Assembly of capsids from bacterially-expressed andIn Vitro translated retroviral Gag precursors

Klikova, M.; Weldon, Robert; Sakalian, Michael; Rhee, Sehun; Ruml, Tomáš; Hunter, Eric

doi:10.1007/bf02559892

Cited by 3 publications

(7 citation statements)

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“…2B, left), confirming previous reports in which retroviral Gag protein (e.g. Mason-Pfizer monkey virus, Rous sarcoma virus, and HIV) assemble into VLPs inside E. coli cells (24,27,28). In contrast, no particles were seen in E. coli cells when maintained at 30°C (Fig.…”

Section: Time Courses Of Hiv Gag Expression and Assembly In Esupporting

confidence: 89%

“…Interestingly, spherical particles with similar morphologies are also produced when purified soluble Gag protein is subjected to in vitro assembly reactions (25,26), although the particle sizes are heterogeneous when compared with those of VLPs produced by eukaryotic cells. Similar findings have been reported for other retroviral Gags (27,28). These data show that the assembly phenotypes of Gag produced by expression in E. coli as well as by in vitro reaction mimic the authentic Gag assembly observed in eukaryotic cells and suggest that the assembly reaction is driven by the intrinsic properties of Gag protein.…”

supporting

confidence: 87%

“…A number of studies have demonstrated that in vitro assembly using purified Gag forms spherical particles, remarkably similar to the immature form of retroviral capsids (25,27,28). We have also shown that, in an in vitro assembly reaction, Gag forms a 60 S multimer that subsequently shifts to 600 S, equivalent to the immature form of HIV capsids, suggesting that the 60 S multimer may represent an assembly intermediate during capsid formation (26).…”

Section: Time Courses Of Hiv Gag Expression and Assembly In Ementioning

confidence: 62%

“…For comparison, we enriched the large Gag complex, corresponding to 600 S, which was observed in E. coli cells (Fig. 2, left) as described previously (28). As expected, the structure of the complex showed a hollow sphere surrounded by a double ring structure (Fig.…”

Section: Time Courses Of Hiv Gag Expression and Assembly In Ementioning

confidence: 75%

“…1B). It has been reported for Mason-Pfizer monkey virus that Gag assembles to spherical particles in E. coli and forms inclusion bodies, which were precipitated by centrifugation under similar conditions (28).…”

Section: Time Courses Of Hiv Gag Expression and Assembly In Ementioning

confidence: 99%

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Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

Morikawa

Goto

Momose

2004

Journal of Biological Chemistry

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Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37°C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30°C resulted in the production of only the 60 S form of Gag multimer, and crescentshaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37°C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30°C reaction produced only the 60 S multimers. However, when the 60 S multimers were crosslinked so as not to allow conformational changes, in vitro assembly reactions at 37°C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37°C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.

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Section: Time Courses Of Hiv Gag Expression and Assembly In Esupporting

confidence: 89%

supporting

confidence: 87%

Section: Time Courses Of Hiv Gag Expression and Assembly In Ementioning

confidence: 62%

Section: Time Courses Of Hiv Gag Expression and Assembly In Ementioning

confidence: 75%

Section: Time Courses Of Hiv Gag Expression and Assembly In Ementioning

confidence: 99%

See 3 more Smart Citations

Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

Morikawa

Goto

Momose

2004

Journal of Biological Chemistry

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Modulation of HIV-like particle assemblyin vitroby inositol phosphates

Campbell

Fisher

Towler

et al. 2001

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

191

195

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HIV-1 Gag protein assembles into 100- to 120-nm diameter particles in mammalian cells. Recombinant HIV-1 Gag protein assembles in a fully defined system in vitro into particles that are only 25–30 nm in diameter and that differ significantly in other respects from authentic particles. However, particles with the size and other properties of authentic virions were obtained in vitro by addition of inositol phosphates or phosphatidylinsitol phosphates to the assembly system. Thus, the interactions between HIV-1 Gag protein molecules are altered by binding of inositol derivatives; this binding is apparently essential for normal HIV-1 particle assembly. This requirement is not seen in a deleted Gag protein lacking residues 16–99 within the matrix domain.

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In Vitro Assembly of Human Immunodeficiency Virus Type 1 Gag Protein

Morikawa

Goto

Sano

1999

Journal of Biological Chemistry

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Retroviral Gag protein is sufficient to produce Gag virus-like particles when expressed in higher eukaryotic cells. Here we describe the in vitro assembly reaction of human immunodeficiency virus Gag protein, which consists of two sequential steps showing the optimal conditions for each reaction. Following expression and purification, Gag protein lacking only the C-terminal p6 domain was present as a monomer (50 kDa) by velocity sedimentation analysis. Initial assembly of the Gag protein to 60 S intermediates occurred by dialysis at 4°C in low salt at neutral to alkaline pH. However, higher order of assembly required incubation at 37°C and was facilitated by the addition of Mg 2؉ . Prolonged incubation under these conditions produced complete assembly (600 S), equivalent to Gag virus-like particles obtained from Gag-expressing cells. Neither form disassembled by treatment with nonionic detergent, suggesting that correct assembly might occur in vitro. Electron microscopic observation confirmed that the 600 S assembly products were spherical particles similar to authentic immature human immunodeficiency virus particles. The latter assembly stage but not the former was accelerated by the addition of RNA although not inhibited by RNaseA treatment. These results suggest that Gag protein alone assembles in vitro, but that additional RNA facilitates the assembly reaction.

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Assembly of capsids from bacterially-expressed andIn Vitro translated retroviral Gag precursors

Cited by 3 publications

References 0 publications

Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

Modulation of HIV-like particle assemblyin vitroby inositol phosphates

In Vitro Assembly of Human Immunodeficiency Virus Type 1 Gag Protein

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