Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Sep;80(18):8940-50.
doi: 10.1128/JVI.00724-06.

Keratinocyte-secreted laminin 5 can function as a transient receptor for human papillomaviruses by binding virions and transferring them to adjacent cells

Timothy D Culp  1 , Lynn R Budgeon, M Peter Marinkovich, Guerrino Meneguzzi, Neil D Christensen
Affiliations

Keratinocyte-secreted laminin 5 can function as a transient receptor for human papillomaviruses by binding virions and transferring them to adjacent cells

Timothy D Culp et al. J Virol. 2006 Sep.

Abstract

Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing alpha6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking alpha6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing alpha6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
HPV capsids bind LN5 in the ECM of cultured keratinocytes. (a) Low-passage primary human vaginal keratinocytes were grown on glass coverslips for 24 h. After fixation and blocking with BSA, cells were first incubated with HPV-11 VLPs for 20 min. Coverslips were next incubated simultaneously with two antibodies, one to indicate bound VLPs (H11.H3) (green) and the other to indicate either α6 integrin (GoH3) or LN5 (ab14509) (red). This incubation was followed by labeled secondary antibodies and nuclear staining with Hoechst (blue). (b) HaCaT cells were removed from coverslips with EDTA, fixed with methanol, and blocked with BSA. Coverslips were first incubated for 2 h at 4°C with anti-LN5 (ab14509) or Rb IgG (all bound rabbit antibodies are indirectly labeled red), followed by binding with authentic HPV-11 virions (green) for 20 min. Coverslips were subsequently treated with H11.H3 and then with secondary antibodies and Hoechst stain. Similar results were obtained using VLPs and the fixed ECM of vaginal keratinocytes. (c) A blocking experiment was performed on ice using the unfixed ECM of HaCaT cells. As indicated in the panels in the first column, BSA-blocked coverslips were incubated initially either with GoH3 (first row), anti-LN5 (ab14509) (third row), rat IgG or rabbit IgG (not shown) or with HPV-11 L1-VLPs (second and fourth rows). After rinsing, coverslips were next incubated with the alternate treatment indicated in the panels of the second column (antibodies or VLPs). All coverslips were then incubated with H11.H3 to indicate bound VLPs followed by appropriate secondary antibodies and Hoechst stain (blue [absent, indicating successful cell removal]). α6 integrin and LN5 are shown as red, while bound VLPs are green. Bars, 50 μm.
FIG. 2.
FIG. 2.
Binding of HPV particles to purified LN5. (a) For ELISA, triplicate wells of a 96-well microtiter plate were precoated with purified LN5, laminin from human placenta (LNmix), BSA, or heparin-BSA (hep-BSA) as described in Materials and Methods. After the wells were blocked, selected wells were incubated for 1 h with anti-LN5 rabbit serum or control Rb serum (shown in brackets). The entire plate was then rinsed and incubated with HPV-11 virions or L1-only VLPs followed by H11.B2 MAb to detect bound capsids. In parallel experiments, the commercially available anti-LN5 (ab14509) effectively blocked virion and VLP adsorption, while the control antibody (anti-LN [AB1953]) had no effect (data not shown). Values are means ± standard errors of the means (S.E.M.) (error bars). (b) For coimmunoprecipitation experiments, HPV-11 L1 VLPs or BSA were preincubated with (+) or without (−) purified LN5 and then mixed with protein A agarose prebound to anti-LN5 or rabbit IgG. Agarose complexes were washed to remove unbound proteins and analyzed by SDS-PAGE and immunoblotting for L1. Lane 1 contains input VLPs (2.5 μg). Lane 2 contains protein A agarose only (A). Lanes 3, 4, 5, and 7 contain protein A agarose conjugated to ab14509 (αLN5). Lane 6 contains protein A agarose conjugated to rabbit IgG (IgG).
FIG. 3.
FIG. 3.
Confocal images of human cervical mucosa bound with HPV particles. HPV-6 VLPs, HPV-11 VLPs, or HPV-11 virions were incubated with sections of cervical mucosa for 45 min followed by H6.B10.5 or H11.B2 MAb (green), and anti-LN5 (ab14509) (red). No-particle control sections were treated with all three primary antibodies. All sections were subsequently incubated with labeled secondary antibodies and Hoechst stain (blue [not shown only in the rightmost panels]). Insert to aid in tissue orientation shows an hematoxylin and eosin stain of a companion cervical section. The location of the outer surface of the mucosa where present (S) is indicated. The leftmost panels show triple staining of mucosa at a magnification of ×45 (bars, 150 μm). The remaining three sets of panels show ×227 image including BM (bars, 30 μm). Hoechst staining is not shown in the fourth column merge for greater clarity.
FIG. 4.
FIG. 4.
Blocking of basement membrane with anti-LN5 prior to capsid binding. Sequential sections of cervical mucosa were incubated for 40 min with anti-LN5 or rabbit IgG antibody (Ab) at 25 μg/ml followed by rinsing and incubation with VLPs or virions for 45 min. Sections were then exposed to H6.B10.5 or H11.B2 together with a low concentration of anti-LN5 (to indicate the position of the BM) followed by fluorescently labeled secondary antibodies and Hoechst stain (not shown). Gray-scale images show green channel only (bound particles). The location and angle of LN5 staining are shown by the white arrows for orientation. The location of the outer surface of the mucosa (S) is indicated. Bars, 50 μm.
FIG. 5.
FIG. 5.
Blocking the binding of HPV-11 virions to cervical sections using HPV-16 VLPs. Sequential sections of cervical mucosa were treated with HPV-16 L1-VLPs or with BSA only prior to incubation with authentic HPV-11 virions. Sections were then incubated with H11.A3.2 (red) and anti-LN5 (green) followed by appropriate secondary antibodies and Hoechst stain (blue). Triple staining is shown in column A. LN5 and HPV-11 virions are shown in column B, and virions alone are shown in column C. The wide hollow arrows in the BSA-blocked control section (top right) show virions bound to the suprabasal mucosa, while the narrow solid arrows indicate virions bound to the BM. Binding to both areas was blocked in the sequential cervical section that was first treated with HPV-16 VLPs. The location of the outer surface of the mucosa (S) is indicated. Bars, 50 μm.
FIG. 6.
FIG. 6.
Infection of cultured cells by HPV-11 virions preadsorbed to various substrates. (a) Wells of a cell culture plate were coated with the ECM of COS-7 cells (C-ECM) or HaCaT cells (H-ECM) using EDTA removal of cells as described in Materials and Methods. Empty control wells were also treated with EDTA (EDTA-PS). All substrates were rinsed two times with PBS following EDTA treatment and prior to immediate incubation with HPV-11 virions suspended in culture medium for 5 h at 37°C. Following this incubation for viral attachment, all wells were rinsed repeatedly with PBS to remove unattached virions. Wells were then seeded with uninfected HaCaT, KH-SV, or BOUA-SV (BO-SV) cells or COS-7 cells or in a separate experiment, with human vaginal keratinocytes (HVK). (b) As described above for panel a, EDTA-resistant ECM substrates produced by COS-7 (C-ECM) or HaCaT cells (H-ECM) or EDTA-treated control wells (EDTA-PS) were incubated with HPV-11 virions in culture medium at 37°C, rinsed, and seeded with uninfected BOUA-SV-neo, BOUA-SV-α6, or HaCaT cells. In panels a and b, viral E1^E4 transcripts were assayed by QRT-PCR. The levels of viral transcripts (means ± standard errors of the means [error bars] of replicates) for each cell line are shown relative to the E1^E4 transcript levels measured in control wells (EDTA-PS) for that same cell line (horizontal dashed line). Values that were significantly different from the values for EDTA-PS wells for that cell line (P values of <0.05 by Student's t test) are indicated by asterisks.
FIG. 7.
FIG. 7.
Effects of anti-LN5 and anti-α6 integrin antibodies on HPV infection. (a) Wells precoated with HaCaT ECM were incubated with medium only (mock), LN5 antiserum (1:25), or control rabbit serum (1:25) (control) for 3.5 h at 4°C. Unbound antibodies were removed by rinsing, and wells were treated with HPV-11 virions for 30 min at 37°C. Unbound particles were removed by rinsing prior to seeding each well with uninfected HaCaT cells. Viral E1^E4 transcripts were assayed by QRT-PCR. (b) 75% confluent HaCaT cells were inoculated with HPV-11 virions (150 particle/cell) in 1 ml growth medium and immediately treated with 5 μg of a control MAb (control), GoH3, or heat-denatured GoH3 (dGoH3), with a companion well left untreated (post). Twelve hours postinfection, all monolayers were rinsed and given fresh medium containing neutralizing MAb (H11.H3). Six hours later, the previously untreated well (post) was treated with 5 μg GoH3 to examine nonspecific effects of this antibody on viral infection. Infection was assayed by QRT-PCR. (c) Wells of a 96-well culture plate were precoated with LN5-rich ECM or left untreated prior to blocking with BSA (see Materials and Methods). Blocked wells were incubated with pseudovirions for 1 h at 37°C, rinsed, and seeded with uninfected 293TT cells. Three days later, the spent medium was analyzed for levels of seAP. BPV-1, bovine papillomavirus type 1; CRPV, cottontail rabbit papillomavirus. (d) Wells of a 96-well plate were precoated with LN5-rich ECM (ECM of HaCaT cells [H-ECM]) or heparin-BSA and then blocked with BSA. Select wells were then treated with 2 μg anti-LN5 or rabbit IgG (in BSA) for 2 additional hours at 4°C. All wells were then rinsed and incubated with HPV-11 pseudovirions for 1 h at 37°C. Wells were rinsed again and seeded with 293TT cells as described above. (e) HPV-11 pseudovirions were incubated for 1 h in BSA-blocked wells which were precoated with either LN5-rich ECM or heparin-BSA. After the wells were rinsed, they were seeded with 293TT cells, and select wells were immediately treated with 0.5 μg GoH3 or rat IgG. Values that were significantly different from the values for wells treated with the control antibody (P values of <0.05 by Student's t test) are indicated by asterisks.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andriessen, M. P., J. van den Born, M. A. Latijnhouwers, M. Bergers, P. C. van de Kerkhof, and J. Schalkwijk. 1997. Basal membrane heparan sulphate proteoglycan expression during wound healing in human skin. J. Pathol. 183:264-271. - PubMed
    1. Aumailley, M., A. El Khal, N. Knoss, and L. Tunggal. 2003. Laminin 5 processing and its integration into the ECM. Matrix Biol. 22:49-54. - PubMed
    1. Becker, C., P. Buttler, and H. G. Graber. 2002. Influence of anti-CD49f and anti-CD29 monoclonal antibodies on mitotic activity of epithelial cells (HaCaT) and gingival fibroblasts in vitro. Eur. J. Oral Sci. 110:137-143. - PubMed
    1. Bobardt, M. D., A. C. Saphire, H. C. Hung, X. Yu, B. Van der Schueren, Z. Zhang, G. David, and P. A. Gallay. 2003. Syndecan captures, protects, and transmits HIV to T lymphocytes. Immunity 18:27-39. - PubMed
    1. Boukamp, P., R. T. Petrussevska, D. Breitkreutz, J. Hornung, A. Markham, and N. E. Fusenig. 1988. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106:761-771. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

-