A putative alpha-helical structure which overlaps the capsid-p2 boundary in the human immunodeficiency virus type 1 Gag precursor is crucial for viral particle assembly

Abstract

The capsid (CA) and nucleocapsid domains of the human immunodeficiency virus type 1 Gag polyprotein are separated by the p2 spacer peptide, which is essential for virus replication. Previous studies have revealed that p2 has an important role in virus morphogenesis. In this paper, we show that a crucial assembly determinant maps to the highly conserved N terminus of p2, which is predicted to form part of an alpha-helix that begins in CA. A mutational analysis indicates that the ability of the N terminus of p2 to adopt an alpha-helical structure is essential for its function during virus assembly. To prevent CA-p2 processing, it was necessary to mutate both the CA-p2 cleavage site and an internal cleavage site within p2. Virions produced by the double mutant lacked a conical core shell and instead contained a thin electron-dense shell about 10 nm underneath the virion membrane. These results suggest that p2 is transiently required for proper assembly, but needs to be removed from the C terminus of CA to weaken CA-CA interactions and allow the rearrangement of the virion core shell during virus maturation.

FIG. 1

(A) Location of mutations. The domain organization of the HIV-1 Gag precursor Pr55^gag is illustrated at the top. The amino acid sequences of wild-type and mutant p2 together with N- and C-terminal flanking residues are shown below. Substitutions are underlined, and blank spaces represent deletions. Numbers refer to the positions of residues counting from the N terminus of Pr55^gag. The CA-p2 and p2-NC cleavage sites are indicated by solid arrows, and a reported internal cleavage site in p2 (25) is indicated by open arrows. (B) Secondary structure analysis with the PHD program (–47). Residues 351 to 377 of Pr55^gag are shown, and amino acids predicted to form an α-helix are boxed. A reliability index for the secondary structure prediction with values from 0 (lowest reliability) to 9 (highest reliability) is given for each of the boxed residues.

FIG. 2

Effects of alterations in different regions of p2 on virus replication. Jurkat cells were transfected with the parental proviral construct HXBH10/R⁺ (wild type [WT]) or with the indicated p2 mutants, and virus replication was monitored by measuring RT activity in the culture supernatants.

FIG. 3

A critical assembly determinant maps to the N terminus of p2. HeLa cells were transfected with wild-type proviral DNA (WT) or with the indicated mutants and metabolically labeled from 48 to 60 h posttransfection. Viral particles released during the labeling period were pelleted through 20% sucrose cushions, disrupted in RIPA buffer, and directly analyzed by SDS-PAGE. Two different exposures of the same gel are shown. 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. Mock, mock transfection.

FIG. 4

Effects of single-amino-acid substitutions in p2 designed to preserve or to disrupt a predicted α-helical structure on virus replication and on particle formation. (A) RT activity in culture supernatants of Jurkat cells transfected with the indicated proviruses. (B) Direct SDS-PAGE analysis of [³⁵S]methionine-labeled particulate material released from HeLa cells transfected with the indicated proviruses. WT, wild type.

FIG. 5

Electron micrographs of HeLa cell cultures transfected with the parental HXBH10/R⁺ provirus (A) or with the p2 mutants E2A (B), E2G (C), and Δ2 (D). The magnification is the same for all micrographs (bars represent 100 nm).

FIG. 6

Effects of cleavage site mutations on virus replication and CA processing. (A) RT activity in culture supernatants of Jurkat cells transfected with HXBH10/R⁺ (WT) or the indicated cleavage site mutants. (B) Direct SDS-PAGE analysis of [³⁵S]methionine-labeled particulate material released from HeLa cells transfected with the parental provirus or the indicated cleavage site mutants. Molecular mass markers (in kilodaltons) are indicated on the right. Mock, mock transfection.

FIG. 7

Electron micrographs of HeLa cells transfected with the CA1/M4I double mutant. Arrows point to examples of immature viral particles. Note the electron-dense layer approximately 10 nm below the viral lipid membrane visible in several particles with a mature appearance. Bars represent 100 nm.