Interaction of human papillomavirus type 16 particles with heparan sulfate and syndecan-1 molecules in the keratinocyte extracellular matrix plays an active role in infection - PubMed (original) (raw)
Interaction of human papillomavirus type 16 particles with heparan sulfate and syndecan-1 molecules in the keratinocyte extracellular matrix plays an active role in infection
Zurab Surviladze et al. J Gen Virol. 2015 Aug.
Abstract
Oncogenic human papillomaviruses (HPVs) attach predominantly to extracellular matrix (ECM) components during infection of cultured keratinocytes and in the rodent vaginal challenge model in vivo. However, the mechanism of virion transfer from the ECM to receptors that mediate entry into host cells has not been determined. In this work we strove to assess the role of heparan sulfate (HS) chains in HPV16 binding to the ECM and determine how HPV16 release from the ECM is regulated. We also assessed the extent to which capsids released from the ECM are infectious. We show that a large fraction of HPV16 particles binds to the ECM via HS chains, and that syndecan-1 (snd-1) molecules present in the ECM are involved in virus binding. Inhibiting the normal processing of snd-1 and HS molecules via matrix metalloproteinases and heparanase dramatically reduces virus release from the ECM, cellular uptake and infection. Conversely, exogenous heparinase activates each of these processes. We confirm that HPV16 released from the ECM is infectious in keratinocytes. Use of a specific inhibitor shows furin is not involved in HPV16 release from ECM attachment factors and corroborates other studies showing only the intracellular activity of furin is responsible for modulating HPV infectivity. These data suggest that our recently proposed model, describing the action of HS proteoglycan processing enzymes in releasing HPV16 from the cell surface in complex with the attachment factor snd-1, is also relevant to the release of HPV16 particles from the ECM to promote efficient infection of keratinocytes.
Figures
Fig. 1.. Effect of furin inhibitor on HPV16 binding to and release from ECM, internalization by HKs and infection. (a, c–e) HPV16 PsVs were bound to cell-free ECM in the presence or absence of 1.3 μM furin inhibitor. Unbound virus particles were intensively washed away; treatments were as indicated. (a–d) The final products were extracted with RIPA buffer, fractionated by SDS-PAGE, and analysed by immunoblot for HPV16 L1 (ns is a non-specific cellular protein). Results in each panel are representative of two or three experiments; error bars represent the sem. (a) HPV16 PsVs bound to the ECM. (b) HPV16 release in the presence of furin. ECM-bound virus was incubated with 1.5 ml complete medium (CM), with or without 3 nM His-furin at 37 °C for 3 h. Virus released into the medium was recovered by pull-down then analysed as in (a). (c) HPV16 PsV release from ECM. ECM-bound virus was incubated with 1.5 ml CM at 37 °C for 4 h with and without furin inhibitor. Virus released into the medium was recovered and analysed as in (a, b). (d) HPV16 entry assay. HaCaT cells were seeded atop ECM-bound HPV16 and incubated for 4 h at 37 °C in the presence or absence of furin inhibitor. Extracellular virus was removed as detailed in Methods, and intracellular HPV levels analysed by immunoblot. Graphed is the mean of four independent experiments. (e) Infectious entry of HPV16. HPV16 PsVs were bound to ECM; infectious transfer of virus to HaCaT cells in the absence or presence of furin inhibitor was measured at 24 h post-infection. HaCaT cells were pre-incubated at 37 °C for 1 h with furin inhibitor. Extracellular furin inhibitor was removed by intensive washing and cells were added to the ECM-bound HPV16. Relative infectivity was scored 24 h post-infection. (f, g) Furin activity in EL fractions. HPV16-exposed HaCaT cells were incubated for 16 h with or without 3 μM furin inhibitor 1. An equal amount of purified EL fraction lysate was fractionated by SDS-PAGE and analysed for capsid proteins using rabbit polyclonal HPV16 antibody. CD63 (an EL fraction-specific protein detected with anti-CD63 mAb) was detected as a loading control (f). (g) Furin activity in EL fractions of HKs growing in the presence or absence of 3 μM furin inhibitor 1 for 3 h.
Fig. 2.. Effect of MMP and heparanase inhibitors on ECM binding and release, and infectious transfer of HPV16 from the ECM to keratinocytes. (a) HaCaT cell ECM was incubated with HPV16 PsVs in the presence of 1 μM batimastat, 20 μM OGT2115, or with a mixture of both. After 1 h at room temperature, unbound PsVs were removed by intensive washing, and ECM-bound virus was solubilized and analysed as described in Fig. 1(a). (b) HPV-bound ECM was incubated at 37 °C for 3 h with complete medium (CM), or with CM supplemented with 1 μM batimastat or 20 μM OGT2115. Conditioned media were analysed for released HPV16 as described in Fig. 1(b). (c) HaCaT cells were seeded atop ECM-bound HPV16 PsVs and incubated at 37 °C in the presence of 1 μM batimastat or 20 μM OGT2115. Relative pseudoinfection was scored after 24 h. Error bars represent the sem of three independent experiments.
Fig. 3.. Effect of ECM-protein cross-linking on HPV16-ECM interactions. ECM proteins were cross-linked with DTSSP prior to HPV16 PsV exposure. Unbound particles were removed by intensive washing and ECM-bound virions were subjected to various treatments. The products were analysed by SDS-PAGE and immunoblot. Relative protein levels (shown below the blots) determined by densitometry. (a) HPV16 PsVs bound to native or DTSSP xECM for 1 h. (b) Native or xECM analysed for levels of intact LN-332. (c) Native or xECM pre-bound with HPV16 were incubated at 37 °C for 20 h. Conditioned medium was subjected to the soluble HPV16 pull-down assay. (d) HPV16 bound to native and xECM was incubated at 37 °C for 2 h in the presence or absence of 0.1 U heparinase III. Virus released into the conditioned medium was analysed for HPV16 L1 content as in (c).
Fig. 4.. Analysis of HK-secreted ECM for shed forms of snd-1.Native or xECM solubilized directly in Laemmli buffer, fractionated by SDS-PAGE and analysed by immunoblot. (a) Snd-1 content in ECM. (b, c) HPV16 PsVs were bound to native ECM for 1 h; unbound virus was removed by intensive washing and extracted with RIPA buffer. IP was performed using anti-snd-1 rabbit polyclonal antibody coupled to Protein A-magnetic beads. Immunoblot was performed for snd-1 (see Fig. S4), HPV16 L1 (b) and LN-332 (c). Three independent experiments verified that PsVs associate with snd-1 in the ECM.
Fig. 5.. Effect of ECM cross-linking on HPV16 release from the ECM, and internalization into and infection of keratinocytes. HaCaT cells were seeded over virus-bound native or xECM in the presence or absence of 0.1 U heparinase III, and incubated at 37 °C. (a) Conditioned media were collected after 20 h and analysed for the content of released virus. (b) HPV16 entry into HaCaT cells grown atop ECM-bound virus was measured after 3.5 h, as described in Figs. 1(d) and S2. The cellular non-specific band (ns) served as a loading control. L1 band intensities were quantified by densitometry (a, b). (c) HPV16 PsVs were bound to native and xECM. Unbound virus was removed and HaCaT cells were seeded atop HPV-ECM in the presence or absence of 0.1 U heparinase III. Infection was assayed 24 h later. Error bars indicate the sem from two independent experiments performed in triplicate.
Fig. 6.. Dose-responsive infection of HKs by HPV16 PsVs released from ECM. HaCaT cell-derived ECM was incubated with ∼3 × 108 viral genome equivalents (vge) per well HPV16 PsVs at 37 °C for 2.5 h. Conditioned medium was collected and quantified for HPV16 PsVs. HaCaT cells were incubated with serial dilutions of the ECM-released HPV16 and infection was scored after 24 h. Raw data were normalized to protein content and infectivity plotted as a function of exposure dose. Error bars represent the sem of three replicates.
Fig. 7.. Schematic presentation of ECM-bound HPV16 release and infectious entry in HKs. (a) Highly charged HPV molecules may bind many ECM constituents (e.g. snd-1 or LN-332) by direct protein–protein interaction; however, a considerable amount of virus binds via the proteoglycan HS chains. Heparanase (i) or proteases (ii) process the ECM, resulting in release of virus attached to fragments of ECM-resident HS or proteins, respectively. Soluble virus can attach to HKs via the specificity of ligands in complex with HPV16 binding to cognate receptors (iii), or less specifically (iv). (b) Cross-linking of ECM-resident proteins with DTSSP (X) does not affect HPV16 binding, but inhibits virus release by preventing protease digestion of the ECM. HPV binds to the HS in the ECM or to GF-decorated HS chain of shed snd-1. Heparinase III catalyses virus particle released still in complex with some ligands (e.g. GFs) attached to HS chains (v) or with fragments of HS (vi). Liberated HPV could attach to HKs by direct protein–protein interaction of charged capsids with non-specific receptors (vii). Virus released by HS processing could bind to HS-binding HK receptors (viii) or cognate receptors of HS-interacting ligands/GFs complexed with HPV16 (ix).
References
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