Simian hemorrhagic fever virus cell entry is dependent on CD163 and uses a clathrin-mediated endocytosis-like pathway

Abstract

Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-β-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component.

Importance: Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

FIG 1

SHFV particles rapidly enter target cells. (A) Time-dependent transmission electron micrographs of SHFV particle entry into MA-104 cells. Shown are representative images of two independent experiments. (B) Growth kinetics of SHFV at various MOIs in MA-104 cells as determined by plaque assay. The error bars indicate the standard deviations of triplicate samples from one of two independent experiments.

FIG 2

SHFV particles enter target cells in a low-pH-dependent manner. (A to D) Effects of pretreatment of MA-104 cells with increasing concentrations of inhibitors affecting pH on SHFV particle yield, as determined by plaque assay. (E) Time-of-addition experiment using chloroquine at a fixed concentration of 150 μM. The error bars indicate the standard deviations of triplicate samples from one of two independent experiments.

FIG 3

SHFV particles enter cells using a clathrin-mediated endocytosis-like pathway. (A to D) Effects of pretreatment of MA-104 cells with increasing concentrations of inhibitors of clathrin-mediated endocytosis on SHFV particle yield as determined by plaque assay. (E) Time-of-addition experiment testing the effects of Dyngo-4a at the same concentration but at different points after cell exposure to SHFV particles. (F) Effect of overexpression of wild-type dynamin 1 or a dominant-negative mutant thereof in MA-104 cells on SHFV progeny production. (G) Evaluation of clathrin HC expression in MA-104 cells treated with clathrin HC-specific gRNAs or control gRNA by Western blotting. (H) Effects of gRNA treatment of MA-104 cells on SHFV progeny production. The error bars indicate the standard deviations of triplicate samples of one of two independent experiments. <, measurement below the threshold of detection (20 PFU/ml); *, P < 0.05 (Student's t test).

FIG 4

SHFV particles do not enter cells by macropinocytosis. (A to C) Effects of pretreatment of MA-104 cells with increasing concentrations of inhibitors of macropinocytosis on the SHFV viral titer, as determined by plaque assay (left), or on the percentage of VACV-GFP-infected cells, as determined by high-content imaging (right). The error bars indicate the standard deviations of triplicate samples from one of two independent experiments.

FIG 5

SHFV cell entry is independent of actin polymerization. (A to C) (Left) Effects of pretreatment of MA-104 cells with increasing concentrations of inhibitors of actin polymerization on SHFV particle yield, as determined by plaque assay. (Right) Immunofluorescence images of the MA-104 cells showing the disruption of actin networks by the inhibitors using Alexa 594-phalloidin staining. (D) Alexa 594-phalloidin staining of untreated MA-104 cells treated with dimethyl sulfoxide (DMSO). The error bars indicate the standard deviations of triplicate samples from one of two independent experiments.

FIG 6

SHFV particles do not enter cells by caveola-mediated endocytosis. (A and B) Effects of pretreatment of MA-104 cells with increasing concentrations of inhibitors of caveola-mediated endocytosis on SHFV particle yield, as determined by plaque assay. (C and D) Effects of pretreatment of MA-104 cells with increasing concentrations of the same inhibitors on RVFV infection, as determined by IFA. (E) Influence of cholesterol depletion on SHFV particle yield. Cells were treated with 5 or 10 mM methyl-β-cyclodextrin (MβCD) and infected with SHFV. The particle yield was determined by plaque assay. Alternatively, the cells were treated with MβCD, and exogenous soluble cholesterol was added to reverse the effect of MβCD. The error bars indicate the standard deviations of triplicate samples from one of two independent experiments.

FIG 7

Cathepsins L and B do not play a role in SHFV cell entry and replication. (A to C) Effects of pretreatment of MA-104 cells with increasing concentrations of cathepsin inhibitors on SHFV and MERS-CoV (positive control) particle yield, as determined by plaque assay. MA-104 cells were pretreated with E-64d (cathepsin L and B inhibitor), FYdmk (cathepsin L inhibitor), or CA-074 (cathepsin B inhibitor) for 4 h and then infected with SHFV or MERS-CoV at an MOI of 5. The error bars indicate the standard deviations of triplicate samples from one of two independent experiments.

FIG 8

SHFV particles use a proteinaceous cell surface receptor to gain entry into target cells. (A to D) Effects of pretreatment of MA-104 cells with increasing concentrations of proteases on SHFV particle yield, as determined by plaque assay. (E and F) Effects of pretreatment of MA-104 cells with increasing concentrations of phospholipase A2 or D on SHFV particle yield. The error bars indicate the standard deviations of triplicate samples from one of two independent experiments.

FIG 9

CD163 is a crucial SHFV cell entry factor. (A and B) Effects of incubation of MA-104 or MARC-145 cells with increasing concentrations of human anti-CD163 or control antibody on SHFV and PRRSV particle yield, as determined by plaque assay. The error bars indicate the standard deviations of triplicate samples from one of two independent experiments.