In vitro assembly properties of purified bacterially expressed capsid proteins of human immunodeficiency virus.
The Gag polyprotein of retroviruses is sufficient for assembly and budding of virus-like particles from the host cell. In the case of human immunodeficiency virus (HIV), Gag contains the domains matrix, capsid (CA), nucleocapsid (NC) and p6 which are separated by the viral proteinase inside the nascent virion, leading to morphological maturation to yield an infectious virus. In the mature virus, CA forms a capsid shell surrounding the ribonucleoprotein core consisting of NC and the genomic RNA. To define requirements for particle assembly and functional contributions of individual domains, we expressed domains of HIV Gag in Escherichia coli and purified the products to near homogeneity. In vitro assembly of CA, with or without the C-terminally adjacent spacer peptide, yielded tubular structures with a diameter of approximately 55 nm and heterogeneous length. Efficient particle formation required high protein concentration, high salt and neutral to alkaline pH. In contrast, in vitro assembly of CA-NC occurred at a 20-fold lower protein concentration and in low salt, but required addition of RNA. These results suggest that hydrophobic interactions of capsid proteins are sufficient for particle formation while the RNA-binding nucleocapsid domain may concentrate and align structural proteins on the viral genome.
Gross
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Heinrich-Pette-Institut, Hamburg, Germany.
Hohenberg
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Kräusslich
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HG
D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D015683:Gene Products, gag / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure; D005814:Genes, Viral; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000648:ultrastructure; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure
D017931:DNA Primers; D015683:Gene Products, gag; D011994:Recombinant Proteins; D015678:Viral Structural Proteins