Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor

Abstract

Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.

FIG. 1.

VOPBA analysis of purified dengue virus serotype 1 on HepG2 cell membrane proteins. HepG2 cell membrane proteins were extracted and separated on denaturing and nondenaturing gels. The membrane proteins were transferred to nitrocellulose membranes and incubated with dengue virus serotype 1. Virus binding protein bands were detected via a panspecific monoclonal antibody directed against the dengue virus E protein. Control reactions were identical, except that no virus was included in the hybridization mix (−ve lanes).

FIG. 2.

Inhibition of dengue virus serotype 1 binding and infection of HepG2 cells. (Top) HepG2 cells were preincubated with either no antibody (control) or 1, 5, 10, or 20 μg of an anti-37/67-kDa laminin receptor antibody, 5 μg of an anti-N-terminal GRP78 antibody, or 0 (control) 5, 10, or 20 μg of soluble laminin prior to infection with dengue virus serotype 1 at an MOI of 1. Levels of viral production were assayed by a plaque assay after 1.5 viral replication cycles. Unbound (postinfection) and intracellular and extracellular (after 1.5 replication cycles) virus levels were also independently assayed at the highest level of antibody used (20 μg) in parallel with control (no-antibody) infections. Results are normalized against the level of virus produced with no antibody preincubation. Each experimental point is the sum of triplicate experiments with two assay of titer. Error bars represent standard errors of the mean. Asterisks represent a significant difference from the control (one-sample t test); a, P = 0.0400; b, P = 0.0065; c, P = 0.0122; d, P = 0.045; e, P < 0.0001; f, P = 0.010. (Bottom) HepG2 cells were pregrown on glass slides and preincubated either without antibody (A) or with antibodies directed against either the 37/67-kDa high-affinity laminin receptor (B) or the N terminus of GRP78 (D) or with soluble laminin (C) prior to infection with dengue virus serotype 1 at a MOI of 10. The binding of the dengue virus to HepG2 cells was visualized by subsequent and successive incubation with a panspecific monoclonal antibody directed against the dengue virus E protein and an FITC-conjugated anti-mouse monoclonal antibody. The slides were viewed under a fluorescence microscope. Magnification, ×200.

FIG. 3.

Effects of enzyme and antibody-enzyme pretreatment on infection of HepG2 cells by dengue virus serotype 1. HepG2 cells were either untreated (controls) or treated with trypsin or a combination of trypsin and heparinase III or preincubated with an antibody against the 37/67-kDa high-affinity laminin receptor or treated with heparinase III and subsequently preincubated with an antibody against the 37/67-kDa high-affinity laminin receptor prior to infection with dengue virus serotype 1 at a MOI of 1. Levels of virus in the medium were assayed either immediately after the period of viral inoculation (unbound virus) or after 1.5 virus replication cycles (extracellular virus). Each experimental point is the sum of triplicate experiments with duplicate assay of titer. Error bars represent standard errors of the mean. Asterisks indicate a significant difference from the control (one-sample t test); a, P = 0.0030; b, P = 0.0024; c, P = 0.0006, d, P < 0.0001. δ indicates a significant difference between experimental groups (P < 0.025, one-sample t test).

FIG. 4.

Expression of the 37/67-kDa high-affinity laminin receptor on dengue virus serotype 1 infection. Cell membrane proteins or RNA were prepared separately from normal HepG2 cells (control), mock-infected HepG2 cells (mock), or dengue virus serotype 1-infected cells (MOI = 1) at the time points indicated. Membrane proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a solid matrix support before being probed sequentially with antibodies directed against the 37/67-kDa high-affinity laminin receptor and actin (Western blots). RNA samples were either slot blotted directly onto a solid matrix (slot blots) or separated by electrophoresis through formaldehyde-agarose gels before being transferred to a solid matrix (Northern blots). Slot blots included a control dilution series of total HepG2 RNA and were probed sequentially with digoxigenin-labeled probes against the 37/67-kDa high-affinity laminin receptor and actin. Northern blots were probed sequentially with α-³²P labeled probes against the 37/67-kDa high-affinity laminin receptor and actin.

FIG. 5.

Serotype specificity of the 37/67-kDa high-affinity laminin receptor. HepG2 cells were incubated with either no antibody or 20 μg of anti-37/67-kDa high-affinity laminin receptor antibody or 20 μg of soluble laminin prior to infection with all four serotypes of dengue virus individually. After an incubation of 1.5 viral replication cycles, the level of virus produced was assayed by plaque assay. Results are normalized against the level of individual serotypes of dengue virus produced with no antibody or ligand preincubation. Each experimental point is the sum of triplicate experiments with duplicate assays of titer. Error bars represent standard errors of the mean. Asterisks represent a significant difference from control (one-sample t test); a, P = 0.0103; b, P < 0.0001