Consideration of the three-dimensional structure of core shells (capsids) in spherical retroviruses.

The problem of three-dimensional organization of retroviral cores has been a matter of interest for the past 30 years. The general opinion in favor of icosahedral symmetry based on electron microscopy observations was questioned when cryo-electron microscopy failed to provide convincing evidence in its favor. More recent studies by cryo-electron microscopy, X-ray crystallography and in vitro assembly of the CA domain of Human immuno deficiency virus (HIV), Murine leukemia virus (MuLV) and Rous sarcoma virus (RSV) threw new light on the organization of retroviral cores. In this communication we report how we produced a three-dimensional (3D) model of MuLV core using data from CA assembly on a lipid film [Ganser, B.K., Cheng, A., Sundquist, W.I., Yeager, M., 2003. Three-dimensional structure of the M-MuLV CA protein on a lipid monolayer: a general model for retroviral capsid assembly. EMBO J. 22, 2886-2892]. The resulting structure revealed that the molecular organization of the core shell is specific and the presence of a 5,3,2 rotational symmetry of the 3D model provides support for icosahedral shape of MuLV cores. The model made it possible to determine the diameter of the cores and calculate the number of CA copies as well as the molecular mass of a core of specific diameter. Thus MuLV cores 68 (or 81.6) nm in diameter consist of 1500 (or 2160) copies of CA. About 12% of molecules from fullerene-like Gag shells versus 71% of molecules of closely packed (core-like). Gag shells were not incorporated into the core shells (capsids). Our 3D models received support from X-ray data of MuLV CA NTD domain published by Mortuza et al. [Mortuza, G., Haire, L.F., Stevens, A., Smerdon, S.J., Stoye, J.P., Taylor, I.A., 2004. High resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431, 481-485].

Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins.

In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (DeltaProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV DeltaProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which DeltaProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into DeltaProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.

Structural analyses of purified human immunodeficiency virus type 1 intracellular reverse transcription complexes.

Retroviruses copy their RNA genome into a DNA molecule, but little is known of the structure of the complex mediating reverse transcription in vivo. We used confocal and electron microscopy to study the structure of human immunodeficiency virus type 1 (HIV-1) intracellular reverse transcription complexes (RTCs). Cytoplasmic extracts were prepared 3, 4, and 16 h after acute infection by Dounce homogenization in hypotonic buffer. RTCs were purified by velocity sedimentation, followed by density fractionation in linear sucrose gradients and dialysis in a large pore cellulose membrane. RTCs had a sedimentation velocity of approximately 350 S and a density of 1.34 g/ml and were active in an endogenous reverse transcription assay. Double labeling of nucleic acids and viral proteins allowed specific visualization of RTCs by confocal microscopy. Electron microscopy revealed that RTCs are large nucleoprotein structures of variable shape consisting of packed filaments ca. 6 nm thick. Integrase and Vpr are associated with discrete regions of the 6-nm filaments. The nucleic acids within the RTC are coated by small proteins distinct from nucleocapsid and are partially protected from nuclease digestion.

Time course of Gag protein assembly in HIV-1-infected cells: a study by immunoelectron microscopy.

Recent biochemical studies have identified high molecular complexes of the HIV Gag precursor in the cytosol of infected cells. Using immunoelectron microscopy we studied the time course of the synthesis and assembly of a HIV Gag precursor protein (pr55gag) in Sf9 cells infected with recombinant baculovirus expressing the HIV gag gene. We also immunolabeled for pr55gag human T4 cells acutely or chronically infected with HIV-1. In Sf9 cells, the time course study showed that the first Gag protein appeared in the cytoplasm at 28-30 h p.i. and that budding started 6-8 h later. Colloidal gold particles, used to visualize the Gag protein, were first scattered randomly throughout the cytoplasm, but soon clusters representing 100 to 1000 copies of pr55gag were also observed. By contrast, in cells with budding or released virus-like particles the cytoplasm was virtually free of gold particles while the released virus-like particles were heavily labeled. Statistical analysis showed that between 80 and 90% of the gold particles in the cytoplasm were seen as singles, as doublets, or in small groups of up to five particles probably representing small oligomers. Clusters of gold particles were also observed in acutely infected lymphocytes as well as in multinuclear cells of chronically infected cultures of T4 cells. In a few cases small aggregates of gold particles were found in the nuclei of T4 lymphocytes. These observations suggest that the Gag polyprotein forms small oligomers in the cytoplasm of expressing cells but that assembly into multimeric complexes takes place predominantly at the plasma membrane. Large accumulations of Gag protein in the cytoplasm may represent misfolded molecules destined for degradation.

Molecular organization of Mason-Pfizer monkey virus capsids assembled from Gag polyprotein in Escherichia coli.

We describe the results of a study by electron microscopy and image processing of Gag protein shells-immature capsids--of Mason-Pfizer monkey virus assembled in Escherichia coli from two truncated forms of the Gag precursor: Deltap4Gag, in which the C-terminal p4Gag was deleted, and Pro(-)CA.NC, in which the N-terminal peptides and proline 1 of the CA domain were deleted. Negative staining of capsids revealed small patches of holes forming a trigonal or hexagonal pattern most clearly visible on occasional tubular forms. The center-to-center spacing of holes in the network was 7.1 nm in Deltap4Gag capsids and 7.4 nm in Pro(-)CA.NC capsids. Image processing of Deltap4Gag tubes revealed a hexagonal network of holes formed by six subunits with a single subunit shared between rings. This organization suggests that the six subunits are contributed by three trimers of the truncated Gag precursor. Similar molecular organization was observed in negatively stained Pro(-)CA.NC capsids. Shadowed replicas of freeze-etched capsids produced by either construct confirmed the presence of a hexagonal network of holes with a similar center-to-center spacing. We conclude that the basic building block of the cage-like network is a trimer of the Deltap4Gag or Pro(-)CA.NC domains. In addition, our results point to a key role of structurally constrained CA domain in the trimeric interaction of the Gag polyprotein.