Identification of Domains in Gag Important for Prototypic Foamy Virus Egress

Abstract

Sequence motifs (L domains) have been described in viral structural proteins. Mutations in these lead to a defect at a late stage in virus assembly and budding. For several viruses, recruitment of an endosomal sorting complexes required for transport 1 subunit (Tsg101), a component of the class E vacuolar protein sorting (EVPS) machinery, is a prerequisite for virion budding. To effect this, Tsg101 interacts with the PT/SAP L domain. We have identified candidate L-domain motifs, PSAP, PPPI, and YEIL, in the prototypic foamy virus (PFV) Gag protein, based on their homology to known viral L domains. Mutation of the PSAP and PPPI motifs individually reduced PFV egress, and their combined mutation had an additive effect. When PSAP was mutated, residual infectious PFV release was unaffected by dominant negative Vps4 (an ATPase involved in the final stages of budding), and sensitivity to dominant negative Tsg101 was dramatically reduced, suggesting that the PSAP motif functions as a conventional class E VPS-dependent L domain. Consistent with this notion, yeast two-hybrid analysis showed a PSAP motif-dependent interaction between PFV Gag and Tsg101. Surprisingly, PFV release which is dependent on the PPPI motif was Vps4-independent and was partially inhibited by dominant negative Tsg101, suggesting that PPPI functions by an unconventional mechanism to facilitate PFV egress. Mutation of the YEIL sequence completely abolished particle formation and also reduced the rate of Gag processing by the viral protease, suggesting that the integrity of YEIL is required at an assembly step prior to budding and YEIL is not acting as an L domain.

Requirements for the Assembly and Release of Newcastle Disease Virus-Like Particles

“…The essential role of these matrix proteins in release is due in part to short motifs, called late domains, in these proteins that interact with components of the host vacuolar protein sorting system (2,17,25,31,35,40,45,49,51). It is thought that these proteins hijack the host vacuolar protein sorting pathway for use in virus budding (10,24,31,44).…”

Subviral Particle Release Determinants of Prototype Foamy Virus

“…Proteins of the VPS machinery were already reported to be important for egress of various viruses, including retroviruses, and perhaps more importantly for release of FV particles; moreover, VPS proteins were recently implicated in the export of infectious HBV particles (5,24,38,48). Our results suggest that late components of the VPS machinery, including CHMP3 (which participates in ESCRT-III complex formation) and the recycling AAA ATPase VPS4, affect PFV SVP release.…”

“…Like HBV and a variety of other viruses, PFV hijacks the cellular VPS machinery for viral particle release. In the case of PFV, this occurs through a PSAP L-domain-dependent interaction between the Gag protein and the ESCRT-I component TSG101 (24,38,48,52). In contrast to HBV viral particle release, HBV SVP release was recently reported to be independent of the ESCRT complexes (24,52).…”

“…The best-studied and -characterized isolate to date is the so-called prototype FV (PFV; formerly known as human FV [HFV]), which was originally isolated from an African patient who presumably was infected zoonotically by a chimpanzee FV (1,22). With respect to particle morphogenesis, it was demonstrated that PFV, similarly to other viruses, including retroviruses and HBV, uses the cellular VPS machinery for budding and release of capsids preassembled at the centrosome of the host cell (24,38,48,52,55). PFV Gag contains a PSAP L-domain motif linking the viral capsid in a TSG101-dependent manner through the ESCRT-I complex to the VPS machinery (38,48).…”

“…With respect to particle morphogenesis, it was demonstrated that PFV, similarly to other viruses, including retroviruses and HBV, uses the cellular VPS machinery for budding and release of capsids preassembled at the centrosome of the host cell (24,38,48,52,55). PFV Gag contains a PSAP L-domain motif linking the viral capsid in a TSG101-dependent manner through the ESCRT-I complex to the VPS machinery (38,48). However, only primate FVs harbor a PSAP L-domain motif, raising the question about the nature of the L domains of nonprimate FVs.…”