Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells

Rhodes, Andrew; Spitali, Mariangela; Hutchinson, Gillian; Rud, Erling W.; Stephens, Paul E.

doi:10.1099/0022-1317-75-1-207

Cited by 12 publications

(6 citation statements)

References 21 publications

(11 reference statements)

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“…Evidence from expression studies of soluble envelope glycoprotein containing gp120 fused to the gp41 ectodomain (2,18,19,43) as well as of membrane-bound full-length gp160 (17,44,45) support the view that the envelope glycoprotein is a trimer or a tetramer. Although the trimeric nature of vesicular stomatitis virus (46) and rabies virus (47) envelope glycoproteins has been determined using virion-derived envelope, relatively little is known about the oligomeric nature of the HIV envelope on virions (5,6).…”

Section: Resultsmentioning

confidence: 97%

Processing, Stability, and Receptor Binding Properties of Oligomeric Envelope Glycoprotein from a Primary HIV-1 Isolate

Staropoli¹,

Chanel²,

Girard³

et al. 2000

Journal of Biological Chemistry

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The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is thought to exist on the virion surface as a trimer of non-covalently associated gp120/gp41 molecules. We expressed trimeric envelope glycoprotein from three primary, macrophage tropic HIV-1 isolates in baby hamster kidney cells and analyzed the furin-mediated cleavage, stability, and receptor binding properties of the oligomers. The envelope glycoprotein was secreted in a soluble form deleted of its transmembrane anchor and the intracytoplasmic domain (gp140). A mixture of trimers, dimers, and monomers of gp140 as well as monomeric gp120 was detected on polyacrylamide gels. Analysis by sucrose gradient centrifugation revealed that trimers and dimers were essentially composed of uncleaved gp140, whereas most of the gp120 was found in the monomeric fraction. To analyze the effect of the cleavage of gp140 to gp120/⌬41 on trimerization, we co-expressed the furin protease along with gp140. Surprisingly, furin expression changed the subcellular localization of the envelope glycoprotein, which became in majority sequestered in the major furin compartment, the trans-Golgi network, as judged by confocal laser microscopy. The envelope glycoprotein secreted from furin-co-expressing cells was almost completely cleaved to gp120 and ⌬gp41, but gp120 was found exclusively in the monomeric fraction, with a few residual oligomers being composed of uncleaved gp140. Secreted uncleaved gp140 trimers were purified to homogeneity and analyzed for their capacity to interact with cellular receptors CD4 and CC chemokine receptor 5 (CCR5). Receptor binding was analyzed on CD4-and CCR5-expressing cells as well as on peripheral blood mononuclear cells. Trimers showed greatly reduced binding to CD4 as compared with monomers. Neither monomers nor trimers bound directly to CCR5. In conclusion, our results show that the cleaved form of the envelope glycoprotein does not form stable trimers, suggesting that gp120/gp41 oligomers on the virion surface might be stabilized by a yet to be identified mechanism and that the virion might attach to CD4 via a monomeric form of gp120. These results are relevant to the development of an envelope-based vaccine against AIDS.

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Section: Resultsmentioning

confidence: 97%

Processing, Stability, and Receptor Binding Properties of Oligomeric Envelope Glycoprotein from a Primary HIV-1 Isolate

Staropoli¹,

Chanel²,

Girard³

et al. 2000

Journal of Biological Chemistry

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“…Two vectors pTH.tat and pTH.rev contain the respective re6 genes into the BamHI site without upstream Ub-R. The SIV macJ5 molecular clone was used as the source of these genes as previously described [2,10,12]. Vector pND14-G1 expressed the SIV mac239 envelope gp120 coding sequence under control of the hCMV IE enhancer/promotor.…”

Section: Dna Expression Vector Based Vaccinesmentioning

confidence: 99%

“…Recombinant MVA [15] in this study were engineered to express the gag/pol, nef, tat, re6, and en6 genes of SIV macJ5 [10,12] under transcriptional control of vaccinia virus-specific promoters. Briefly, the gag/pol, nef, tat and re6 coding sequences from the SIV mac32HJ5 molecular clone [10,12] were placed under control of vaccinia virus early/late promoter P7.5 by insertion into the unique SmaI site of the MVA vector plasmid pUCII-LZ-P7.5 [16]. The SIV mac32HJ5 en6 coding cDNA was cloned into the MVA vector plasmids pIII-sP and pIIIgpt-sP to allow for recombinant gene expression regulated by a strong synthetic vaccinia virus promoter (Table 1) [17].…”

Section: Mva Based Vaccinesmentioning

confidence: 99%

A vaccine strategy utilizing a combination of three different chimeric vectors which share specific vaccine antigens

Heeney

Koopman

Rosenwirth

et al. 2000

J of Medical Primatology

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A large number of recombinant of viral and bacterial systems have been engineered as vectors to express foreign genes for vaccination and/or gene therapy. A common problem is the immune response to the vector itself. The presence of anti-vector immune responses may preclude sufficient 'priming' or immunogenicity if pre-existing immune responses are present, or they may impair optimal 'boosting' upon repeated immunization or delivery with the same vector. To circumvent this problem we developed a strategy using different chimeric vectors which share only the expression of common specific antigens desired for immunization. This approach not only has the advantage of avoiding increased anti-vector responses, but allows the use of combinations of vectors which could subsequently present the same or related antigen differently to the immune system as well as at alternative sites to induce the optimal type of immunity against the pathogen of interest.

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“…SIV-infected cells have been reported to produce dimers of full-length precursor and TM subunit env (8). Similarly, a soluble uncleaved recombinant SIV env lacking the transmembrane domain and cytoplasmic tail (gp140) was reported to form tetramers and dimers (9). Chemical crosslinking and sucrose gradient sedimentation suggested that full-length or soluble uncleaved HIV-1 precursor occurred as dimers and higher-order oligomers (10).…”

mentioning

confidence: 99%

Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation

Schuck¹,

Leapman²,

Arthur³

et al. 2001

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

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The envelope proteins (env) of simian immunodeficiency virus (SIV) and HIV type 1 assemble to form noncovalently associated oligomers in the endoplasmic reticulum. After cleavage in a Golgi compartment, oligomeric env complexes are transported to the surface of infected cells, where incorporation into budding virions can occur. Difficulties in obtaining adequate quantities of virions retaining env, as well as the unstable nature and hydrophobicity of the oligomer, may account for the absence of previous biophysical studies to determine the oligomeric valency of membrane-associated env. The aim of this study was to evaluate the oligomeric state of SIV env before membrane-fusion activation. Virion-associated env, obtained by crosslinking and detergent extraction, and noncrosslinked secreted env ectodomain (recombinant gp140) were purified by lentil-lectin chromatography and gel filtration as single predominant species. Sedimentation equilibrium-derived mass values for both forms of SIV env were close to those predicted for trimeric assemblies. Determination of the mass of individual molecules by scanning transmission electron microscopy confirmed that SIV virion-associated env and gp140 formed largely homogeneous populations of trimers. Furthermore, a triangular or tri-lobed morphology was clearly visualized in a subset of the trimers.

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Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells

Cited by 12 publications

References 21 publications

Processing, Stability, and Receptor Binding Properties of Oligomeric Envelope Glycoprotein from a Primary HIV-1 Isolate

Processing, Stability, and Receptor Binding Properties of Oligomeric Envelope Glycoprotein from a Primary HIV-1 Isolate

A vaccine strategy utilizing a combination of three different chimeric vectors which share specific vaccine antigens

Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation

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