Role of heparan sulfate in attachment to and infection of the murine female genital tract by human papillomavirus - PubMed (original) (raw)

Role of heparan sulfate in attachment to and infection of the murine female genital tract by human papillomavirus

Katherine M Johnson et al. J Virol. 2009 Mar.

Abstract

The host factors required for in vivo infection have not been investigated for any papillomavirus. Using a recently developed murine cervicovaginal challenge model, we evaluated the importance of heparan sulfate proteoglycans (HSPGs) in human papillomavirus (HPV) infection of the murine female genital tract. We examined HPV type 16 (HPV16) as well as HPV31 and HPV5, for which some evidence suggests that they may differ from HPV16 in their utilization of HSPGs as their primary attachment factor in vitro. Luciferase-expressing pseudovirus of all three types infected the mouse genital tract, although HPV5, which normally infects nongenital epidermis, was less efficient. Heparinase III treatment of the genital tract significantly inhibited infection of all three types by greater than 90% and clearly inhibited virion attachment to the basement membrane and cell surfaces, establishing that HSPGs are the primary attachment factors for these three viruses in vivo. However, the pseudoviruses differed in their responses to treatment with various forms of heparin, a soluble analog of heparan sulfate. HPV16 and HPV31 infections were effectively inhibited by a highly sulfated form of heparin, but HPV5 was not, although it bound the compound. In contrast, a N-desulfated and N-acylated variant preferentially inhibited HPV5. Inhibition of infection paralleled the relative ability of the variants to inhibit basement membrane and cell surface binding. We speculate that cutaneous HPVs, such as HPV5, and genital mucosal HPVs, such as HPV16 and -31, may have evolved to recognize different forms of HSPGs to enable them to preferentially infect keratinocytes at different anatomical sites.

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Figures

FIG. 1.

Visualization of luciferase-expressing pseudovirus infection in vivo. Quantification of luciferase expression was performed on an IVIS 100 imaging system at 48 h postinfection. Representative animals are shown for the three PV types. The color scale represents expression levels. Note that the scales vary among the three virus types, indicating different luciferase expression levels.

FIG. 2.

Inhibition of in vivo infection with heparin. The luciferase signal following infection with the pseudoviruses, either untreated or with heparin coinstillation, is shown at 48 h postinfection. Each group was composed of 10 animals. The percent inhibition is indicated above the bars. The significance is noted when relevant. Error bars indicate standard errors.

FIG. 3.

Inhibition of in vitro infection with heparin. The effect of heparin (H4784) on pseudovirus infection was determined on HaCaT cells. Pseudovirions that contained an encapsidated RFP-expressing reporter plasmid were utilized. The heparin was serially diluted from 256 μg/ml to 1 μg/ml, and infection was compared to samples with no exogenous heparin. All infections were performed in triplicate. Each of the untreated pseudovirus inocula infected approximately 20% of the cells. Infection was evaluated at 48 h. Error bars indicate standard errors .

FIG. 4.

Inhibition of in vivo infection with polysaccharides. Infection, as measured by luciferase expression, was determined following coinstillation of pseudoviruses with chondroitin-6 sulfate or different heparin formulations as indicated. Each group was composed of three mice. Mice were examined at 48 h postinfection. Error bars indicate standard errors.

FIG. 5.

Pseudovirion binding in the presence of heparin. Genital tissue was excised 4 hours after instillation of the pseudoviruses to evaluate the effect of heparin on tissue binding. Pseudovirions were detected with rabbit polyclonal antisera and Alexa Fluor 488-conjugated donkey anti-rabbit secondary antiserum. HPV16 is shown in panels A and B. HPV31 is shown in panels C and D. HPV5 is shown in panels E and F. The leftmost image in each case (A, C, and E) shows the detection of untreated virus. The rightmost image (B, D, and F) shows detection of virus in the presence of heparin.

FIG. 6.

Interaction of pseudovirions with heparin. The ability of pseudovirions to bind to a heparin HiTrap column was evaluated by Western analyses. Lanes 1, input pseudovirions; lanes 2, pseudovirions present in the flowthrough fraction; lanes 3, pseudovirions present following 0.8 M NaCl elution. untx., profile of untreated pseudovirions; +hep., profile of pseudovirions following incubation with heparin (H4784).

FIG. 7.

Heparinase inhibition of in vivo infection. The luciferase signal at 48 h following infection with the pseudoviruses either of untreated animals or following heparinase treatment (hepx) is shown. Each group was composed of five animals. The percent inhibition is indicated above the bars. Error bars indicate standard errors.

FIG. 8.

Detection of HPV16 binding and laminin 5 following heparinase treatment. Genital tissue was excised 4 hours after instillation of HPV16 pseudovirus to evaluate the effect of heparinase treatment on tissue binding. Detection of HPV16 binding is shown in panel A (untreated) and panel B (heparinase treated). Laminin 5 staining is shown in panel C (untreated) and panel D (heparinase treated).

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Role of heparan sulfate in attachment to and infection of the murine female genital tract by human papillomavirus - PubMed (original) (raw)