Redundant roles for nucleocapsid and matrix RNA-binding sequences in human immunodeficiency virus type 1 assembly

Abstract

RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.

FIG. 1.

Proviral constructs. Cartoons of the Gag regions of the various constructs used in this study are presented. The shaded area indicates the MA basic region, and mutations in that region are indicated by a white X. The NC region is hatched, and the deletion is represented by a thick black line. Inserted sequences are indicated by a black box and are either presented under the respective cartoon or identified in the box. For the MX cartoon, wild-type and mutated MA sequences are given under the region altered. The NC sequences remaining after deletion of the majority of NC are displayed in single-amino-acid code. Starting or ending points in Gag truncations are indicated by arrows pointing upward.

FIG. 2.

Relative RNA content of NX/PX. Bar graphs of total viral RNA as measured by a Ribogeen assay in the NX/PX mutant relative to that measured in the PX protease mutant are presented. Values are from three independent preparations. Error bars, standard deviations.

FIG. 3.

Immunoblots of MX and MX/NX preparations. p17^MA immunoblots of equal volumes of virion preparations are presented. Samples are identified above the lanes. Molecular masses, as calculated by relative mobility, and identities of bands are given on the left and right, respectively.

FIG. 4.

[³⁵S]Met-Cys metabolic radiolabeling and immunoprecipitation of Gag. (A) Phosphorimages of SDS-PAGE gels from a typical p24^CA immunoprecipitation analysis of metabolically labeled Gag from virion and cytoplasmic lysates. The lysates examined are given above each panel, and samples are identified above their respective lanes. (B) Graph of the release factor determined by [³⁵S] radioimmunoprecipitation with p24^CA antiserum. The release factor is the phosphorimage intensity volume of Gag immunoprecipitates from virion lysates divided by that of Gag immunoprecipitated from both virion and cell lysates. (C) p24^CA immunoblot of equal volumes of virion preparations isolated from a 48-h harvest of the cultures just prior to radiolabeling. Positions of the molecular mass standards are given on the left, and reactive bands are identified on the right.

FIG. 5.

Immunoblots of MX/NX rescued with wild-type Gag. Ap17 immunoblot of equal volumes of virion preparations produced by cotransfection is presented. Samples are identified above the lanes. Molecular masses, as calculated by relative mobility, and identities of bands are given on the left and right, respectively.

FIG. 6.

Immunoblots of MX/NX rescued with the single mutants. Immunoblots of equal volumes of virion preparations produced by cotransfection are presented. (A) p17 immunoblot of virion samples produced from cotransfection of the MX/NX/PX and MX/PX constructs. (B) p17 immunoblot of virion samples produced from cotransfection of the MX/NX/PX and NX/PX constructs. (C) p24 immunoblot of virion samples produced from cotransfection of MX/NX and NX/PX constructs. Samples are identified above the lanes. Molecular masses (in kilodaltons), as calculated by relative mobility, and identities of bands are given on the left and right, respectively.

FIG. 7.

Immunoblots of MX/NX rescued with portions of Gag, basic residues, or multimerization domains. Immunoblots of equal volumes of virion preparations produced by cotransfection are presented. (A) p17 immunoblot of virion samples from cotransfections of 293T cells with portions of Gag. Lanes: 1, MX plus sssDNA; 2, MX/NX plus sssDNA; 3, MX/NX plus LNX; 4, MX/NX plus PX; 5, MX/NX plus MA-p6; 6, MX/NX/PX plus sssDNA; 7, MX/NX/PX plus MA-CA; 8, MX/NX/PX plus PX. (B) p17 immunoblot of virion samples from transfection of 293T cells with the MKK, MRR, Mp10, or MZip construct. Samples are identified above the lanes. Molecular masses, as calculated by relative mobility, and identities of bands are given on the left and right, respectively. (C) Graph of the release factor determined by [³⁵S] radioimmunoprecipitation with p24^CA antiserum. The release factor is the phosphorimage intensity volume of Gag immunoprecipitates from virion lysates divided by that of Gag immunoprecipitated from both virion and cell lysates.