The Rous Sarcoma Virus Env Glycoprotein Contains a Highly Conserved Motif Homologous to Tyrosine-Based Endocytosis Signals and Displays an Unusual Internalization Phenotype

The envelope (Env) protein of Moloney murine leukemia virus (MoMuLV) is a homotrimeric complex whose monomers consist of linked surface (SU) and transmembrane (TM) proteins cleaved from a precursor protein by a cellular protease. In addition, a significant fraction of virion-associated TM is further processed by the viral protease to remove the C-terminal 16 amino acids of the cytoplasmic domain, the R peptide. This cleavage greatly enhances the fusogenicity of the protein and is necessary for the formation of a fully functional Env protein complex. We have previously proposed that R peptide cleavage enhances fusogenicity by altering the conformation of the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Using a series of truncation and point mutants of MoMuLV Env, we now provide direct biochemical and immunological evidence that the cytoplasmic tail and the membrane-spanning region of Env can influence the overall structure of the ectodomain of the protein and alter the strength of the SU-TM interaction. The R-peptidetruncated form of the protein, in particular, exhibits a markedly different conformation than the full-length protein.Retroviral envelope (Env) glycoproteins are expressed on the surface of infected cells and are incorporated into the viral lipid envelope during budding of a virion from a cell. Following binding to a specific cell surface receptor, Env promotes fusion between the viral and host cell membranes and thereby initiates a new infection. For the murine leukemia viruses (MuLVs), Env is initially translated as a precursor protein, Pr85, which oligomerizes in the endoplasmic reticulum, probably as a homotrimer (17). This precursor is cleaved by a cellular protease into two subunits, the surface (SU) protein gp70 and the transmembrane (TM) protein p15E, which remain associated in a labile interaction which may include a disulfide bond (48,51).MuLV Env also undergoes a second cleavage event in the cytoplasmic tail of TM that is essential for protein function. The C-terminal 16 amino acids of the cytoplasmic tail, the R peptide, is removed from a subset of the virion TM proteins by the viral protease (22,27,60). This results in an Env protein that is inherently more fusogenic than the full-length protein, causing, for example, cell-cell fusion to occur when it is expressed in NIH 3T3 cells (29,53,54). It has been suggested that the virus has adopted this strategy of regulating Env fusogenicity in order to limit the expression of a potentially cytotoxic molecule on the cell surface (53, 54). Similar processing of Env cytoplasmic tails has been observed in the MasonPfizer monkey virus (M-PMV), the gibbon ape leukemia virus (GaLV), and the equine infectious anemia virus (4, 7, 56); in addition, truncated M-PMV and GaLV Env proteins are more fusogenic than their full-length counterparts (3, 7). Truncations of the long cytoplasmic domains of lentiviral Env proteins occur under certain culture conditions (5,31,32,59), and an increase in Env fusogenicity has been reported for...

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confidence: 99%

Cytoplasmic Tail of Moloney Murine Leukemia Virus Envelope Protein Influences the Conformation of the Extracellular Domain: Implications for Mechanism of Action of the R Peptide

Aguilar

Anderson

Cannon

2003

“…In contrast, palmitoylation of the transmembrane (TM) protein of Rous sarcoma virus was shown to be required for protein stability and virus infectivity (42). Nevertheless, acylated Env glycoproteins of this virus are not sequestered into lipid raft domains (43). The TM proteins of HIV-1 and simian immunodeficiency virus are also posttranslationally modified by the addition of palmitate to the Cys residues located in the cytoplasmic domains of these viruses through a thioester bond (68).…”

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confidence: 99%

Wild-Type-Like Viral Replication Potential of Human Immunodeficiency Virus Type 1 Envelope Mutants Lacking Palmitoylation Signals

Chan

Lin

Chen

2005

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4 ؉ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T-and M-tropic mutants also productively replicated in human primary CD4 ؉ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.

“…The recombinant simian virus 40 (SV40) expression vector for RSV EnvC, pSVenvKX, used for Env expression in CV-1 cells has been described previously (10,39). To generate pCB6-EnvC, the 1.86-kb KpnI/BamHI env fragment from pSVenvKX was inserted into pCB6.…”

Section: Methodsmentioning

confidence: 99%

“…Env is synthesized as a precursor molecule (Pr95) that is proteolytically processed in the Golgi into two disulfide-linked subunits, SU/gp85 (surface) and TM/gp37 (transmembrane) (18). The TM region is divided into an ectodomain involved in membrane fusion, a membrane-spanning domain anchoring the protein, and a cytoplasmic C terminus containing endocytosis and potentially other trafficking signals (39). The ectodomains of both SU and TM are extensively glycosylated.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Novel Monoclonal Antibody Directed at the Receptor Binding Site on the Avian Sarcoma and Leukosis Virus Env Complex

Ochsenbauer-Jambor

Delos

Accavitti

et al. 2002

Self Cite

We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptorbinding site on a prototypical retroviral envelope.Not only are the avian retroviruses (alpharetroviruses, or avian sarcoma and leukosis viruses [ASLV]) relevant pathogens in poultry with economic impact, but they also have long served as an important model system for studying the biological functions of retroviruses and other enveloped viruses. Attachment to and infection of target cells is mediated by the ASLV envelope (Env) glycoproteins. Env is synthesized as a precursor molecule (Pr95) that is proteolytically processed in the Golgi into two disulfide-linked subunits, SU/gp85 (surface) and TM/gp37 (transmembrane) (18). The TM region is divided into an ectodomain involved in membrane fusion, a membrane-spanning domain anchoring the protein, and a cytoplasmic C terminus containing endocytosis and potentially other trafficking signals (39). The ectodomains of both SU and TM are extensively glycosylated. SU contains two hypervariable regions, hr1 and hr2, that are determinants of host range and thus of the interaction of SU with its respectiv...