Characterization of the R572T Point Mutant of a Putative Cleavage Site in Human Foamy Virus Env

Abstract

A putative cleavage site of the human foamy virus (HFV) envelope glycoprotein (Env) was altered. Transient env expression revealed that the R572T mutant Env was normally expressed and modified by asparagine-linked oligosaccharide chains. However, this single-amino-acid substitution was sufficient to abolish all detectable cleavage of the gp130 precursor polyprotein. Cell surface biotinylation demonstrated that the uncleaved mutant gp130 was transported to the plasma membrane. The uncleaved mutant protein was incapable of syncytium formation. Glycoprotein-driven virion budding, a unique aspect of HFV assembly, occurred despite the absence of Env cleavage. We then substituted the R572T mutant env into a replication-competent HFV molecular clone. Transfection of the mutant viral DNA into BHK-21 cells followed by viral titration with the FAB (foamy virus-activated ␤-galactosidase expression) assay revealed that proteolysis of the HFV Env was essential for viral infectivity. Wild-type HFV Env partially complemented the defective virus phenotype. Taken together, these experimental results established the location of the HFV Env proteolytic site; the effects of cleavage on Env transport, processing, and function; and the importance of Env proteolysis for virus maturation and infectivity.Foamy viruses (FV) are grouped together as the spumavirus family of complex retroviruses and are ubiquitous in several mammalian species (7). The genomes of these complex retroviruses are comprised of the three typical retrovirus genes (i.e., gag, pol, and env) in addition to the accessory bel genes. The Env of FV is a type 1 membrane-spanning protein that possesses the general structural features observed in all retroviral glycoproteins (20). Two forms of the HFV envelope proteins have been reported (9, 10, 16). The predominant form of HFV Env is synthesized as a polyprotein precursor (gp130) that is proteolytically cleaved to yield two mature subunits, SU (gp80) and TM (gp47) (10). The second form, expressed at 30 to 50% of the level of gp130, is a 170-kDa Env-Bet fusion protein. This is generated by an alternative splicing mechanism and currently its biological role is unknown (9, 16). One feature unique to FV Env relative to all other retroviral glycoproteins is the presence of the dilysine motif, or KKXX, an endoplasmic reticulum (ER) retrieval signal at the TM C terminus (13). It was demonstrated that this protein sorting signal is responsible for ER localization of the HFV glycoprotein (12) and the partitioning of HFV budding to intracellular membranes (11). A putative proteolytic processing site within Env (20) was conserved among all six FV sequences available for analysis (Table 1). This consensus tetrabasic motif, R/K-X-R/K-R, was previously identified as a putative proteolytic cleavage site for retroviral envelope glycoproteins (4). Processing of the polyprotein at this basic amino acid cluster is performed by subtilisin-like cellular endoproteases in late Golgi compartments (15). The polyprotein becomes cleaved at the carbo...

Prototype Foamy Virus Envelope Glycoprotein Leader Peptide Processing Is Mediated by a Furin-Like Cellular Protease, but Cleavage Is Not Essential for Viral Infectivity

“…1C. In contrast, the SU/TM cleavage site fits the requirements of an optimal furin recognition site (18), as previously predicted and implicated by mutagenesis analysis (1,15,19). This processing site was confirmed in this study by N-terminal sequencing of the immunoadhesin TM-IgG subunit.…”

“…Taken together, these data suggested that PFV Env LP/SU cleavage occurs after R 126 of the gp130 Env precursor protein. Furthermore, they indicated that the PFV Env SU/TM furin cleavage site predicted by sequence (19) and mutagenesis (1,15) analysis is indeed used and the PFV Env SU subunit is processed after R 571 . These two processing events of the precursor protein generate PFV Env SU and TM subunits with N-terminal amino acid sequences of SLRMQH and SVDNNY, respectively.…”

Foamy Virus Envelope Protein Is a Substrate for Signal Peptide Peptidase-like 3 (SPPL3)

“…2A), whereas the more C-terminal PC* cleavage is hardly observed upon cellular expression of FVenv containing the ER retrieval signal (25). Thus, processing of FVenv at the N-terminal PC cleavage site is comparable with a classical shedding event of single-span transmembrane proteins required for subsequent intramembrane proteolysis.…”

Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein

“…Furin and other members of the Golgi-resident protein convertase protease family are known to cleave retroviral and nonretroviral glycoprotein precursors (28). In retroviruses, this cleavage separates the SU and TM domains of Env, an event that is absolutely required for induction of membrane fusion by TM after SU-mediated receptor recognition (3,8,13).…”