Ubiquitination of the Prototype Foamy Virus Envelope Glycoprotein Leader Peptide Regulates Subviral Particle Release

Abstract

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...

Subviral Particle Release Determinants of Prototype Foamy Virus

“…Even though ubiquitination of PFV Gag does not seem to occur and is not required for viral particle egress, PFV interacts with the ubiquitination machinery during particle morphogenesis, as we have reported previously (49). However, it is the viral glycoprotein, not the viral capsid, which is posttranslationally modified by ubiquitin.…”

“…This includes the major interaction domain for the viral capsid, which is required during the Env-dependent particle release process, and posttranslational modification by ubiquitin, which influences the level of SVP release (27,49). An additional potential function of subdomains within gp18 LP for SVP egress was investigated here by quantifying the SVP release of various N-terminal truncation mutants, again in the wild-type or ubiquitination-deficient (⌬Ubi) context (Fig.…”

“…The replication-deficient PFV Gag/Pol-expressing vector pczDWP001 and the Gag/Pol/Env-expressing proviral expression vector pDL01, containing an internal spleen focus-forming virus U3 region-driven enhanced green fluorescent protein (EGFP)-Neo fusion protein marker gene cassette, have been described previously (11,41). All PFV envelope expression constructs generated for this study are based on either the wild type or the ubiquitination-deficient expression constructs pczHFVenvEM002 or pczHFVenvEM140, respectively (40,49 (40). They exhibit successive deletions, ranging from the C-terminal CyD to the entire membrane-spanning domain (MSD) of the Env TM subunit.…”

“…Interestingly, glycoprotein ubiquitination seemingly has no effect on FV particle egress. However, it apparently suppresses the intrinsic ability of the PFV glycoprotein to induce release of SVPs, as a PFV Env mutant having all potential cytoplasmic ubiquitin attachment sites in gp18 LP inactivated by lysine-to-arginine exchange was characterized by a massive induction of SVPs upon expression in cells (49).…”

“…LP not only contains the major interaction domain recognized by the viral capsid, which is essential for Env-dependent particle egress, but is also the target for posttranslational modification by ubiquitin (27,49). Interestingly, glycoprotein ubiquitination seemingly has no effect on FV particle egress.…”