Kay Grünewald

Simian Virus 40 Depends on ER Protein Folding and Quality Control Factors for Entry into Host Cells

Cell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated viruses and found that SV40 makes use of the thiol-disulfide oxidoreductases, ERp57 and PDI, as well as the retrotranslocation proteins Derlin-1 and Sel1L. ERp57 isomerizes specific interchain disulfides connecting the major capsid protein, VP1, to a crosslinked network of neighbors, thus uncoupling about 12 of 72 VP1 pentamers. Cryo-electron tomography indicated that loss of interchain disulfides coupled with calcium depletion induces selective dissociation of the 12 vertex pentamers, a step likely to mimic uncoating of the virus in the cytosol. Thus, the virus utilizes the protein folding machinery for initial uncoating before exploiting the ER-associated degradation machinery presumably to escape from the ER lumen into the cytosol.

Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ

The envelope glycoprotein (Env) complexes of the human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate viral entry and are a target for neutralizing antibodies. The receptor binding surfaces of Env are in large part sterically occluded or conformationally masked prior to receptor binding. Knowledge of the unliganded, trimeric Env structure is key for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. We have used cryo-electron tomography and averaging to obtain the structure of the SIV Env complex prior to fusion. Our result reveals novel details of Env organisation, including tight interaction between monomers in the gp41 trimer, associated with a three-lobed, membrane-distal gp120 trimer. A cavity exists at the gp41–gp120 trimer interface. Our model for the spike structure agrees with previously predicted interactions between gp41 monomers, and furthers our understanding of gp120 interactions within an intact spike.

Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis

Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release--akin to its role in vesicle formation--and is not restricted to severing the thin membrane tether.

Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry

The concerted action of four viral glycoproteins and at least one cellular receptor is required to catalyze herpes simplex virus 1 entry into host cells either by fusion at the plasma membrane or intracellularly after internalization by endocytosis. Here, we applied cryo electron tomography to capture 3D intermediates from Herpes simplex virus 1 fusion at the plasma membrane in their native environment by using two model systems: adherent cells and synaptosomes. The fusion process was delineated as a series of structurally different steps. The incoming capsid separated from the tegument and was closely surrounded by the cortical cytoskeleton. After entry, the viral membrane curvature changed concomitantly with a reorganization of the envelope glycoprotein spikes. Individual glycoprotein complexes in transitional conformations during pore formation and dilation revealed the complex viral fusion mechanism in action. Snapshots of the fusion intermediates provide unprecedented details concerning the overall structural changes occurring during herpesvirus entry. Moreover, our data suggest that there are two functional “poles” of the asymmetric herpesvirion: one related to cell entry, and the other formed during virus assembly.

Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: implications of crowding

Electron cryotomography has unique potential for three-dimensional visualization of macromolecular complexes at work in their natural environment. This approach is based on reconstructing three-dimensional volumes from tilt series of electron micrographs of cells preserved in their native states by vitrification. Resolutions of 5-8 nm have already been achieved and the prospects for further improvement are good. Since many intracellular activities are conducted by complexes in the megadalton range with dimensions of 20-50 nm, current resolutions should suffice to identify many of them in tomograms. However, residual noise and the dense packing of cellular constituents hamper interpretation. Recently, tomographic data have been collected on vitrified eukaryotic cells (Medalia et al., Science (2002) in press). Their cytoplasm was found to be markedly less crowded than in the prokaryotes previously studied, in accord with differences in crowding between prokaryotic and eukaryotic cells documented by other (indirect) biophysical methods. The implications of this observation are twofold. First, complexes should be more easily identifiable in tomograms of eukaryotic cytoplasm. This applies both to recognizing known complexes and characterizing novel complexes. An example of the latter-a 5-fold symmetric particle is-given. Second, electron cryotomography offers an incisive probe to examine crowding in different cellular compartments.

The Three-Dimensional Organization of Polyribosomes in Intact Human Cells

Structural studies have provided detailed insights into different functional states of the ribosome and its interaction with factors involved in nascent peptide folding, processing, and targeting. However, how the translational machinery is organized spatially in native cellular environments is not yet well understood. Here we have mapped individual ribosomes in electron tomograms of intact human cells by template matching and determined the average structure of the ribosome in situ. Characteristic features of active ribosomes in the cellular environment were assigned to the tRNA channel, elongation factors, and additional densities near the peptide tunnel. Importantly, the relative spatial configuration of neighboring ribosomes in the cell is clearly nonrandom. The preferred configurations are specific for active polysomes and were largely abrogated in puromycin-treated control cells. The distinct neighbor orientations found in situ resemble configurations of bacterial polysomes in vitro, indicating a conserved supramolecular organization with implications for nascent polypeptide folding.

Electron Cryotomography of Tula Hantavirus Suggests a Unique Assembly Paradigm for Enveloped Viruses

ABSTRACT Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses that cause a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, GN and GC, are embedded in the viral membrane and form protrusions, or “spikes.” The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein, and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryomicroscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane, and ribonucleoprotein are organized. The structure of the GN-GC spike complex was solved to 3.6-nm resolution by averaging tomographic subvolumes. Each spike complex is a square-shaped assembly with 4-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses.

Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for GN-GC Glycoprotein Heterodimers

ABSTRACT Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins GN and GC, organized on a T=12 icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either GN and GC homodimers or GN-GC heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five- and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a GN-GC heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens.

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

Bunyaviridae is a large family of viruses that have gained attention as “emerging viruses” because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or “spikes,” were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry.

Cryo Electron Tomography of Native HIV-1 Budding Sites

The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.