Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude
. Heavily glycosylated S trimers bind to the ...angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells
. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes
. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy
, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization and mechanism of uncoating are under debate. We adapted a strategy to visualize individual ...reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.
...processed CA and the mature CA lattice are not needed for virus assembly or release, pointing toward a function of the mature lattice structure during or after virus entry. ...early schemes of ...HIV-1 replication assumed rapid and complete disintegration (uncoating) of the CA lattice after membrane fusion with subsequent cytoplasmic conversion of the genomic RNA into double-stranded cDNA in the released reverse transcription complex (RTC) (reviewed in 4). Rapid uncoating after membrane fusion appeared to be supported by the rapid and complete dissociation of the capsid when the membrane of cell-free virions was stripped by detergent and by the failure to detect cone-shaped objects in the cytosol of infected cells by electron microscopy (EM) (reviewed in 5). ...early replication complexes purified from extracts of infected cells contained little or no CA 5. Adding deoxynucleoside triphosphates (dNTPs) to this stabilized structure in vitro even allowed complete reverse transcription of the endogenous viral genome and revealed breakage, but not complete disassembly of the lattice, when synthesis of the double-stranded cDNA approached completion 8. Since IP6 is present in high concentration in the cytosol, these in vitro data clearly indicate that the capsid structure could remain intact for prolonged periods of time after membrane fusion and could support the full process of reverse transcription once dNTPs become available.
HIV-1 infects host cells by fusion at the plasma membrane, leading to cytoplasmic entry of the viral capsid encasing the genome and replication machinery. The capsid eventually needs to disassemble, ...but time and location of uncoating are not fully characterized and may vary depending on the host cell. To study the fate of the capsid by fluorescence and superresolution (STED) microscopy, we established an experimental system that allows discrimination of subviral structures in the cytosol from intact virions at the plasma membrane or in endosomes without genetic modification of the virus. Quantitative microscopy of infected SupT1-R5 cells revealed that the CA signal on cytosolic HIV-1 complexes corresponded to ∼50% of that found in virions at the cell surface, in agreement with dissociation of nonassembled CA molecules from entering capsids after membrane fusion. The relative amount of CA in postfusion complexes remained stable until they reached the nuclear pore complex, while subviral structures in the nucleus of infected cells lacked detectable CA. An HIV-1 variant defective in binding of the host protein cleavage and polyadenylation specificity factor 6 (CPSF6) exhibited accumulation of CA-positive subviral complexes close to the nuclear envelope without loss of infectivity; STED microscopy revealed direct association of these complexes with nuclear pores. These results support previous observations indicating capsid uncoating at the nuclear pore in infected T-cell lines. They suggest that largely intact HIV-1 capsids dock at the nuclear pore in infected SupT1-R5 cells, with CPSF6 being a facilitator of nucleoplasmic entry in this cell type, as has been observed for infected macrophages.
The HIV-1 capsid performs essential functions during early viral replication and is an interesting target for novel antivirals. Thus, understanding molecular and structural details of capsid function will be important for elucidating early HIV-1 (and retroviral in general) replication in relevant target cells and may also aid antiviral development. Here, we show that HIV-1 capsids stay largely intact during transport to the nucleus of infected T cells but appear to uncoat upon entry into the nucleoplasm. These results support the hypothesis that capsids protect the HIV-1 genome from cytoplasmic defense mechanisms and target the genome toward the nucleus. A protective role of the capsid could be a paradigm that also applies to other viruses. Our findings raise the question of how reverse transcription of the HIV-1 genome is accomplished in the context of the capsid structure and whether the process is completed before the capsid is uncoated at the nuclear pore.
Abstract
Chicxulub impact (66 Ma) event resulted in deposition of spheroids and melt glass, followed by deposition of diamectite and carbonate ejecta represented by large polished striated rounded ...pebbles and cobbles, henceforth, called Albion Formation
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Pook’s Pebbles, name given from the first site identified in central Belize, Cayo District. Here we report that magnetic analysis of the Pook’s Pebbles samples revealed unique electric discharge signatures. Sectioning of Pook’s Pebbles from the Chicxulub ejecta from the Albion Formation at Belize showed that different parts of Pook’s Pebbles had not only contrasting magnetization directions, but also sharply different level of magnetizations. Such behavior is indicative of electric discharge taking place sometimes during the formation of the Chicxulub ejecta blanket. In addition, some of the Pook’s Pebbles’ surface had recrystallized down to 0.2 mm depth. This is evidence of localized extreme pressures and temperatures during the fluidized ejecta formation which was imprinted in the outer layer of Pook’s Pebbles. Recrystallization caused formation of nanophase iron along the surface, which was revealed by mapping of both natural remanent magnetization and of saturation remanence magnetization signatures. While the spheroids’ magnetization orientation is consistent with reversed magnetic field at the time of impact, the study of the Pook’s Pebbles provided, in addition, new evidence of electric charging during the vapor plume cloud processes.
The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules toward the nucleus. ...While it was initially believed that the reverse transcribed genome is released from the capsid in the cytosol, recent observations indicate that a high amount of capsid protein (CA) remains associated with subviral complexes during import through the nuclear pore complex (NPC). Observation of postentry events via microscopic detection of HIV-1 CA is challenging, since epitope shielding limits immunodetection and the genetic fragility of CA hampers direct labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes revealed an amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells consistent with a complete capsid and showed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes. IMPORTANCE The cone-shaped capsid of HIV-1 has recently been recognized as a master organizer of events from cell entry of the virus to the integration of the viral genome into the host cell DNA. Fluorescent labeling of the capsid is essential to study its role in these dynamic events by microscopy, but viral capsid proteins are extremely challenging targets for the introduction of labels. Here we describe a minimally invasive strategy that allows us to visualize the HIV-1 capsid protein in infected cells by live-cell imaging and superresolution microscopy. Applying this strategy, we confirmed that, contrary to earlier assumptions, an equivalent of a complete capsid can enter the host cell nucleus through nuclear pores. We also observed that entering capsids cluster in the nucleus in a dose-dependent manner, suggesting that they may have followed a common entry route to a site suitable for viral genome release.
Infection of non-enveloped polyomaviruses depends on an intact microtubular network. Here we focus on mouse polyomavirus (MPyV). We show that the dynamics of MPyV cytoplasmic transport reflects the ...characteristics of microtubular motor-driven transport with bi-directional saltatory movements. In cells treated with microtubule-disrupting agents, localization of MPyV was significantly perturbed, the virus was retained at the cell periphery, mostly within membrane structures resembling multicaveolar complexes, and at later times post-infection, only a fraction of the virus was found in Rab7-positive endosomes and multivesicular bodies. Inhibition of cytoplasmic dynein-based motility by overexpression of dynamitin affected perinuclear translocation of the virus, delivery of virions to the ER and substantially reduced the numbers of infected cells, while overexpression of dominant-negative form of kinesin-1 or kinesin-2 had no significant impact on virus localization and infectivity. We also found that transport along microtubules was important for MPyV-containing endosome sequential acquisition of Rab5, Rab7 and Rab11 GTPases. However, in contrast to dominant-negative mutant of Rab7 (T22N), overexpression of dominant-negative mutant Rab11 (S25N) did not affect the virus infectivity. Altogether, our study revealed that MPyV cytoplasmic trafficking leading to productive infection bypasses recycling endosomes, does not require the function of kinesin-1 and kinesin-2, but depends on functional dynein-mediated transport along microtubules for translocation of the virions from peripheral, often caveolin-positive compartments to late endosomes and ER - a prerequisite for efficient delivery of the viral genome to the nucleus.
Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid ...is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.
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•Nuclear translocation of HIV-1 capsids is captured by 3D CLEM/cryo-ET•Nuclear pore complexes in T cells are sufficiently dilated to accommodate HIV-1 capsids•Cone-shaped HIV-1 capsids translocate through nuclear pore complexes•Inside the nucleus HIV-1 capsids rupture and release their interior
Visualization of nuclear translocation of HIV-1 capsids by 3D correlative fluorescence light and electron microscope, combined with cryoelectron tomography, demonstrates that nuclear pore complexes in infected T cells are sufficiently dilated to allow cone-shaped HIV-1 capsids to pass through.
Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing ...structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.
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