Role of murine leukemia virus nucleocapsid protein in virus assembly

Abstract

The retroviral nucleocapsid protein (NC) originates by cleavage of the Gag polyprotein. It is highly basic and contains one or two zinc fingers. Mutations in either the basic residues or the zinc fingers can affect several events of the virus life cycle. They frequently prevent the specific packaging of the viral RNA, affect reverse transcription, and impair virion assembly. In this work, we explore the role of NC in murine leukemia virus (MLV) particle assembly and release. A panel of NC mutants, including mutants of the zinc finger and of a basic region, as well as truncations of the NC domain of Gag, were studied. Several of these mutations dramatically reduce the release of virus particles. A mutant completely lacking the NC domain is apparently incapable of assembling into particles, although its Gag protein is still targeted to the plasma membrane. By electron microscopy on thin sections of virus-producing cells, we observed that some NC mutants exhibit various stages of budding defects at the plasma membrane and have aberrant particle morphology; electron micrographs of cells expressing some of these mutants are strikingly similar to those of cells expressing "late-domain" mutants. However, the defects of NC mutants with respect to virus release and infectivity could be complemented by an MLV lacking the p12 domain. Therefore, the functions of NC in virus budding and infectivity are completely distinct from viral late-domain function.

FIG. 1.

MLV NC mutants used in this study. The NC domain is located at the C terminus of the MLV Gag polyprotein. The amino acid sequence of MLV NC, which contains only 60 residues, is shown in 1-letter code. The location of the zinc finger is indicated. The NC mutants used in this study are the C39S mutant, carrying a point mutation in the zinc finger (22); the NC(Δ16-23) mutant, with a deletion of residues R16 through R23 (35); and the C37- mutant, which is truncated after R23. These mutants fail to encapsidate genomic RNA efficiently. Another mutant, C17- (19), is missing the 17 C-terminal amino acids of the NC domain of Gag but still packages some viral RNA.

FIG. 2.

Virus release by MLV NC mutants. MLV Pr65^Gag and p30^CA released from transfected cells into culture fluid in pelletable form (V) and Pr65^Gag in cell lysates (C) are analyzed by immunoblotting with an anti-p30^CA antiserum. Lanes 1 and 2, wild type; lanes 3 and 4, C39S; lanes 5 and 6, NC(Δ16-23); lanes 7 and 8, PR⁻; lanes 9 and 10, C17-; lanes 11 and 12, C37-; lanes 13 and 14, C60-; lane 15, lysate of mock-transfected cells.

FIG. 3.

Localization and quantitation of mutant Gag proteins by immunofluorescence and immunoblotting. Cells were first transfected with the indicated viral clones and then fixed and stained for detection of CA as described in Materials and Methods. (A) Immunofluorescence images. Bar, 10 μm. (B) Relative concentrations of anti-CA-reactive material in the cells, measured either by integrating immunofluorescence intensity over the cells as described in Materials and Methods (microscope) or by immunoblotting (Western). wt, wild type.

FIG. 4.

Morphology of NC mutant particles. (A) Thin sections of cells transfected with the indicated viral clones. (B) Thin section of cells transfected with the NC(Δ16-23) mutant. Bar in panel B, 200 nm.

FIG. 5.

Immunoelectron microscopy of the C60- Gag mutant. Cells transfected with the indicated viral clones were fixed in 4% paraformaldehyde and stained with anti-p30^CA as described in Materials and Methods. Wild-type MLV (wt), C60-, and negative-control (mock) cells are shown. Bars, 100 nm.

FIG. 6.

Complementation between NC mutants and the Δp12 mutant. (A) Cells were transfected with the indicated viral clones. Pelletable material in culture fluids was analyzed by immunoblotting with an anti-p30^CA antiserum. An equal volume of culture fluid was loaded in each lane. Numbers in parentheses above lanes 1, 2, and 8 are micrograms of wild-type MLV DNA transfected. (B) Infectivity titers of culture fluids from cells transfected with the indicated viral clones.