Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.

Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.

Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.

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.

Correction: Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.

[This corrects the article DOI: 10.1371/journal.ppat.1005860.].

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.

A unique spumavirus Gag N-terminal domain with functional properties of orthoretroviral matrix and capsid.

The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.

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.

The cooperative function of arginine residues in the Prototype Foamy Virus Gag C-terminus mediates viral and cellular RNA encapsidation.

BACKGROUND: One unique feature of the foamy virus (FV) capsid protein Gag is the absence of Cys-His motifs, which in orthoretroviruses are irreplaceable for multitude functions including viral RNA genome recognition and packaging. Instead, FV Gag contains glycine-arginine-rich (GR) sequences at its C-terminus. In case of prototype FV (PFV) these are historically grouped in three boxes, which have been shown to play essential functions in genome reverse transcription, virion infectivity and particle morphogenesis. Additional functions for RNA packaging and Pol encapsidation were suggested, but have not been conclusively addressed. RESULTS: Here we show that released wild type PFV particles, like orthoretroviruses, contain various cellular RNAs in addition to viral genome. Unlike orthoretroviruses, the content of selected cellular RNAs in capsids of PFV vector particles was not altered by viral genome encapsidation. Deletion of individual GR boxes had only minor negative effects (2 to 4-fold) on viral and cellular RNA encapsidation over a wide range of cellular Gag to viral genome ratios examined. Only the concurrent deletion of all three PFV Gag GR boxes, or the substitution of multiple arginine residues residing in the C-terminal GR box region by alanine, abolished both viral and cellular RNA encapsidation (>50 to >3,000-fold reduced), independent of the viral production system used. Consequently, those mutants also lacked detectable amounts of encapsidated Pol and were non-infectious. In contrast, particle release was reduced to a much lower extent (3 to 20-fold). CONCLUSIONS: Taken together, our data provides the first identification of a full-length PFV Gag mutant devoid in genome packaging and the first report of cellular RNA encapsidation into PFV particles. Our results suggest that the cooperative action of C-terminal clustered positively charged residues, present in all FV Gag proteins, is the main viral protein determinant for viral and cellular RNA encapsidation. The viral genome independent efficiency of cellular RNA encapsidation suggests differential packaging mechanisms for both types of RNAs. Finally, this study indicates that analogous to orthoretroviruses, Gag - nucleic acid interactions are required for FV capsid assembly and efficient particle release.

Prototype foamy virus protease activity is essential for intraparticle reverse transcription initiation but not absolutely required for uncoating upon host cell entry.

Foamy viruses (FVs) are unique among retroviruses in performing genome reverse transcription (RTr) late in replication, resulting in an infectious DNA genome, and also in their unusual Pol biosynthesis and encapsidation strategy. In addition, FVs display only very limited Gag and Pol processing by the viral protease (PR) during particle morphogenesis and disassembly, both thought to be crucial for viral infectivity. Here, we report the generation of functional prototype FV (PFV) particles from mature or partially processed viral capsid and enzymatic proteins with infectivity levels of up to 20% of the wild type. Analysis of protein and nucleic acid composition, as well as infectivity, of virions generated from different Gag and Pol combinations (including both expression-optimized and authentic PFV open reading frames [ORFs]) revealed that precursor processing of Gag, but not Pol, during particle assembly is essential for production of infectious virions. Surprisingly, when processed Gag (instead of Gag precursor) was provided together with PR-deficient Pol precursor during virus production, infectious, viral DNA-containing particles were obtained, even when different vector or proviral expression systems were used. Although virion infectivity was reduced to 0.5 to 2% relative to that of the respective parental constructs, this finding overturns the current dogma in the FV literature that viral PR activity is absolutely essential at some point during target cell entry. Furthermore, it demonstrates that viral PR-mediated Gag precursor processing during particle assembly initiates intraparticle RTr. Finally, it shows that reverse transcriptase (RT) and integrase are enzymatically active in the Pol precursor within the viral capsid, thus enabling productive host cell infection.

Foamy virus for efficient gene transfer in regeneration studies.

BACKGROUND: Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders. RESULTS: We show that replication-deficient foamy virus (FV) vectors efficiently transduce cells in two different regeneration models in cell culture and in vivo. Injection of EGFP-expressing FV but not lentivirus vector particles into regenerating limbs and tail resulted in widespread expression that persisted throughout regeneration and reamputation pointing to the utility of FV for analyzing adult phenotypes in non-mammalian models. Furthermore, tissue specific transgene expression is achieved using FV vectors during limb regeneration. CONCLUSIONS: FV vectors are efficient mean of transferring genes into axolotl limb/tail and infection persists throughout regeneration and reamputation. This is a nontoxic method of delivering genes into axolotls in vivo/ in vitro and can potentially be applied to other salamander species.

Basic residues in the foamy virus Gag protein.

Foamy virus (FV) capsid proteins have few lysines. Basic residues are almost exclusively represented by arginines indicating positive selective pressure. To analyze the possible functions of this peculiarity, we mutated an infectious molecular clone of the prototypic FV (PFV) to harbor lysines in the Gag protein at arginine-specifying positions and analyzed various aspects of the FV replication cycle. The majority of mutants replicated equally as well in permanent cell cultures as the original wild-type (wt) virus and were genetically stable in gag upon 10 cell-free passages. With respect to the features of late reverse transcription, nucleic acid content, and infectiousness of the virion DNA genome, the majority of mutants behaved like the wt. Several mutants of PFV were ubiquitinated in Gag but unable to generate virus-like particles (VLPs) or to undergo pseudotyping by a heterologous envelope. Using primary cells, however, a replicative disadvantage of the majority of mutants was disclosed. This disadvantage was enhanced upon interferon (IFN) treatment. We found no evidence that the lysine-bearing gag mutants showed more restriction than the wt virus by tetherin (CD317) or Trim5α. A single lysine in PFV Gag was found to be nonessential for transient replication in permanent cell culture if replaced by an arginine residue. Upon replication in primary cells, even without IFN treatment, this mutant was severely impaired, indicating the importance of specifying at least this lysine residue in PFV Gag. The paucity of lysines in FV Gag proteins may be a consequence of preventing proteasomal Gag degradation.