p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease

TLDR

The C‑terminal p6 domain of HIV‑1 Gag is a proline‑rich 6‑kDa region implicated in Vpr incorporation and virus particle production. The study aimed to define p6’s role and map its functional domains by introducing targeted nonsense and amino‑acid substitution mutations. Mutations were introduced into the full‑length pNL4‑3 clone, and the resulting effects on Gag expression, processing, particle production, infectivity, and ultrastructure were assessed. p6 is essential for efficient particle production, with the Pro‑Thr‑Ala‑Pro motif being critical; mutations outside this motif have minimal impact, the defect appears at a late budding stage and impairs infection in T cells, while protease inactivation rescues the defect, linking p6 to Gag processing.

Abstract

The human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55Gag, contains at its C-terminal end a proline-rich, 6-kDa domain designated p6. Two functions have been proposed for p6: incorporation of the HIV-1 accessory protein Vpr into virus particles and virus particle production. To characterize the role of p6 in the HIV-1 life cycle and to map functional domains within p6, we introduced a number of nonsense and single and multiple amino acid substitution mutations into p6. Following the introduction of the mutations into the full-length HIV-1 molecular clone pNL4-3, the effects on Gag protein expression and processing, virus particle production, and virus infectivity were analyzed. The production of mutant virus particles was also examined by transmission electron microscopy. The results indicate that (i) p6 is required for efficient virus particle production from a full-length HIV-1 molecular clone; (ii) a Pro-Thr-Ala-Pro sequence, located between residues 7 and 10 of p6, is critical for virus particle production; (iii) mutations outside the Pro-Thr-Ala-Pro motif have little or no effect on virus assembly and release; (iv) the p6 defect is manifested at a late stage in the budding process; and (v) mutations in p6 that severely reduce virion production in HeLa cells also block or significantly delay the establishment of a productive infection in the CEM (12D-7) T-cell line. We further demonstrate that mutational inactivation of the viral protease reverses the p6 defect, suggesting a functional linkage between p6 and the proteolytic processing of the Gag precursor protein during the budding of progeny virions.