Identification of an ER retrieval signal in a retroviral glycoprotein

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.Foamy viruses (FVs), also known as spumaviruses, are widespread complex retroviruses which have been isolated from nonhuman primates, cattle, and cats. Although highly lytic in vitro, these viruses, which are innocuous in vivo, are known to induce a persistent infection in their hosts (20). All FVs characterized to date have very large genomes (between 12 to 13 kb) with the classical gag, pol, and env structural genes and additional regulatory open reading frames (ORFs) located at the 3Ј end of the env gene which are under the control of both the 5Ј long terminal repeat (LTR) and an internal promoter (IP) (14). In the case of the human foamy virus (HFV), the prototype FV, two accessory proteins, Tas and Bet, have been described. While Tas (originally called Bel1) is the potent DNA binding transactivator of viral gene expression of both the LTR and the IP, Bet has been shown to play a key role in the establishment and control of viral persistence in vitro (1,19).The molecular biology of retroviruses was highly focused on human T-cell leukemia virus (HTLV) and human immunodeficiency virus (HIV), clearly associated with human pathologies. However, recent findings regarding FVs raise important issues of general interest. Indeed, some of their features, such as the formation of a specific pol mRNA and the infectivity of the incoming viral DNA contained in extracellular virions (26, 28), set FVs apart from all other retroviruses. By virtue of these two features, FVs resemble pararetroviruses.By analogy with lentiviruses, which were isolated from cattle, cats, primates, goats, and horses, we decided to look for the presence of an FV in horses. Here, we report the isolation of a new nonprimate FV from blood samples of naturally infected horses. The equine foamy virus (EFV) has been characterized by molecular cloning and nucleotide sequence analysis. The ultrastructure of the extracellular virion and the genomic organization were investigated and compared to those of other cloned FVs. Although displaying limited sequence similarities with primate FVs, EFV is phylogenetically closer to nonprimate FVs, especially to the bovine foamy virus (BFV). Interestingly, in contrast to other...

Section: Resultssupporting

confidence: 65%

Section: Discussionmentioning

confidence: 91%

Isolation and Characterization of an Equine Foamy Virus

Tobaly-Tapiero

Bittoun

Neves

et al. 2000

“…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).…”

mentioning

confidence: 99%

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

Bansal

Shaw

Edwards³

et al. 2000

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...

“…Similar to the intracisternal A-type particles but distinct from all other retroviruses, FV capsids bud through the endoplasmic reticulum (ER) membrane. The FV envelope (Env) protein is also retained in the ER by means of a trilysine motif located at the C-terminal cytoplasmic tail of Env (18,19). FV morphogenesis requires the presence of the Env protein to allow release of virus from the cell, a mechanism also employed by hepatitis B virus, such that capsid budding is completely inhibited in the absence of Env expression (2,5,15).…”

mentioning

confidence: 99%

Identification of a Conserved Residue of Foamy Virus Gag Required for Intracellular Capsid Assembly

Eastman

Linial

2001

In contrast to all retroviruses but similar to the hepatitis B virus, foamy viruses (FV) require expression of the envelope protein for budding of intracellular capsids from the cell, suggesting a specific interaction between the Gag and Env proteins. Capsid assembly occurs in the cytoplasm of infected cells in a manner similar to that for the B-and D-type viruses; however, in contrast to these retroviruses, FV Gag lacks an N-terminal myristylation signal and capsids are not targeted to the plasma membrane (PM). We have found that mutation of an absolutely conserved arginine (Arg) residue at position 50 to alanine (R50A) of the simian foamy virus SFV cpz(hu) inhibits proper capsid assembly and abolishes viral budding even in the presence of the envelope (Env) glycoproteins. Particle assembly and extracellular release of virus can be restored to this mutant with the addition of an N-terminal Src myristylation signal (Myr-R50A), presumably by providing an alternate site for assembly to occur at the PM. In addition, the strict requirement of Env expression for capsid budding can be bypassed by addition of a PM-targeting signal to Gag. These results suggest that intracellular capsid assembly may be mediated by a signal akin to the cytoplasmic targeting and retention signal CTRS found in Mason-Pfizer monkey virus and that FV Gag has the inherent ability to assemble capsids at multiple sites like conventional retroviruses. The necessity of Env expression for particle egress is most probably due to the lack of a membrane-targeting signal within FV Gag to direct capsids to the PM for release and indicates that Gag-Env interactions are essential to drive particle budding.