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

Abstract

BackgroundThe 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.ResultsIn 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.ConclusionsWe 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.

Prototype Foamy Virus Gag Nuclear Localization: a Novel Pathway among Retroviruses

“…A 4-component PFV vector system consisting of the expression vectors pcoPG4 (PFV Gag), pcoPE (PFV Env), pcoPP or pcziPol, (PFV Pol), and the enhanced green fluorescent protein (eGFP)-expressing PFV transfer vector pMD9 has been described previously (20,33,50). Due to biosafety issues, a previously described variant PFV Pol expression construct with catalytically inactive reverse transcriptase, pcoPP2 (Pol iRT) (32,33), was used for live-cell imaging experiments employing the 4-component PFV vector system.…”

“…Additionally, the original attR1 site, the chloramphenicol resistance gene, the ccdB gene, the attR2 site, and the V5 epitope were replaced by multiple cloning sites (SpeI, SwaI, NheI, XhoI, PmeI, and EcoRI). The pLV7 PG construct was generated by excision of the expression-optimized Gag gene from pcoPG4 (50) and subsequent ligation of the insert into pLV7 (EF1a MCS ZeoW) using the SwaI and XhoI restriction sites. The pLV7 PG CeYFP construct was generated by excision of the expression-optimized Gag gene with a C-terminal enhanced yellow fluorescent protein (eYFP) (CeYFP) tag from pcoPG4 CeYFP (50) and subsequent ligation of the insert into pLV7 (EF1a MCS ZeoW) using the SwaI and XhoI restriction sites.…”

Prototype Foamy Virus Protease Activity Is Essential for Intraparticle Reverse Transcription Initiation but Not Absolutely Required for Uncoating upon Host Cell Entry

“…Furthermore, a PR-inactive variant (D 24 A mutation), pcziPol iPR (ncoPP iPR), based on the pcziPol packaging construct was generated. Plasmid puc2MD9 (PTV), encoding a packageable PFV vector RNA containing a spleen focus-forming virus U3 region (SFFV U3)-driven egfp marker gene expression cassette, was used as a transfer vector (37).…”

Foamy Virus Nuclear RNA Export Is Distinct from That of Other Retroviruses

“…Plasmid pcHSRV2 (50), the pol ATG-to-CTG mutant plasmid pcHSRV2-M54 (22), the gag ATG-to-TTG mutant plasmid pcHSRV2-M78 (30), pNLS-NES (73), pNLS-NES-M90 (73), p3CANc (57), the codon-optimized gag expression plasmid pcoPG4 (69), and pEGFP (Invitrogen) have been described previously.…”