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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
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Cumulative mutations of ubiquitin acceptor sites in human immunodeficiency virus type 1 gag cause a late budding

Eva Gottwein1, Stefanie Jäger, Anja Habermann

  • 1Abteilung Virologie, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany.

Journal of Virology
|June 16, 2006
PubMed

Insights

Ubiquitination of human immunodeficiency virus type 1 (HIV-1) Gag proteins is crucial for efficient virus release. Mutating lysine residues near the viral late domain significantly impairs HIV-1 budding, suggesting a role in protein interactions.

Area of Science:

  • Virology
  • Molecular Biology
  • Cell Biology

Background:

  • Monoubiquitination of the p6 domain in human immunodeficiency virus type 1 (HIV-1) Gag has been established.
  • Previous studies indicated low-level monoubiquitination in MA, CA, and NC domains, with a potential role in virus release, though its precise relevance remained unclear.

Purpose of the Study:

  • To investigate the functional significance of Gag ubiquitination in HIV-1 replication.
  • To elucidate the role of specific lysine residues within Gag domains (NC, SP2, p6) in virus release and budding.

Main Methods:

  • Construction of HIV-1 Gag variants with lysine-to-arginine mutations in NC, SP2, and p6 domains, individually and in combination.
  • Analysis of ubiquitination status of Gag domains using mutagenesis and biochemical assays.
  • Assessment of virus release kinetics and budding structures via electron microscopy and quantitative assays.

Main Results:

  • SP2 domain was identified as a site for mono- or di-ubiquitination, similar to other Gag domains.
  • Mutations in single domains showed minor effects on virus release.
  • Cumulative mutations in NC/SP2 or NC/p6 domains led to accumulation of late budding structures.
  • Comprehensive mutation of lysine residues downstream of CA significantly reduced virus release and increased budding structures.

Conclusions:

  • Ubiquitination of lysine residues in Gag, particularly near the viral late domain, is essential for efficient HIV-1 budding.
  • No single lysine residue is indispensable; individual domains appear functionally redundant.
  • Gag ubiquitination likely facilitates transient protein interactions at the viral budding site, crucial for the release process.

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