Structural criteria for regulation of membrane fusion and virion incorporation by the murine leukemia virus TM cytoplasmic domain

Taylor, Gwen M.; Sanders, D.A.R.

doi:10.1016/s0042-6822(03)00297-6

Cited by 32 publications

(50 citation statements)

References 68 publications

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“…This has been suggested to stabilize the prefusion structure of the spike. The model has been supported by the phenotypes of mutants designed to perturb the proposed coiled-coil interaction (20,24). In such a structure the cleavage of the R peptide of one TM subunit is expected to disturb the coiled-coil interaction, possibly resulting in the release of the uncleaved R peptide tails of the other two subunits in a protease-sensitive form.…”

Section: Discussionmentioning

confidence: 97%

“…The structure of the endodomain of the Env trimer with the R peptide is not known, but it has been modeled as a coiled coil (20). This has been suggested to stabilize the prefusion structure of the spike.…”

Section: Discussionmentioning

confidence: 99%

“…This is characterized in particular by the preference of Leu over Phe and Tyr at the P1 position and by the exclusion of charged amino acids and amino acids with side chains branched at the beta-carbon (Ile and Val) at this position. Consequently, we along with others have created L 649 R (LR), L 649 I (LI), and L 649 V (LV) Env mutants and shown that these were inhibited in R peptide cleavage when assembled into virus particles, which were also shown to be noninfectious (8,15,20). Earlier, we used such mutant virus to characterize the mechanism of fusion facilitation by the R peptide cleavage (9).…”

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confidence: 99%

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Cooperative Cleavage of the R Peptide in the Env Trimer of Moloney Murine Leukemia Virus Facilitates Its Maturation for Fusion Competence

Löving

Kronqvist

Sjöberg

et al. 2011

J Virol

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The spike protein of murine leukemia virus, MLV, is made as a trimer of the Env precursor. This is primed for receptor-induced activation of its membrane fusion function first by cellular furin cleavage in the ectodomain and then by viral protease cleavage in the endodomain. The first cleavage separates the peripheral surface (SU) subunit from the transmembrane (TM) subunit, and the latter releases a 16-residue-long peptide (R) from the TM endodomain. Here, we have studied the distribution of R peptide cleavages in the spike TM subunits of Moloney MLV preparations with partially R-peptide-processed spikes. The spikes were solubilized as trimers and separated with an R peptide antibody. This showed that the spikes were either uncleaved or cleaved in all of its TM subunits. Further studies showed that R peptide cleavage-inhibited Env mutants, L 649 V and L 649 I, were rescued by wild-type (wt) Env in heterotrimeric spikes. These findings suggested that the R peptide cleavages in the spike are facilitated through positive allosteric cooperativity; i.e., the cleavage of the TM subunit in one Env promoted the cleavages of the TMs in the other Envs. The mechanism ensures that protease cleavage in newly released virus will generate R-peptide-cleaved homotrimers rather than heterotrimeric intermediates. However, using a cleavage site Env mutant, L 649 R, which was not rescued by wt Env, it was possible to produce virus with heterotrimers. These were shown to be less fusion active than the R-peptidecleaved homotrimers. Therefore, the cooperative cleavage will speed up the maturation of released virus for fusion competence.The spike protein of murine leukemia virus (MLV) is assembled in the endoplasmic reticulum (ER) of the producer cell from three copies of the Env precursor protein gp80 (10, 18). The trimeric spike undergoes two proteolytic cleavage events to prepare it for receptor-induced activation of the membrane fusion process. The first one is mediated by the furin of the host cell and takes place when the spike passes the trans-Golgi network on its way from the ER to the cell surface (3,6). This cleavage separates the polypeptide of the peripheral surface (SU) subunit from that of the transmembrane (TM) subunit (7). It also releases the Env fusion peptide at the membrane-distal amino-terminal region of the TM (26). The second cleavage is mediated by the viral protease and takes place inside newly formed particles. Here, the protease cleaves not only the Gag and the Gag-Pol precursors into the mature internal proteins (matrix, p12, capsid, and nucleocapsid proteins) and the viral enzymes (protease, polymerase, and integrase) but also the endodomain of the TM subunit of Env. A 16-residue-long C-terminal peptide, the R peptide, is cleaved off, generating p15E from the TM precursor, Pr15E (5, 7, 17). By studying cell-associated Env with an R peptide truncation, it was shown that the R peptide inhibited the Env from becoming activated for membrane fusion by the receptor (14, 15).The R peptide cleavage site QAL 649 /VL...

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Cooperative Cleavage of the R Peptide in the Env Trimer of Moloney Murine Leukemia Virus Facilitates Its Maturation for Fusion Competence

Löving

Kronqvist

Sjöberg

et al. 2011

J Virol

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“…Most of the ∼32-residue-long endodomains of the TM subunits, including their C-terminal 16-residue-long R-peptides, have been modeled as trimeric coiled coils (18). This has been suggested to inhibit the receptor triggering of the R-peptide Env precursor.…”

Section: Discussionmentioning

confidence: 99%

“…It was suggested that the R-peptide ties the TM subunits together in the endodomain and that this prevents conformational changes in the Env that are necessary for fusion. Indeed, the endodomain with the R-peptide has been modeled as a trimeric coiled coil (18). This model has been supported by mutational studies using Moloney (Mo) MLV.…”

mentioning

confidence: 93%

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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Amphotropic retrovirus transduction is inhibited by high doses of particle‐associated envelope proteins

Landázuri

Doux

2007

Biotech & Bioengineering

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Using a panel of amphotropic murine leukemia virus packaging cell lines that differed only in their levels of envelope protein (gp70) expression, we examined the relationship between transduction and the number of envelope proteins per virus. We generated virus stocks that contained different levels of virus-associated envelope proteins, purified them from gp70 that was not associated with the viruses, quantified their titers, and measured the efficiency with which they transduced NIH 3T3, TE671, and HeLa cells. As expected, titers increased monotonically with viral envelope protein number. Titers are measured using highly dilute virus, however, and are often not predictive of gene transfer when high doses of virus are used, as is done in gene therapy protocols. Interestingly, when we used high doses of virus, we observed significantly different trends: gene transfer increased, reached a maximum, and then declined sharply as the number of envelope proteins per virus increased. The highest levels of gene transfer occurred when cells were transduced with a moderate dose of virus that contained low levels of envelope protein. Our results indicate that transduction is inhibited when viruses that contain large numbers of envelope proteins are used. This is most likely because each virus, when it binds to a cell, delivers a large payload of envelope proteins that occupy or inactivate multiple virus receptors, reducing or eliminating the susceptibility of the cell to being transduced by additional viruses. The implications of our findings for the design of improved retroviral vectors for human gene therapy are discussed.

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Structural criteria for regulation of membrane fusion and virion incorporation by the murine leukemia virus TM cytoplasmic domain

Cited by 32 publications

References 68 publications

Cooperative Cleavage of the R Peptide in the Env Trimer of Moloney Murine Leukemia Virus Facilitates Its Maturation for Fusion Competence

Cooperative Cleavage of the R Peptide in the Env Trimer of Moloney Murine Leukemia Virus Facilitates Its Maturation for Fusion Competence

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

Amphotropic retrovirus transduction is inhibited by high doses of particle‐associated envelope proteins

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