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Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
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.

Place, publisher, year, edition, pages
2003. Vol. 310, no 1, 163-172 p.
Keyword [en]
Papillomavirus; CFDA SE; Heparan sulfate; Uptake, Receptor
Identifiers
URN: urn:nbn:se:umu:diva-2394DOI: 10.1016/S0042-6822(03)00114-4PubMedID: 12788640OAI: oai:DiVA.org:umu-2394DiVA: diva2:140371
Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2011-04-05Bibliographically approved
In thesis
1. Human papillomavirus tropism: determinants of viral tissue specificity
Open this publication in new window or tab >>Human papillomavirus tropism: determinants of viral tissue specificity
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cervical cancer is the second most common cancer among women worldwide and human papillomavirus (HPV) is a prerequisit for the development of this cancer. HPV belongs to the Papillomaviridae family and infects the basal layer of epithelial cells where it generally progresses into warts or condylomas. HPV can only reproduce in differentiating epithelia and it is therefore difficult to study the natural infection of HPV. More than 100 HPV types exist and they are divided into different genera based on their L1 open reading frame sequence. Most of the HPV types in the alpha-papillomavirus genus infect the mucosal epithelium while HPVs from the beta-papillomavirus genus usually infect cutaneous epithelial cells. Presently, it is not known what decides the anatomical tropism and our aim was to study determinants of this tropism.

By using HPV virus like particles (VLP) and pseudovirus we found that VLPs from the two alpha-papillomaviruses HPV-6 and HPV-16 interacted with cell-surface heparan sulfate (HS) for initial attachment. When we labelled HPV VLPs with a fluorescent dye to study internalization HPV-6 was more strongly inhibited than HPV-16. Furthermore, a pseudovirus infection assay demonstrated that the beta-papillomavirus HPV-5 was less dependent on HS for infection than HPV-16. By analyzing the isoelectric point (p1) of the HPV L1 capsid protein we found that alpha HPV types were more positively charged than beta HPV types. Also, HPV-6 had a higher positive charge than HPV-16. Thus, the inhibition of the negatively charged heparin against HPV infection was clearly related to the charge of the HPV L1 capsid. This suggested that the initial interaction could be one of the determinants of tropism although not the sole factor.

Lactoferrin is a protein found in milk, saliva, semen, tear fluid and endocervical secretions that has antiviral activities. Both human and bovine lactoferrin inhibited HPV infection but we found no significant differences in inhibition of alpha- and beta-papillomavirus infection. We could however demonstrate that different lactoferricins, small peptide derivates from the N-terminal part of lactoferrin, were able to inhibit HPV infection. This antiviral activity depended on lactoferricin peptide, HPV type and cell origin.

The regulation of HPV gene expression in the host cell could also determine HPV tropism. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. It has been hypothesized that the combination of transcription factors in the host cell determines the cell-type-specific expression. In cells with a skin origin the HPV-5 LCR was twice as efficient in transcriptional activation compared to HPV-16 LCR, while in cervical cells the HPV-16 LCR was almost twice as effective in activating transcription compared to HPV-5 LCR.

To conclude, alpha- and beta-papillomaviruses differed regarding their ability to infect cells and regulate viral gene expression. These abilities corresponded with their natural host cells and suggested that HPV anatomical tropism could be determined at several steps in the HPV life cycle.

Place, publisher, year, edition, pages
Umeå: Klinisk mikrobiologi, 2007. 57 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1105
Keyword
papillomavirus, receptor, heparan sulfate, pI, lactoferrin, lactoferricin, transcription, long control region, tropism
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-1149 (URN)978-91-7264-332-1 (ISBN)
Public defence
2007-06-05, Astrid Fagraeus Salen, 6A 103, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2009-06-15Bibliographically approved
2. Papillomavirus binding and entry: The heparan sulfate receptor and inhibition by lactoferrin
Open this publication in new window or tab >>Papillomavirus binding and entry: The heparan sulfate receptor and inhibition by lactoferrin
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.

Publisher
62 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 963
Identifiers
urn:nbn:se:umu:diva-550 (URN)
Public defence
2005-05-20, 00:00 (English)
Available from: 2005-06-08 Created: 2005-06-08 Last updated: 2009-11-11Bibliographically approved

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