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Papillomavirus binding and entry: The heparan sulfate receptor and inhibition by lactoferrin
Umeå University, Faculty of Medicine, Clinical Microbiology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Papillomaviruses (PVs) infect epithelial cells and are the main causative agent of cervical carcinoma. There are today more than a hundred different PV types and these can be divided into high risk and low risk types. They infect differentiating epithelial cells which make it cumbersome to propagate and produce human papillomavirus (HPV) virions. A common method to study HPV infection is to use HPV virus like particles (VLPs) produced in recombinant eukaryotic expression systems. Glycosaminoglycans (GAGs) have been described as an initial attachment receptor for several viruses. Our aim was to study the interactions between HPV VLPs and different GAGs to determine how these could affect binding and internalization. We found that soluble heparin was the best GAG inhibitor of HPV-16 VLP binding followed by heparan sulfate of mucosal origin. We could also see that CHO cells deficient in GAG expression had a reduced ability to bind VLPs, as did cells pretreated with heparinase III. Our results suggested a primary interaction between HPV and heparin sulfate. To be able to study the early steps of internalization we developed a method where we conjugated the CFDA-SE dye to the surface of VLPs. CFDA-SE is activated by cellular esterases inside the cell. This renders the particle fluorescent and thereby visible in flow cytometry analysis. With this new technique we found that entry of the mucosal HPV-6 and HPV-16 was inhibited by heparin. We could also detect differences between mucosal HPV-16 and cutaneous HPV-5 when these where pre-incubated together with GAGs. The cutaneous PV type was not inhibited by heparin to as high degree as the mucosal type. This might be explained by charge differences in the capsid. The mucosal capsid seems to be more positively charged than the epithelial type which should result in a higher affinity for the negatively charged GAGs. Also, we report for the first time that HPV-5 uses a clathrin mediated internalization process. It has been reported for other viruses such as herpes simplex virus (HSV) that lactoferrin, a protein found in high concentrations in breast milk and vaginal fluids could inhibit infection. Interestingly, HSV also use heparan sulfate as a primary attachment molecule. We wanted to investigate if lactoferrin and lactoferricin could have an effect on HPV binding and internalization. We pre-treated HPV-16 VLPs with lactoferrin of bovine or human origin before infection. After incubation we could detect reduced levels of both bound and internalized HPV VLPs to HaCaT cells. In this case, lactoferrin of bovine origin proved to be more efficient in inhibiting both binding and internalization. To further investigate this we used the N-terminal part of lactoferrin, lactoferricin, to study any possible inhibitory effects. Here we found that lactoferricin of bovine origin was a more potent inhibitor of binding, while human lactoferricin was more effective in inhibiting internalization. This could in part be explained by folding differences between these two related proteins. This work further strengthens the proposed interaction between HS and PV for initial interaction and for the first time show charge depending differences between cutaneous and mucosal type binding and internalization and also that lactoferrin and lactoferricin, parts of the innate immune system inhibit PV binding and internalization in vivo.

Place, publisher, year, edition, pages
2005. , 62 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 963
Identifiers
URN: urn:nbn:se:umu:diva-550OAI: oai:DiVA.org:umu-550DiVA: diva2:143794
Public defence
2005-05-20, 00:00 (English)
Available from: 2005-06-08 Created: 2005-06-08 Last updated: 2009-11-11Bibliographically approved
List of papers
1. Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry
Open this publication in new window or tab >>Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry
2003 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 310, no 1, 163-172 p.Article in journal (Refereed) Published
Abstract [en]

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC(50) (50% inhibition) was only 0.5 microg/ml for heparin, 1 microg/ml for dextran sulfate, and 5-10 microg/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate.

Keyword
Papillomavirus; CFDA SE; Heparan sulfate; Uptake, Receptor
Identifiers
urn:nbn:se:umu:diva-2394 (URN)10.1016/S0042-6822(03)00114-4 (DOI)12788640 (PubMedID)
Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2017-12-14Bibliographically approved
2. Mucosal and cutaneous papillomaviruses interact differently with the attachment receptor heparan sulfate on the cell surface.
Open this publication in new window or tab >>Mucosal and cutaneous papillomaviruses interact differently with the attachment receptor heparan sulfate on the cell surface.
Show others...
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4618 (URN)
Available from: 2005-06-08 Created: 2005-06-08 Last updated: 2011-04-05Bibliographically approved
3. Lactoferrin inhibits human papillomavirus binding and uptake in vitro.
Open this publication in new window or tab >>Lactoferrin inhibits human papillomavirus binding and uptake in vitro.
2004 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 64, no 1, 63-68 p.Article in journal (Refereed) Published
Abstract [en]

Lactoferrin (LF), a member of the transferrin family, is a bi-globular protein secreted in milk, saliva, tears, seminal fluid, endocervix and vaginal secretions. LF is an important player in the defence against pathogenic microorganisms and has also been shown to have activity against several viruses including herpesvirus, adenovirus, rotavirus and poliovirus. The antiviral activity of LF is directed against the early steps of viral infection and the LF antiviral effect against herpesvirus is mediated through LF binding to the herpesvirus receptor heparan sulfate. Human papillomavirus (HPV) causes genital warts and is a prerequisite for cervical cancer. HPV can also use heparan sulfate on the cell surface as a receptor. We studied the inhibition by LF on HPV entry by incubating HaCaT cells and HPV-16 virus-like particles (VLPs) with either human (HLF) or bovine lactoferrin (BLF). LF inhibited internalization of HPV-16 particles using CFDA-SE-labelled VLPs that only fluoresce after internalisation. By using a western blot assay we also found dose-dependent LF inhibition of HPV-16 VLP binding to the HaCaT cell surface. BLF was a more potent inhibitor of HPV entry than human LF. It was also clear that LF acted early in the HPV uptake process.

Keyword
Lactoferrin, Human papillomavirus, Heparan sulfate, Antiviral activity
Identifiers
urn:nbn:se:umu:diva-20707 (URN)10.1016/j.antiviral.2004.05.005 (DOI)15451180 (PubMedID)
Available from: 2009-03-24 Created: 2009-03-24 Last updated: 2017-12-13
4. The anti-HPV activity of human and bovine lactoferricin.
Open this publication in new window or tab >>The anti-HPV activity of human and bovine lactoferricin.
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4620 (URN)
Available from: 2005-06-08 Created: 2005-06-08 Last updated: 2011-04-05Bibliographically approved

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