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Analysis of Prototype Foamy Virus particle-host cell interaction with autofluorescent retroviral particles

Kristin Stirnnagel1, Daniel Lüftenegger15, Annett Stange1, Anka Swiersy1, Erik Müllers1, Juliane Reh1, Nicole Stanke1, Arend Große1, Salvatore Chiantia2, Heiko Keller2, Petra Schwille2, Helmut Hanenberg3, Hanswalter Zentgraf4 and Dirk Lindemann1*

Author Affiliations

1 Institut für Virologie, Medizinische Fakultät "Carl Gustav Carus", Technische Universität Dresden, Dresden, Germany

2 Biophysics, BIOTEC, Technische Universität Dresden, Dresden, Germany

3 Department of Pediatric Oncology, Hematology & Clinical Immunology, Children's Hospital, Heinrich Heine University, Düsseldorf, Germany

4 Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany

5 Current Address: ViroLogik GmbH, Henkestr. 91, 91052 Erlangen, Germany

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Retrovirology 2010, 7:45  doi:10.1186/1742-4690-7-45

Published: 17 May 2010

Abstract

Background

The foamy virus (FV) replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive.

Results

In order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP) fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction.

Conclusions

We have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV - host cell interactions using modern fluorescent imaging techniques. Furthermore, we succeeded for the first time in identifying two cell lines resistant to Prototype Foamy Virus Env-mediated gene transfer. Interestingly, both cell lines still displayed FV Env-dependent attachment of fluorescent retroviral particles, implying a post-binding block potentially due to lack of putative FV entry cofactors. These cell lines might ultimately lead to the identification of the currently unknown ubiquitous cellular entry receptor(s) of FVs.