A role for heparan sulfate in viral surfing

Abstract

Heparan sulfate (HS) moieties on cell surfaces are known to provide attachment sites for many viruses including herpes simplex virus type-1 (HSV-1). Here, we demonstrate that cells respond to HSV-1 infection by enhancing filopodia formation. Filopodia express HS and are subsequently utilized for the transport of HSV-1 virions to cell bodies in a surfing-like phenomenon, which is facilitated by the underlying actin cytoskeleton and is regulated by transient activation of a small Rho GTPase, Cdc42. We also demonstrate that interaction between a highly conserved herpesvirus envelope glycoprotein B (gB) and HS is required for surfing. A HSV-1 mutant that lacks gB fails to surf and quantum dots conjugated with gB demonstrate surfing-like movements. Our data demonstrates a novel use of a common receptor, HS, which could also be exploited by multiple viruses and quite possibly, many additional ligands for transport along the plasma membrane.

Figure 1. Exogenous HSV-1 can induce filopodia formation

(A) Increase in the number of filopodia upon exposure to HSV-1 were counted for cells indicated. Counting was done 15–30 min before and after the addition of virus. Numbers are represented as percentages to better compare different cell lines. 3 independent experiments were counted for each cell type (n=25). Numbers of filopodia are presented as means with error bars showing standard deviation. (B) Western Blot analysis confirms activation of Cdc42 at the time points indicated. (C) Down-regulation of Cdc42 inhibits HSV-1 entry. HeLa cells were transfected with siRNA against Cdc42 or control siRNA and exposed to a β-galactosidase expressing HSV-1(KOS) virus.

Figure 2. HSV-1 binds and surfs along filopodia

(A) Time course of K26GFP virus (green) surfing (arrow) on a single Vero cell at indicated time points. Cells were infected at an approximate MOI of 200. (B) Representative binding of K26GFP virus to filopodia. To quantify virus binding, the average fluorescent intensity of a selected region of interest on filopodia was plotted over time. (C) Tracking of viral particles demonstrates an average particle speed of 1.5 μm/min. To quantify viral surfing speed, 8 to 10 particles per cell were tracked every 30 s and the distance traveled was plotted over time.

Figure 3. Heparan sulfate and gB are important for attachment to filopodia and surfing

(A) Anti-Heparan sulfate staining of Vero and CHO-K1 cells. As a negative control, primary antibody was omitted. FITC conjugated secondary anti-mouse IgG (SIGMA) was used in 1:200 dilution. (B) Western Blot Analysis confirming gB and gD presence in cell lysates. (C) Quantification of the average green intensity around the cell membrane and filopodia for gB and gD. 20 μg of total protein from gB and gD lysates were taken and incubated with monoclonal antibodies (Virusys Corp and Abcam, respectively) before using as probes on cells. The figure represents 3 independent experiments (n=11). Data are presented as means with error bars showing standard deviations. (D) Quantification of Heparinase treated cells and gB null virus. Average green intensity at filopodia was measured after 1 h of treatment with the enzyme or incubation with the mutant virions. Error bars show standard deviations. (E) Quantum dots (red) conjugated to gB via an antibody were used for demonstration of binding of gB to filopodia and its ability to surf on HeLa cells.