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.

The foamy virus (FV) glycoprotein precursor gp130Env undergoes a highly unusual biosynthesis, resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM). Little structural and functional information on the extracellular domains of FV Env is available. In this study, we characterized the prototype FV ( Viral envelope glycoproteins initiate entry of membraneenveloped viruses into cells by binding to cell surface receptors, followed by conformational changes leading to membrane fusion and delivery of the genome containing viral capsid to the cytoplasm (7). The envelope (Env) glycoproteins of foamy viruses (FVs) are no exception and mediate attachment to host cells through binding to yet unknown cellular receptor molecules. Viral particles are then taken up by endocytosis, and a pH-controlled glycoprotein-mediated fusion of viral and cellular lipid membranes leads to the release of FV capsids into the cytoplasm of the host cell (35).The principal domain structure of the prototype FV (PFV) Env, comprising an 18-kDa N-terminal signal or leader peptide (LP), a central 80-kDa surface (SU) subunit, and a C-terminal 48-kDa transmembrane (TM) subunit, is similar to other retroviral glycoproteins, although the 126-amino-acid (aa) LP is unusually long (reviewed in reference 24). Biosynthesis and maturation of the FV glycoprotein precursor gp130Env is also unusual in several aspects. First, gp130 Env is translated as a full-length precursor protein into the rough endoplasmic reticulum, where it initially adopts a type III membrane topology with both its N and C termini located intracytoplasmically (12,25). Second, only during its transport to the cell surface is it posttranslationally processed by cellular, most likely furin-like, proteases and not by the signal peptidase complex. Processing results in formation of at least three subunits (10, 11). In the heterotrimeric FV glycoprotein complex, the N-terminal LP has a type II membrane topology, whereas the C-terminal TM subunit has a type I membrane topology. The internal SU subunit presumably associates with extracellular domains of TM on the luminal side (25, 42). Processing of the SU/TM but not the LP/SU cleavage site is essential for generation of infectious viral particles in the supernatant (1,36). Third, all three subunits are incorporated into FV particles, and interactions of the gp18 LP subunit with the viral capsid are essential for FV budding and particle release (25,42).Image reconstruction analysis from electron micrographs of negatively stained virions revealed the characteristic, prominent Env spike structures on FV particles, indicating that the FV Env glycoprotein, similar to other viral glycoproteins, forms trimeric complexes containing three copies of each of the three individual subunits (41). However, additional highresolution structural information of FV glycoprotein subunits is not available and little functional analysis of the extracellular domains has been performed. In addi...

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particleassociated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.Secreted or membrane-anchored glycoproteins contain signal sequences targeting them to the secretory pathway (reviewed in reference 12). These so-called signal peptides (SP) can be removed co-or posttranslationally by the cellular membrane-bound signal peptidase complex (SPC). If not cleaved, they may serve as membrane anchors for proteins with distinct membrane orientations. In most cases SP cleavage is thought to occur cotranslationally. However, for some proteins, in particular retroviral glycoproteins (e.g., the human immunodeficiency virus type 1), SP cleavage occurs very late after translation (7).Spumaretroviruses, or foamy viruses (FVs), use a replication pathway with features distinctive from orthoretroviruses (reviewed in reference 17). The particle-associated glycoprotein of FV is unique compared to other retroviral envelope proteins because its coexpression is strictly required for the FV particle release process and its function cannot be replaced by heterologous viral glycoproteins (reviewed in reference 9). The FV envelope precursor protein seems to initially have a type III protein configuration with both its N and C terminus located intracytoplasmically (10). During its transport to the cell surface, it is posttranslationally processed by cellular proteases into at least three subunits. The N-terminal signal or ...

Foamy virus (FV) particle egress is unique among retroviruses because of its essential requirement for Gag and Env coexpression for budding and particle release. The FV glycoprotein undergoes a highly unusual biosynthesis resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM), derived from a precursor protein by posttranslational proteolysis mediated by furin or furinlike proteases. Previously at least three LP products of different molecular weights were detected in purified FV particles. Here we demonstrate that the higher-molecular-weight forms gp28 LP and gp38LP are ubiquitinated variants of the major gp18 LP cleavage product, which has a type II membrane topology. Furthermore, we show that all five lysine residues located within the N-terminal 60-amino-acid cytoplasmic domain of gp18 LP can potentially be ubiquitinated, however, there seems to be a preference for using the first three. Inactivation of ubiquitination sites individually resulted in no obvious phenotype. However, simultaneous inactivation of the first three or all five ubiquitination sites in gp18 LP led to a massive increase in subviral particles released by these mutant glycoproteins that were readily detectable by electron microscopy analysis upon expression of the ubiquitination-deficient glycoprotein by itself or in a proviral context. Surprisingly, only the quintuple ubiquitination mutant showed a two-to threefold increase in singlecycle infectivity assays, whereas all other mutants displayed infectivities similar to that of the wild type. Taken together, these data suggest that the balance between viral and subviral particle release of FVs is regulated by ubiquitination of the glycoprotein LP.Retroviral glycoproteins (Env proteins) are usually translated as precursor proteins and go through a series of modifications before reaching their mature functional state enabling them to interact with specific cellular receptors and to fuse viral and cellular lipid membranes. In general the Env proteins are cotranslationally inserted into the rough endoplasmic reticulum membrane. During their transport to the cell surface the precursor proteins oligomerize and are proteolytically cleaved by cellular proteases into at least two mature subunits, surface (SU), mainly involved in receptor recognition, and transmembrane (TM), harboring the membrane fusion machinery. In addition, the Env proteins undergo different kinds of posttranslational modifications during their biosynthesis and intracellular transport. For example, retroviral Env proteins are generally modified by attachment of N-and sometimes O-linked carbohydrate chains at asparagine and serine or threonine residues, respectively, although the extent and subunit distribution show variation between individual glycoproteins (reviewed in reference 40). Furthermore, for some retroviral Env proteins the addition of fatty acids via linkage to cysteine residues have been reported (reviewed in reference 31).Incorporation of t...