Alex de Marco

Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.

Poring Over the Nuclear Pore The nuclear pore is a macromolecular complex that traverses the paired membranes of the nuclear envelope through which a variety of nuclear protein and RNA cargoes must traffic. Szymborska et al. (p. 655, published online 11 July) combined super-resolution microscopy with single-particle averaging to localize the proteins that make up the structural scaffold of the nuclear pore complex with a precision well below one nanometer. These molecular positional constraints clarified contradictory models for the structure of the nuclear pore and demonstrate that the structural organization of protein complexes can be studied by light microscopy in situ in whole cells. The localization of individual components of the nuclear pore complex was dissected using information from thousands of pores. Much of life’s essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.

Structural Analysis of HIV-1 Maturation Using Cryo-Electron Tomography

HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA.

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.

Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging.

Cryo-electron tomography combined with image processing by sub-tomogram averaging is unique in its power to resolve the structures of proteins and macromolecular complexes in situ. Limitations of the method, including the low signal to noise ratio within individual images from cryo-tomographic datasets and difficulties in determining the defocus at which the data was collected, mean that to date the very best structures obtained by sub-tomogram averaging are limited to a resolution of approximately 15 Å. Here, by optimizing data collection and defocus determination steps, we have determined the structure of assembled Mason-Pfizer monkey virus Gag protein using sub-tomogram averaging to a resolution of 8.5 Å. At this resolution alpha-helices can be directly and clearly visualized. These data demonstrate for the first time that high-resolution structural information can be obtained from cryo-electron tomograms using sub-tomogram averaging. Sub-tomogram averaging has the potential to allow detailed studies of unsolved and biologically relevant structures under biologically relevant conditions.

Fast transcription rates of RNA polymerase II in human cells

Averaged estimates of RNA polymerase II (RNAPII) elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb min−1. In this work, nascent RNAs from an integrated human immunodeficiency virus type 1‐derived vector were detectable at the single living cell level by fluorescent RNA tagging. At steady state, a constant number of RNAs was measured corresponding to a minimal density of polymerases with negligible fluctuations over time. Recovery of fluorescence after photobleaching was complete within seconds, indicating a high rate of RNA biogenesis. The calculated transcription rate above 50 kb min−1 points towards a wide dynamic range of RNAPII velocities in living cells.

Conserved and Variable Features of Gag Structure and Arrangement in Immature Retrovirus Particles

ABSTRACT The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.

Role of the SP2 domain and its proteolytic cleavage in HIV-1 structural maturation and infectivity

ABSTRACT HIV-1 buds as an immature, noninfectious virion. Proteolysis of its main structural component, Gag, is required for morphological maturation and infectivity and leads to release of four functional domains and the spacer peptides SP1 and SP2. The N-terminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, while the roles of the two C-terminal cleavages and the role of SP2, separating the NC and p6 domains, are less well defined. We have analyzed HIV-1 variants with defective cleavage at either or both sites flanking SP2, or largely lacking SP2, regarding virus production, infectivity, and structural maturation. Neither the presence nor the proteolytic processing of SP2 was required for particle release. Viral infectivity was almost abolished when both cleavage sites were defective and severely reduced when the fast cleavage site between SP2 and p6 was defective. This correlated with an increased proportion of irregular core structures observed by cryo-electron tomography, although processing of CA was unaffected. Mutation of the slow cleavage site between NC and SP2 or deletion of most of SP2 had only a minor effect on infectivity and did not induce major alterations in mature core morphology. We speculate that not only separation of NC and p6 but also the processing kinetics in this region are essential for successful maturation, while SP2 itself is dispensable.

Intragenic transcriptional cis-activation of the human immunodeficiency virus 1 does not result in allele-specific inhibition of the endogenous gene

BackgroundThe human immunodeficiency virus type 1 (HIV-1) favors integration in active genes of host chromatin. It is believed that transcriptional interference of the viral promoter over the endogenous gene or vice versa might occur with implications in HIV-1 post-integrative transcriptional latency.ResultsIn this work a cell line has been transduced with a HIV-based vector and selected for Tat-inducible expression. These cells were found to carry a single silent integration in sense orientation within the second intron of the HMBOX1 gene. The HIV-1 Tat transactivator induced the viral LTR and repressed HMBOX1 expression independently of vector integration. Instead, single-cell quantitative in situ hybridization revealed that allele-specific transcription of HMBOX1 carrying the integrated provirus was not affected by the transactivation of the viral LTR in cis.ConclusionA major observation of the work is that the HIV-1 genome has inserted in genes that are also repressed by Tat and this could be an advantage for the virus during transcriptional reactivation. In addition, it has also been observed that transcription of the provirus and of the endogenous gene in which it is integrated may coexist at the same time in the same genomic location.

In Vitro Assembly of Virus-Like Particles of a Gammaretrovirus, the Murine Leukemia Virus XMRV

ABSTRACT Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus of CA to fold back to form a β-hairpin, anchored by an internal salt bridge between the N-terminal proline and the inner aspartate. Using an in vitro assembly system of capsid-nucleocapsid protein (CANC), we studied the formation of virus-like particles (VLP) of a gammaretrovirus, the xenotropic murine leukemia virus (MLV)-related virus (XMRV). We show here that, unlike other retroviruses, XMRV CA and CANC do not assemble tubular particles characteristic of mature assembly. The prevention of β-hairpin formation by the deletion of either the N-terminal proline or 10 initial amino acids enabled the assembly of ΔProCANC or Δ10CANC into immature-like spherical particles. Detailed three-dimensional (3D) structural analysis of these particles revealed that below a disordered N-terminal CA layer, the C terminus of CA assembles a typical immature lattice, which is linked by rod-like densities with the RNP.

Subcellular localization of the interaction between the human immunodeficiency virus transactivator Tat and the nucleosome assembly protein 1

The histone chaperone nucleosome assembly protein, hNAP-1, is a host cofactor for the activity of the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. The interaction between these two proteins has been shown to be important for Tat-mediated transcriptional activation and for efficient viral infection. Visualization of HIV-1 transcription and fluorescence resonance energy transfer experiments performed in this work demonstrate that hNAP-1 is not recruited to the site of Tat activity but the two proteins interact at the nuclear rim. These data are consistent with a mechanism that requires hNAP-1 for the transport of Tat within the nucleus rather than for the remodeling of nucleosomes on the provirus. Protein–protein docking and molecular modeling of the complex suggest that this interaction occurs between the basic domain of Tat and the histone-binding domain. The combination of theoretical and whole cell studies provided new insights into the functional significance of the Tat:hNAP-1 recognition.