The C-terminal half of the human immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly

Abstract

Human immunodeficiency virus type 1 particle assembly is directed by the Gag polyprotein Pr55(gag), the precursor for the matrix (MA), capsid (CA), and nucleocapsid proteins of the mature virion. We now show that CA sequences N terminal to the major homology region (MHR), which form a distinct domain, are dispensable for particle formation. However, slightly larger deletions which extend into the MHR severely impair particle production. Remarkably, a deletion which removed essentially all MA and CA sequences between the N-terminal myristyl anchor and the MHR reduced the yield of extracellular particles only moderately. Particle formation even exceeded wild-type levels when additional MA sequences, either from the N or the C terminus of the domain, were retained. We conclude that no distinct region between the myristyl anchor and the MHR is required for efficient particle assembly or release.

FIG. 1

Schematic representation of Gag deletion mutants. The domain structure of the Gag polyprotein and the position of the MHR within the CA domain are indicated. Numbers refer to the positions of the deleted amino acids relative to the initiating methionine.

FIG. 2

Effects of large deletions in CA on particle production. HeLa cells were transfected with wild-type proviral DNA (WT) or with the indicated mutants. (A) The cells were metabolically labeled with [³⁵S]methionine from 48 to 60 h posttransfection, and viral proteins were immunoprecipitated from the cell lysates. (B) Viral particles released during the labeling period were pelleted through 20% sucrose cushions, and the protein profile of the pelleted material was directly analyzed by SDS-PAGE. The positions of specific viral proteins are indicted on the left. The Δ126-285 and Δ126-304 mutants were analyzed in duplicate.

FIG. 3

Efficient particle production in the absence of up to 50% of Pr55^gag. HeLa cells transfected with the indicated proviral constructs were labeled with [³⁵S]methionine, and viral proteins were immunoprecipitated from the cell lysates with patient serum (A). Particles released into the supernatant were pelleted through sucrose and directly analyzed by SDS-PAGE (B). The positions of specific viral proteins are indicated on the left. The positions of migration of molecular mass markers (in kilodaltons) are indicated on the right.

FIG. 4

Thin-section electron microscopy of HeLa cells transfected with the wild type provirus (A) or with the indicated Gag deletion mutants (B, C). Bars indicate a length of 100 nm.