Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specifically interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa. The genomes of spumaretroviruses (or foamy viruses [FV])include the classical gag, pro-pol, and env genes that are the hallmark of the retrovirus family (9). Despite the familial relationship implied by their genome organization, FV differ from retroviruses such as oncoviruses or lentiviruses in basic aspects of replication and gene expression (30-32, 39, 53). The differences between FV and the more widely studied retroviruses, such as human immunodeficiency virus (HIV) or murine leukemia virus, provide an opportunity to identify the fundamental mechanisms of processes such as particle assembly and maturation.The shared gene order of the FV genomes and those of other retroviruses allows the identification of common structural proteins. Unfortunately, this relationship does not extend to the structure and function of FV Gag proteins, since no obvious homologies are detectable between FV Gag and MA, CA, and NC domains of other retroviruses for which structures have been determined to high resolution (10, 43). Furthermore, the proteolytic processing of Gag that produces the familiar mature proteins in other retroviruses is unusual, incomplete, and/or delayed in FV (12,23,29,37).The available data indicate that the morphology and morphogenesis of FV are distinct from those of other retroviruses. FV Gag proteins preassemble to form in the cytoplasm spherical capsids that bud through cellular membranes (55). This process contrasts with what occurs with lentiviruses and C-type retroviruses that assemble their capsids at the site of budding but is similar to what occurs with B-and D-type retroviruses (40). Budding of human foamy virus (HFV) i...

Previous studies have shown that foamy virus (FV) particle budding, especially the involvement of the viral env glycoprotein is different from that of other (ortho) retroviruses: the N-terminal Env leader protein Elp is a constituent of released FV particles. A defined sequence in Elp required for particle budding binds to the MA domain of Gag. To extend these findings, we show that feline FV Elp is a membrane-anchored protein with the N-terminus located inside the particle. Thus, the internal/cytoplasmic domain of Elp has the correct topology for interacting with Gag during budding. In addition to Elp, an Elp-related protein of about 9 kDa was shown to be virion associated and is probably generated by cellular signal peptidases. Besides the function of Elp binding, the N-terminal domain of Gag was shown to be required for proper localization of feline FV Gag to the cytoplasm and the perinuclear/nuclear region.

The molecular biology of spuma or foamy retroviruses is different from that of the other members of the Retroviridae. Among the distinguishing features, the N-terminal domain of the foamy virus Env glycoprotein, the 16-kDa Env leader protein Elp, is a component of released, infectious virions and is required for particle budding. The transmembrane protein Elp specifically interacts with N-terminal Gag sequences during morphogenesis. In this study, we investigate the mechanism of Elp release from the Env precursor protein. By a combination of genetic, biochemical, and biophysical methods, we show that the feline foamy virus (FFV) Elp is released by a cellular furin-like protease, most likely furin itself, generating an Elp protein consisting of 127 amino acid residues. The cleavage site fully conforms to the rules for an optimal furin site. Proteolytic processing at the furin cleavage site is required for full infectivity of FFV. However, utilization of other furin proteases and/or cleavage at a suboptimal signal peptidase cleavage site can partially rescue virus viability. In addition, we show that FFV Elp carries an N-linked oligosaccharide that is not conserved among the known foamy viruses.In retroviruses, proteolytic processing of the structural Gag and enzymatic Pol proteins is executed by the virus-encoded protease (6). In contrast, the Env glycoprotein precursors are considered to be processed by two cellular proteases (8,13,19). The first cleavage occurring in the endoplasmic reticulum (ER) removes the signal peptide (SP). In analogy to cellular secretory or transmembrane (TM) proteins, proteolytic removal of the SP which targets the Env precursor to the secretory pathway via the ER and Golgi apparatus is considered to be performed by cellular signal peptidases (SPases) (for a review, see references 17 and 19). The second cleavage, which separates the surface (SU) and TM domains, is catalyzed by a member of the furin convertase protease family, often the furin protease itself (8,28). Related glycoprotein processing mechanisms also apply to a wide variety of other membrane viruses such as influenza virus and Ebola virus (4).The processing of cellular type I membrane proteins (or even polyprotein precursors) by ER-resident SPases has been exploited by many viruses leading to SPs of about 15 to more than 50 amino acid residues independent of whether the protein is of viral or cellular origin (17). In addition, the SP is rapidly degraded by signal peptide peptidases, a process which is often directly linked to the presentation of the corresponding cleavage products by the major histocompatibility complex I for purposes of immune surveillance (17).However, for two distinct foamy retroviruses, namely human and feline foamy virus (HFV and FFV), the 14-to 18-kDa N-terminal Env cleavage product was clearly detectable as a component of released virus particles (11,14,15,35). We showed that the N terminus of FFV Env, termed the Env leader protein (Elp), is located inside particles, confirming that specific interact...