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Elife. 2016 Jun 25;5. pii: e14663. doi: 10.7554/eLife.14663.

Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies.

Carlson LA1,2, Bai Y1, Keane SC3,4, Doudna JA1,2,5,6, Hurley JH1,2,6.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
2
California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States.
3
Howard Hughes Medical Institute, Baltimore, United States.
4
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, United States.
5
Howard Hughes Medical Institute, University of California, Berkeley, Baltimore, United States.
6
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States.

Abstract

HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5' untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another.

KEYWORDS:

HIV-1; SHAPE; biophysics; giant unilamellar vesicles; human; structural biology; viral genome packaging; virus assembly

PMID:
27343348
PMCID:
PMC4946900
DOI:
10.7554/eLife.14663
[Indexed for MEDLINE]
Free PMC Article

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