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Background: It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs., Results: N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Monitoring the fraction of viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured., Conclusions: The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques.

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.

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold. [ABSTRACT FROM AUTHOR]

Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells. [ABSTRACT FROM AUTHOR]

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis. [ABSTRACT FROM AUTHOR]