An N-terminal domain helical motif of Prototype Foamy Virus Gag with dual functions essential for particle egress and viral infectivity.

BACKGROUND: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. RESULTS: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag - Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. CONCLUSIONS: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity.

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.

Subviral particle release determinants of prototype foamy virus.

Glycoproteins of several viruses have the capacity to induce release of noninfectious, capsidless particulate structures containing only the viral glycoprotein. Such structures are often called subviral particles (SVP). Foamy viruses (FVs), a special type of retroviruses with a replication strategy combining features of both orthoretroviruses and hepadnaviruses, express a glycoprotein (Env) which has the ability to induce SVP release. However, unlike human hepatitis B virus, prototype FV (PFV) naturally secretes only small amounts of SVPs, because ubiquitination of the Env protein seems to suppress the intrinsic capacity for induction of SVP release. In this study, we characterized the structural determinants influencing PFV SVP release, examined the role of specific Env ubiquitination sites in the regulation of this process, and analyzed the requirement of the cellular vacuolar protein sorting (VPS) machinery for SVP egress. We observed that the cytoplasmic and membrane-spanning domains of both the leader peptide (LP) and the transmembrane (TM) subunit harbor essential as well as inhibitory domains. Furthermore, only ubiquitination at the most N-terminal lysine residues (K(14) and K(15)) in LP reduced cell surface expression and suppressed SVP release to wild-type levels. This suggests that interaction of Env with cellular components required for SVP release suppression is effective only when Env is ubiquitinated at these lysine residues but not at others. Finally, SVP release was sensitive to dominant-negative mutants of late components, but not early components, of the cellular VPS machinery. PFV therefore differs from hepatitis B virus in using the same cellular pathway for egress of both virions and SVPs.

Correct capsid assembly mediated by a conserved YXXLGL motif in prototype foamy virus Gag is essential for infectivity and reverse transcription of the viral genome.

Unlike other retrovirus Gag proteins, the prototype foamy virus (PFV) p71(g)(ag) protein is not processed into mature matrix (MA), capsid (CA), and nucleocapsid (NC) subunits. Little information about sequence motifs involved in FV capsid assembly and release is available. The recent analysis of candidate L-domain motifs in PFV Gag identified an evolutionarily conserved YXXL sequence motif with a potential function in capsid assembly. Here we provide support for the hypothesis that this motif does not function like a conventional L domain, by demonstrating that, unlike the PFV Gag PSAP L-domain motif, it cannot be functionally replaced by heterologous L-domain sequences. Furthermore, mutation of individual amino acids Y(464), I(466), L(467), and L(469), but not E(465), to alanine led to reduced particle release and production of noninfectious, aberrant capsid structures, although relative structural protein incorporation and processing were not affected. In contrast, mutation of G(468) to alanine resulted in an intermediate, temperature-sensitive phenotype characterized by reduced particle release and reduced infectivity. Despite similar relative RNA genome incorporation for all mutants, analysis and quantification of particle-associated viral nucleic acids demonstrated defects in genomic reverse transcription for all the noninfectious mutants, a process that, unlike that of orthoretroviruses, in the case of FVs takes place in the virus-producing cell. In correlation with the reduced infectivity, the G(468)A mutant displayed an intermediate level of genomic reverse transcription. Taken together, these results demonstrate that the conserved YXXLGL motif in PFV Gag is involved in correct capsid assembly, which in turn is essential for reverse transcription of the FV genome.

Ubiquitination of the prototype foamy virus envelope glycoprotein leader peptide regulates subviral particle release.

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 gp28LP and gp38LP are ubiquitinated variants of the major gp18LP 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 gp18LP 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 gp18LP 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 single-cycle 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.

Characterization of prototype foamy virus gag late assembly domain motifs and their role in particle egress and infectivity.

Foamy viruses (FV) are unusual among retroviruses since they require both Gag and Env structural proteins for particle egress. Recently significant progress has been made towards the mechanistic understanding of the viral release process, in particular that of retroviruses, and the viral domains and cellular pathways involved. However little is currently known about domains of FV structural proteins and cellular proteins engaged in this process. By mutational analysis of sequence motifs in prototype FV (PFV) Gag, bearing homology to known late assembly (L) domains, a PSAP motif with L domain function that was functionally interchangeable by heterologous L domains was identified. In contrast the inactivation of a PPPI motif had no significant influence on PFV particle release, although mutant viral particles displayed reduced infectivity. Similarly mutation of an evolutionary conserved YXXL motif revealed no classical L-domain function but resulted in release of noninfectious viruslike particles. Biochemical and electron microscopy analysis demonstrated that these mutant particles incorporated all viral structural proteins but contained aberrantly capsid structures, suggesting a role in capsid assembly for this PFV Gag sequence motif. In line with the mutational analysis, overexpression of dominant negative (DN) mutants and wild-type TSG101 but not the DN mutant of AIP-1/ALIX reduced PFV particle release and infectivity. Furthermore, DN mutants of Vps4A, Vps4B, and CHMP3 inhibited PFV egress and infectivity. Taken together these results demonstrate that PFV, like other viruses, requires components of the vacuolar protein sorting (VPS) machinery for egress and enters the VPS pathway through interaction with TSG101.

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity.

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 particle-associated 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.