HSV-1 exploits the innate immune scavenger receptor MARCO to enhance epithelial adsorption and infection

Abstract

Herpes simplex virus type 1 is an important epithelial pathogen and has the potential for significant morbidity in humans. Here we demonstrate that a cell surface scavenger receptor, macrophage receptor with collagenous structure (MARCO), previously thought to enhance antiviral defense by enabling nucleic acid recognition, is usurped by herpes simplex virus type 1 and functions together with heparan sulphate proteoglycans to mediate adsorption to epithelial cells. Ligands of MARCO dramatically inhibit herpes simplex virus type 1 adsorption and infection of human keratinocytes and protect mice against infection. Herpes simplex virus type 1 glycoprotein C closely co-localizes with MARCO at the cell surface, and glycoprotein C binds directly to purified MARCO with high affinity. Increasing MARCO expression enhances herpes simplex virus type 1 infection while MARCO(-/-) mice have reduced susceptibility to infection by herpes simplex virus type 1. These findings demonstrate that herpes simplex virus type 1 binds to MARCO to enhance its capacity for disease, and suggests a new therapeutic target to alter pathogenicity of herpes simplex virus type 1 in skin infection.

Figure 1. Scavenger receptor ligands inhibit HSV-1 infection of keratinocytes

a, HaCats were treated with 100μg/ml Poly(I:C), or 50μg/ml of Poly(I) or Poly(C) 20 minutes before the addition of HSV-1 at an MOI of 0.01. Cells were incubated for 5.5 hours before quantification of HSV-1 mRNA by qPCR. b, NHEK were treated with 10μg/ml Poly(I), Poly(C) and Poly(I:C) for 3 hours before qPCR analysis of gene expression. c, NHEK were treated with 10μg/ml of the indicated compounds for 20 min prior to the addition of HSV-1 at an MOI of 0.0005. PFU were quantified 48 hours after infection. d, NHEK were treated with 10μg/ml Poly(I) or heparin 20 min before infection with WT HSV-1 at an MOI of 0.0005 or an equivalent amount of HSV-1 gCΔC5/P (HSV-1 possessing gC with a deletion of the heparin binding domain, amino acids 33-123) viral particles. PFU were quantified 48 hours after infection. PFU are displayed as relative % compared to untreated cells for each virus. NS, not significant. a-d, all data are means ± s.e.m, n=3 from representative experiments repeated at least two times. One-way ANOVA with Tukey post-tests were used for statistical analysis, ** P<0.01; *** P<0.001. b,c, P values were derived from comparisons to vehicle treated samples.

Figure 2. Effect of scavenger receptor ligands on adsorption of HSV-1 and purified gC

a, HaCat keratinocytes were incubated with HSV-1 at an MOI of 100 at 4°C with the indicated concentrations of Poly(I) and Poly(C). Bound HSV-1 was visualized by fluorescence microscopy. Five images of bound HSV-1 per concentration were captured and the number of HSV-1 particles quantified using ImageJ. Each image contained 15-20 cells. Error bars indicate s.e.m. Two-way ANOVA with bonferroni post-tests was used to compare the effect of Poly(I) to Poly(C). n=5, *** P<0.001. b, purified gC was incubated with NHEK in the presence of 100μg/ml heparin, Poly(I), or Poly(C) at 4°C. Unbound gC was removed by multiple wash steps, then cells were fixed and bound gC was detected and quantified using an on-cell western assay. Error bars indicate s.e.m. One-way ANOVA with Tukey post-tests were used for statistical analysis with comparisons made to vehicle treated cells, n=2, ** P<0.01. ND, not detectable, NS, not significant. c,d, 100μg of purified gC was bound to a Heparin column, and eluted with a linear gradient (broken line) of Poly(I) (c), or NaCl (d). Eluted gC was quantified from 1ml fractions by dot blot and plotted as solid black circles representing each individual fraction connected by a solid line.

Figure 3. MARCO co-localizes with HSV-1 gC on the keratinocyte cell surface

NHEK were incubated in the presence of purified HSV-1 gC for 2 hours at 4°C to allow binding of the glycoprotein to the cell surface. Unbound gC was removed by multiple wash steps before cells were fixed and incubated with both a mouse monoclonal antibody targeting HSV-1 gC, and rabbit polyclonal antibodies targeting MARCO (a), syndecan-1 (b), OLR1 (c) or SCARA3 (d). Physical proximity of MARCO, syndecan-1, OLR1 or SCARA3 to HSV-1 gC bound to the cell surface was determined using a fluorescence-based proximity ligation assay that produces a red fluorescent signal only when the antigens recognized by the antibodies utilized in the assay reside within less than 40nm of each other. Nuclei (blue) are stained with Hoescht. Scale bar = 100μm. All data are from representative experiments repeated 2-4 times.

Figure 4. MARCO binds directly to HSV-1 gC

Plate-bound purified MARCO protein was incubated with increasing concentrations of purified gC (a), gB (c) or gCΔC5/P (gC with a deletion of the heparin binding domain, amino acids 33-123) (e). gC, gB, and gCΔC5/P remaining bound after multiple wash steps was detected and quantified by ELISA. The dissociation constant (K_D) and the maximum number of receptor binding sites (B_max) were determined using nonlinear regression with background subtracted using Graphpad Prism. In (b) and (f), data in (a) and (e), respectively, were transformed to create double-reciprocal plots to show linear binding kinetics. d, plate-bound recombinant OLR-1 was incubated with purified gC. gC remaining bound after washing was detected and quantified by ELISA. a-f, all individual replicate values plotted with offset overlapping points. All data are from representative experiments repeated at least two times.

Figure 5. HSV-1 infection correlates with MARCO expression in cells and in mice and is reduced by Poly(I)

a-d, HaCat cells were transfected with a control plasmid, pcDNA3 or a pcDNA3-backbone plasmid with the human MARCO gene inserted (labeled as [MARCO] on graphs) and selected with G418 to enable stable expression. MARCO and GAPDH protein levels were visualized in the same sample of 20μg total protein using two-color western blot (a). The image in (a) is from one representative Western blot of two blots, and the image of the full-length blot is presented in Supplementary Fig. S4. MARCO protein levels relative to GAPDH were quantified using Licor Odyssey software (b), n=2, error bars indicate the s.e.m. c,d, pcDNA3 and [MARCO] cells were infected with WT or HSV-1 gC null (HSV-1 lacking gC) at an MOI of 0.0005. PFU were quantified 48 hours after infection (c) and viral DNA was quantified 24 hours after infection (d). c,d, comparisons between pcDNA3 and [MARCO] cells were made using two-tailed T-tests, n=3. Error bars indicate the s.e.m. Data presented are from one representative experiment of at least two independent experiments. e, 16 week old female mice were immunocompromised with cyclophosphamide prior to infection with HSV-1. Mice were injected subcutaneously with PBS, or 125μg Poly(C) or Poly(I) twice daily at the site of infection beginning the first day of infection. Photographs were taken once daily beginning the first day post-infection. Lesions were quantified using ImageJ. Error bars indicate the s.e.m. Two-way ANOVA with bonferroni post-tests was used to compare wound sizes in Poly(I) and Poly(C) treated mice to PBS treated mice, n=4, ** P<0.01; *** P<0.001. f, 11 week old sex-matched wildtype (WT) and MARCO^-/- mice were immunocompromised with cyclophosphamide prior to infection with HSV-1. Mice were photographed daily until 8 days post-infection when WT mice began to exhibit symptoms of systemic and neurological infection, necessitating euthanasia. Lesion formation was quantified using ImageJ. Error bars indicate the s.e.m. Two-way ANOVA with bonferroni post-tests was used to compare wound sizes in WT and MARCO^-/- mice, n=5; ** P<0.01. Data presented are from one representative experiment of two independent experiments.