Proteolytic activity in vivo and encapsidation of recombinant human immunodeficiency virus type 1 proteinase expressed in baculovirus-infected cells.

Royer, M; Tournier, J; Gay, Bernard; Boulanger, Pierre; Bardy, Martine

doi:10.1099/0022-1317-78-1-131

Cited by 11 publications

(33 citation statements)

References 39 publications

(32 reference statements)

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“…Using a baculovirus expression system, Royer et al (1997) created recombinant HIV-1 Gag-Pol constructs containing various lengths of PR-inactivated Pol sequence fused inframe to the C-terminal p6 of Pr55gag; they reported that the constructs were barely detectable in culture supernatants. They also reported that the p6-containing Gag-Pol constructs were capable of trans-dominantly inhibiting Gag particle release.…”

Section: Discussionmentioning

confidence: 99%

“…may counteract the promoting effect of p6gag on GagPR release. However, in a separate study using an insect cell system, p6gag within PR-inactivated Gag-Pol (similar to the Gag(p6)Pol construct) was observed as markedly reducing VLP yields, as well as quantities of VLP-associated Gag-Pol (Royer et al, 1997). Royer et al therefore suggest that p6gag within Gag-Pol exerts a negative impact on VLP production in addition to impeding Gag(p6)Pol viral incorporation.…”

Section: Introductionmentioning

confidence: 95%

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p6gag domain confers cis HIV-1 Gag-Pol assembly and release capability

Guo

Huang³

et al. 2016

Journal of General Virology

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During virus assembly, HIV-1 Gag-Pol is packaged into virions via interaction with Pr55gag. Studies suggest that Gag-Pol by itself is incapable of virus particle assembly or cell release, perhaps due to the lack of a budding domain in the form of p6gag, which is truncated within Gag-Pol because of a ribosomal frameshift during Gag translation. Additionally (or alternatively), large molecular size may not support Gag-Pol assembly into virus-like particles (VLPs) or release from cells. To test these hypotheses, we constructed Gag-Pol expression vectors retaining and lacking p6gag, and then reduced Gag-Pol molecular size by removing various lengths of the Pol sequence. Results indicate that Gag-Pol constructs retaining p6gag were capable of forming VLPs with a WT HIV-1 particle density. Gag-Pol molecular size reduction via partial removal of the Pol sequence mitigated the Gag-Pol assembly defect to a moderate degree. Our results suggest that the Gag-Pol assembly and budding defects are largely due to a lack of p6gag, but also in part due to size limitation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

p6gag domain confers cis HIV-1 Gag-Pol assembly and release capability

Guo

Huang³

et al. 2016

Journal of General Virology

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“…On the other hand, virus load assays based on genome copy number determination explore the genome encapsidation process and not specifically the capsid assembly, although RNA encapsidation and virus assembly have been shown to be two linked phenomena [56]–[59]. Several laboratories, including ours, have fused an enzyme to the Gag precursor, e.g bacterial beta-galactosidase [31], [60] or firefly luciferase [22], to quantitate VLP in cell culture supernatants using enzymatic assays. Other methods have preconized the fusion of Gag to enhanced green fluorescent protein from jellyfish Aequorea (eGFP), to be able to monitor Gag assembly and budding by flow cytometry, or by fluorescence microscopy in situ in cell compartments [61], [62].…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Novel Type of HIV-1 Particle Assembly Inhibitor Using a Quantitative Luciferase-Vpr Packaging-Based Assay

et al. 2011

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The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.

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“…In addition, retroviral PR domains are extremely insoluble (6,10,21). Expression of an HIV-1 Gag protein with an inactivated PR domain at its C terminus (creating a molecule equivalent to RSV Gag) resulted in a reduction of extracellular VLP production by 5-to 10-fold compared with wild-type Gag, and the particles produced had a lower density and were irregular in shape (19). These data suggest that the PR domain might lead to unnatural aggregation or precipitation of RSV Gag in the baculovirus overexpression system, in effect preventing proper budding.…”

mentioning

confidence: 99%

PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells

Johnson

Scobie

Vogt

2001

J Virol

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While baculovirus expression of Gag proteins from numerous retroviruses has led reliably to production of virus-like particles (VLPs), we observed that expression of Rous sarcoma virus Gag failed to produce VLPs. Transmission and scanning electron microscopy analysis revealed that the Gag protein reached the plasma membrane but was unable to correctly form particles. Addition of a myristylation signal had no effect on the budding defect, but deletion of the PR domain of Gag restored normal budding. The resulting VLPs were morphologically distinct from human immunodeficiency virus type 1 VLPs expressed in parallel.

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Proteolytic activity in vivo and encapsidation of recombinant human immunodeficiency virus type 1 proteinase expressed in baculovirus-infected cells.

Cited by 11 publications

References 39 publications

p6gag domain confers cis HIV-1 Gag-Pol assembly and release capability

p6gag domain confers cis HIV-1 Gag-Pol assembly and release capability

Characterization of a Novel Type of HIV-1 Particle Assembly Inhibitor Using a Quantitative Luciferase-Vpr Packaging-Based Assay

PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells

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