleaved Hendra virus F was digested with 10 nM human cathepsin L at 37°C for 2 to 8 h. An uncleaved Hendra virus F control sample was incubated in buffer without enzyme for 8 h at 37°C. Samples were analyzed as described in the other figure legends. F₀, F₁, and F₂ are designated by arrows on the right.