Volker M. Vogt

The stoichiometry of Gag protein in HIV-1

The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000–1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the immature virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete immature HIV particle contains ∼5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the immature virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.

Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle

BACKGROUND Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and the subject of intense study. The immature HIV-1 particle is traditionally described as having a well ordered, icosahedral structure made up of uncleaved Gag protein surrounded by a lipid bilayer containing envelope proteins. Expression of the Gag protein in eukaryotic cells leads to the budding of membranous virus-like particles (VLPs). RESULTS We have used cryo-electron microscopy of VLPs from insect cells and lightly fixed, immature HIV-1 particles from human lymphocytes to determine their organization. Both types of particle were heterogeneous in size, varying in diameter from 1200-2600 A. Larger particles appeared to be broken into semi-spherical sectors, each having a radius of curvature of approximately 750 A. No evidence of icosahedral symmetry was found, but local order was evidenced by small arrays of Gag protein that formed facets within the curved sectors. A consistent 270 A radial density was seen, which included a 70 A wide low density feature corresponding to the carboxy-terminal portion of the membrane attached matrix protein and the amino-terminal portion of the capsid protein. CONCLUSIONS Immature HIV-1 particles and VLPs both have a multi-sector structure characterized, not by an icosahedral organization, but by local order in which the structures of the matrix and capsid regions of Gag change upon cleavage. We propose a model in which lateral interactions between Gag protein molecules yields arrays that are organized into sectors for budding by RNA.

Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide.

Abstract The four major internal structural proteins (the group-specific antigens) of avian myeloblastosis virus are formed by sequential cleavage of a precursor polypeptide with M r = 76,000 (Pr76). The evidence for this conclusion is based on analysis of immune precipitates from lysates of AMV § -infected cells treated with a multivalent antiserum directed against these proteins. Sodium dodecyl sulfate gel electrophoresis of such immune precipitates from cells pulse-labeled with [ 35 S]-methionine reveals five metabolically unstable radioactive polypeptides. These polypeptides behave kinetically as precursors to virion proteins. Double-label ion-exchange chromatography of tryptic digests of the unstable polypeptides demonstrates that the largest precursor, Pr76, contains the amino acid sequences of all four virion proteins. It appears not to contain the sequence of the fifth and smallest internal virion protein. The four smaller precursors are intermediate cleavage products of Pr76. The arrangement of the virion proteins in Pr76 was determined by labeling cells shortly after inhibiting polypeptide chain initiation. The relative amounts of radioactivity both in completed virion proteins and in the tryptic peptides of Pr76 implies the same order for three of the four proteins. The exact position of one protein remains uncertain. On the basis of these experiments, we propose a cleavage pathway for the generation of the structural proteins of AMV. We also demonstrate that cleavage of precursors can proceed in crude extracts of AMV-infected cells. This proteolysis, while resistant to several protease inhibitors, is completely blocked by addition of agents that disrupt membranes.

Structure of ribosomal DNA in Physarum polycephalum.

Abstract The sequences coding for Physarum ribosomal RNA are localized on idependently replicating, linear DNA molecules of a discrete size, M r = 37 × 10 6 . Restriction endonucleases Eco RI and Hin dIII each cut ribosomal DNA into one large and two small fragments. The latter are represented twice per intact molecule, once at each end. Sedimentation and electron microscopic analyses of intact rDNA † that has been neutralized from alkaline solution indicate that the entire rDNA molecule has a rotational axis of symmetry near the center. Blocks of short, inverted repetitious sequences appear to be located at the center of the native rDNA and also 3.7 × 10 6 to 11 × 10 6 daltons flanking the center.

Structure and self-association of the Rous sarcoma virus capsid protein.

BACKGROUND The capsid protein (CA) of retroviruses, such as Rous sarcoma virus (RSV), consists of two independently folded domains. CA functions as part of a polyprotein during particle assembly and budding and, in addition, forms a shell encapsidating the genomic RNA in the mature, infectious virus. RESULTS The structures of the N- and C-terminal domains of RSV CA have been determined by X-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy, respectively. The N-terminal domain comprises seven alpha helices and a short beta hairpin at the N terminus. The N-terminal domain associates through a small, tightly packed, twofold symmetric interface within the crystal, different from those previously described for other retroviral CAs. The C-terminal domain is a compact bundle of four alpha helices, although the last few residues are disordered. In dilute solution, RSV CA is predominantly monomeric. We show, however, using electron microscopy, that intact RSV CA can assemble in vitro to form both tubular structures constructed from toroidal oligomers and planar monolayers. Both modes of assembly occur under similar solution conditions, and both sheets and tubes exhibit long-range order. CONCLUSIONS The tertiary structure of CA is conserved across the major retroviral genera, yet sequence variations are sufficient to cause change in associative behavior. CA forms the exterior shell of the viral core in all mature retroviruses. However, the core morphology differs between viruses. Consistent with this observation, we find that the capsid proteins of RSV and human immunodeficiency virus type 1 exhibit different associative behavior in dilute solution and assemble in vitro into different structures.

Visualization of retrovirus budding with correlated light and electron microscopy.

We have used correlated scanning EM (SEM) and multiphoton fluorescence microscopy to visualize budding of virus-like particles (VLPs) of Rous sarcoma virus (RSV) and HIV type 1 (HIV-1). When the Gag structural protein was expressed alone as a GFP fusion, most budding particles appeared morphologically aberrant, but normal assembly could be rescued by coexpression of untagged Gag protein. Imaging of live cells allowed budding to be seen in real time as the disappearance of fluorescent spots from the dorsal cell surface. The disappearance of very bright spots containing clusters of VLPs often occurred in a stepwise fashion. Even after imaging times >1 h, only a minority of the spots disappeared, suggesting that some might be budding-incompetent complexes. On individual cells, we enumerated both the fluorescent puncta and the budding structures visible by SEM and compared these numbers for WT Gag proteins and for Gag proteins that were blocked at the last step in budding by a late domain mutation. For the mutant HIV-1 and RSV proteins, almost all of the fluorescent spots corresponded to budding structures. For WT RSV, the dorsal side of cells showed 3-fold more fluorescent spots than budding structures, suggesting that formation of the polymerized Gag shell precedes bulging out of the membrane. For WT HIV-1, most fluorescent spots corresponded with budding structures, consistent with the slower budding rate of this virus. Combining these two types of microscopy will allow innovative approaches for elucidating the mechanism of retrovirus budding.

A mobile group I intron in the nuclear rDNA of physarum polycephalum

We have shown that a strain-specific group I intron (intron 3) in the nuclear extrachromosomal rDNA or Physarum polycephalum is a mobile element. Shortly after mating of amoebae from intron-lacking and intron-containing strains, intron 3 transposes in a site-specific manner into all available recipient molecules. The transposition appears to occur by gene conversion, as evidence by the co-conversion of adjacent sequences and by double strand breakage observed in some of the recipient rDNA molecules. We infer that the double strand break is induced by an endonuclease encoded by intron 3, since in vitro transcription and translation of the cloned intron leads to the synthesis of an enzymatically active, site-specific nuclease. This is the first demonstration of the transposition of a nuclear intron in an experimental setting, and provides a rare example of a protein encoded by an RNA polymerase I transcript.

Nucleic Acid-Independent Retrovirus Assembly Can Be Driven by Dimerization

ABSTRACT The Gag protein of retroviruses alone can polymerize into regular virus-like particles (VLPs) both in vitro and in vivo. In most circumstances the capsid (CA) and nucleocapsid (NC) domains of Gag as well as some form of nucleic acid are required for this process. The mechanism by which NC-nucleic acid interaction promotes assembly has remained obscure. We show here that while deletion of the NC domain of Rous sarcoma virus Gag abolishes formation and budding of VLPs at the plasma membranes of baculovirus-infected insect cells, replacement of NC with a dimer-forming leucine zipper domain restores budding of spherical particles morphologically similar to wild-type VLPs. The positioning of the dimerization domain appears to be critical for proper assembly, as the insertion of a 5-amino-acid flexible linker upstream of the zipper domain leads to budding of tubular rather than spherical particles. Similar tubular particles are formed when the same linker is inserted upstream of NC. The tubes are morphologically distinct from tubes formed when the p10 domain upstream of CA is deleted. The fact that a foreign dimerization domain can functionally mimic NC suggests that the role of nucleic acid in retroviral assembly is not to serve as a scaffold but rather to promote the formation of Gag dimers, which are critical intermediates in the polymerization of the Gag shell.

Characterization of Rous sarcoma virus Gag particles assembled in vitro.

ABSTRACT Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 × 107 Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.

Mobile introns: definition of terms and recommended nomenclature

A number of introns in mitochondrial, chloroplast, nuclear or prokaryotic genes have recently been shown to encode double-strand sequence-specific endonucleases. Such introns are mobile genetic elements that insert themselves at or near the cleaved sites. A uniform nomenclature to designate the molecular elements involved in the phenomenon of intron mobility is proposed.