Identification of an Intermediate Step in Foamy Virus Fusion.

Viral glycoprotein-mediated membrane fusion is an essential step for productive infection of host cells by enveloped viruses; however, due to its rarity and challenges in detection, little is known about the details of fusion events at the single particle level. Here, we have developed dual-color foamy viruses (FVs) composed of eGFP-tagged prototype FV (PFV) Gag and mCherry-tagged Env of either PFV or macaque simian FV (SFVmac) origin that have been optimized for detection of the fusion process. Using our recently developed tracking imaging correlation (TrIC) analysis, we were able to detect the fusion process for both PFV and SFVmac Env containing virions. PFV Env-mediated fusion was observed both at the plasma membrane as well as from endosomes, whereas SFVmac Env-mediated fusion was only observed from endosomes. PFV Env-mediated fusion was observed to happen more often and more rapidly than as for SFVmac Env. Strikingly, using the TrIC method, we detected a novel intermediate state where the envelope and capsids are still tethered but separated by up to 400 nm before final separation of Env and Gag occurred.

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid.

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.

Efficient transient genetic manipulation in vitro and in vivo by prototype foamy virus-mediated nonviral RNA transfer.

Vector systems based on different retroviruses are widely used to achieve stable integration and expression of transgenes. More recently, transient genetic manipulation systems were developed that are based on integration- or reverse transcription-deficient retroviruses. Lack of viral genome integration is desirable not only for reducing tumorigenic potential but also for applications requiring transient transgene expression such as reprogramming or genome editing. However, all existing transient retroviral vector systems rely on virus-encoded encapsidation sequences for the transfer of heterologous genetic material. We discovered that the transient transgene expression observed in target cells transduced by reverse transcriptase-deficient foamy virus (FV) vectors is the consequence of subgenomic RNA encapsidation into FV particles. Based on this initial observation, we describe here the establishment of FV vectors that enable the efficient transient expression of various transgenes by packaging, transfer, and de novo translation of nonviral RNAs both in vitro and in vivo. Transient transgene expression levels were comparable to integrase-deficient vectors but, unlike the latter, declined to background levels within a few days. Our results show that this new FV vector system provides a useful, novel tool for efficient transient genetic manipulation of target tissues by transfer of nonviral RNAs.

Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles.

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.

Heparan sulfate is an attachment factor for foamy virus entry.

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.

A small-molecule-controlled system for efficient pseudotyping of prototype foamy virus vectors.

Foamy virus (FV) vector systems have recently demonstrated their power as efficient gene transfer tools for different target tissues. Unfortunately, FVs cannot be naturally pseudotyped by heterologous viral glycoproteins due to an unusual particle morphogenesis involving a FV Env-dependent particle release process. Therefore, current FV vector systems are constrained to the broad host cell range provided by the cognate viral glycoprotein. We evaluated different approaches for pseudotyping of FV vectors, in which the specific FV Gag-Env interaction, essential for particle egress, is substituted by a small-molecule controlled heterodimerization (HD) system. In one system developed, one HD-domain (HDD) is fused to a membrane-targeting domain (MTD), such as the human immunodeficiency virus (HIV) Gag matrix (MA) subunit, with a second fused to the FV capsid protein. Coexpression of both components with different heterologous viral glycoproteins allowed an efficient, dimerizer-dependent pseudotyping of FV capsids. With this system FV vesicular stomatitis virus glycoprotein (VSV-G) pseudotype titers greater than 1 × 10(6) IU/ml were obtained, at levels comparable to authentic FV vector particles. As a proof-of-principle we demonstrate that Pac2 cells, naturally resistant to FV vectors, become permissive to FV VSV-G pseudotypes. Similar to other retroviral vectors, this FV pseudotyping system now enables adaptation of cell-specific targeting approaches for FVs.

Prototype foamy virus gag nuclear localization: a novel pathway among retroviruses.

Gag nuclear localization has long been recognized as a hallmark of foamy virus (FV) infection. Two required motifs, a chromatin-binding site (CBS) and a nuclear localization signal (NLS), both located in glycine-arginine-rich box II (GRII), have been described. However, the underlying mechanisms of Gag nuclear translocation are largely unknown. We analyzed prototype FV (PFV) Gag nuclear localization using a novel live-cell fluorescence microscopy assay. Furthermore, we characterized the nuclear localization route of Gag mutants tagged with the simian vacuolating virus 40-NLS (SV40-NLS) and also dissected the respective contributions of the CBS and the NLS. We found that PFV Gag does not translocate to the nucleus of interphase cells by NLS-mediated nuclear import and does not possess a functional NLS. PFV Gag nuclear localization occurred only by tethering to chromatin during mitosis. This mechanism was found for endogenously expressed Gag as well as for Gag delivered by infecting viral particles. Thereby, the CBS was absolutely essential, while the NLS was dispensable. Gag CBS-dependent nuclear localization was neither essential for infectivity nor necessary for Pol encapsidation. Interestingly, Gag localization was independent of the presence of Pol, Env, and viral RNA. The addition of a heterologous SV40-NLS resulted in the nuclear import of PFV Gag in interphase cells, rescued the nuclear localization deficiency but not the infectivity defect of a PFV Gag ΔGRII mutant, and did not enhance FV's ability to infect G(1)/S-phase-arrested cells. Thus, PFV Gag nuclear localization follows a novel pathway among orthoretroviral Gag proteins.

Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis.

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.

Analysis of prototype foamy virus particle-host cell interaction with autofluorescent retroviral particles.

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.