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, Hector C.; Anderson, W. French; Cannon, Paula M.

doi:10.1128/jvi.77.2.1281-1291.2003

Cited by 70 publications

(63 citation statements)

References 81 publications

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“…The structural alteration in the Env endodomain seems also to transmit changes into the ectodomain. Although in our structure reconstructions we were unable to find significant differences between the ectodomains of the precursor and the mature Env, biochemical mapping using antibodies and biotinylation have shown that the R-peptide cleavage is associated with ectodomain changes (20).…”

Section: Discussionmentioning

confidence: 83%

“…This has been suggested to inhibit the receptor triggering of the R-peptide Env precursor. The model was tested by analyzing the fusion phenotype of an Env with a mutation of Leu651 into Ala (19,20). Leu651 is positioned just behind the Leu649/Val650 cleavage site and is predicted to be one key residue in the heptad repeat of hydrophobic residues specifying the trimeric coiled-coil structure.…”

Section: Discussionmentioning

confidence: 99%

“…This model has been supported by mutational studies using Moloney (Mo) MLV. In particular, an L651A mutation, just downstream of the R-peptide cleavage site, which should violate the heptad repeat of hydrophobic residues supporting coiled-coil formation, has been shown to rescue the inhibited fusion capacity of cellassociated Env (19,20). Here we have produced wild-type and L651A Env mutant Mo-MLV in the presence of an inhibitor of the viral protease, at concentrations that preferentially inhibit Rpeptide cleavage.…”

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confidence: 99%

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Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering

Löving

Wu²,

Sjöberg

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU-TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.three-dimensional structure | retrovirus | spike protein T he spike protein Env on the surface of the retrovirus murine leukemia virus (MLV) matures by two proteolytic cleavage events mediated by cellular furin and the viral protease (1-3). Env is composed of an 80-kDa transmembrane precursor protein in the rough endoplasmic reticulum of the infected cell (4, 5). Here it trimerizes before it is routed to the cell surface for assembly with the internal Gag and GagPol precursors into virus particles in a budding process (6). The furin cleavage of Env occurs in the trans-Golgi, and it separates the surface (SU) subunit from the transmembrane (TM) (Pr15E) subunit. This cleavage also releases the viral fusion peptide at the N-terminal end of the TM. The viral protease cleavage occurs after virus assembly in the newly formed particle. It removes a 16-residuelong peptide, the R-peptide from the C terminus of the TM, forming p15E (7). Not until this second cleavage is completed is Env primed for receptor-mediated triggering into further conformations that can direct virus entry through fusion of the viral membrane with the cell membrane (8, 9). The viral protease also cleaves the Gag and GagPol precursors into their mature proteins and enzymes.The structure of the MLV Env has been studied by cryoelectron microscopy (cryo-EM) both in intact particles and as solubilized trimers (10, 11). It reveals a remarkably open structure with separated legs. The protomer of the solubilized Env is formed from three protrusions-upper, middle, and lower. Together, they encage a large central cavity, with the top pro...

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Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering

Löving

Wu²,

Sjöberg

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Many examples in the literature describe the cytoplasmic domain as important for the function of the ectodomains of glycoproteins in a variety of virus families [16][17][18][19][20]. Studies examining conformation dependent binding of monoclonal antibodies showed that alterations in the cytoplasmic domain led to structural changes in the ectodomain [21,22]. Moreover, our data highlight that the cytoplasmic domain is an important factor for a correct quaternary structure, which subsequently is required for proper proteolytic processing and function of the viral glycoprotein.…”

Section: The Cytoplasmic Domain Stabilizes Non-cleaved Gp-c For Maturmentioning

confidence: 99%

Maturation cleavage within the ectodomain of Lassa virus glycoprotein relies on stabilization by the cytoplasmic tail

2010

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a b s t r a c tThe Lassa virus glycoprotein consists of an ectodomain, a transmembrane anchor, and a cytoplasmic domain. It is synthesized as an inactive precursor and cleaved within the ectodomain to yield the mature form. Here, we show that this maturation cleavage can be abolished by mutation of single conserved amino acids within the cytoplasmic domain at the carboxy-terminus of the glycoprotein. Moreover, substitutions and deletions of multiple amino acids result in destabilization of the glycoprotein oligomers. These results indicate that conformation changes in the cytoplasmic domain travel across the membrane and subsequently abolish the maturation cleavage. Therefore, we postulate that the cytoplasmic domain is an important maturation factor stabilizing the overall conformation of the glycoprotein. Structured summary:MINT-7997004: LASV GP (uniprotkb:P08669) and LASV GP (uniprotkb:P08669) physically interact (MI:0915) by cosedimentation through density gradient (MI:0029)

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“…For the paramyxovirus SV5 F protein, isolates with a short (20-residue) CT (W3A and WR) cause extensive cell-cell fusion, whereas isolates with an extended CT (T1 and SER) cause little or no cell-cell fusion, and truncation of the CT restores fusion to levels seen in W3A and WR isolates (28,65). For MoMuLV, SIV, and SV5, the hyperfusogenicity caused by truncation of the CT is linked to overall conformational changes in the ectodomain of the protein (2,61,67). In MoMuLV and the Mason-Pfizer monkey virus, the CT is even protease cleaved during viral maturation to "prime" the fusion protein for fusogenicity (2,13).…”

mentioning

confidence: 99%

Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

Aguilar

Matreyek

Choi

et al. 2007

J Virol

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185

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The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membraneadjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3-to 5-fold. These results were corroborated in a reversepseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper-or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ϳ20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P ‫؍‬ 0.007, r 2 ‫؍‬ 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.

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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

Cited by 70 publications

References 81 publications

Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering

Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering

Maturation cleavage within the ectodomain of Lassa virus glycoprotein relies on stabilization by the cytoplasmic tail

Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

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