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Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro

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HIV-1 replication and integration in vitro

To infect a host cell, HIV-1 must reverse transcribe its single-stranded RNA genome into a double-stranded DNA copy and integrate that copy into a host chromosome. Reverse transcription and integration have been characterized separately but have not been reconstituted together outside of the cell. Christensen et al. now report that viral core particles can complete full reverse transcription and integration in a cell-free system. The external capsid shell of the core is required for efficient reverse transcription, and the replicating DNA can loop out of capsid openings. Integration requires cell extract, and this cell-free system should be useful for analyzing how host factors contribute to the first half of the HIV life cycle.

Science, this issue p. eabc8420

Structured Abstract

INTRODUCTION

Reverse transcription and integration are the signature events of retrovirus replication. Reverse transcription creates a double-stranded DNA (dsDNA) copy of the positive-sense viral RNA genome, and integration archives that copy within the genome of the infected cell. Both processes are targets of successful HIV-1 antiretroviral therapies, and the associated enzymatic activities have been characterized by elegant structural, biochemical, and molecular virological analyses. Nevertheless, mechanistic studies of these processes remain challenging because they are performed by viral core particles deep within the infected cell cytoplasm and nucleus. Of particular interest is defining whether the conical capsid that surrounds the viral RNA genome participates in the process of viral replication.

RATIONALE

In principle, informative mechanistic and imaging analyses of HIV-1 replication could be performed in vitro, but the coupled processes of reverse transcription and concerted integration have not yet been recapitulated outside of the cell. To address this limitation, we reconstituted these processes in a cell-free system, using purified HIV-1 virions as the source of viral genomes and enzymes.

RESULTS

We recapitulated the sequential processes of endogenous reverse transcription (ERT) and integration from viral core particles, which were released from purified HIV-1 virions by gently permeabilizing the viral membrane using a pore-forming peptide. ERT was initiated through addition of deoxynucleotide triphosphates (dNTPs), and the DNA products—early, intermediate, and late transcripts—appeared in high yields and in the expected temporal order, with late dsDNA products accumulating maximally after 8 to 10 hours. Integration of the resulting viral DNA into an exogenous target DNA was also recapitulated, provided that cell extract was added to the reaction mix. Deep sequencing and cloning confirmed that the 3′ and 5′ termini of the viral DNA were integrated in a concerted fashion, with the expected 5–base pair target site duplications in the target DNA, and with target site sequence preferences that resembled those reported for HIV-infected cells.

HIV-1 capsids are composed of hexagonal lattices of the viral CA protein, arrayed in a “fullerene cone” structure. Analyses of the requirements for efficient ERT and integration in our cell-free system revealed that these processes require the presence of capsid lattices of appropriate stability and geometry, as determined by using site-directed CA mutations, capsid-stabilizing factors, and potent new capsid inhibitors. Imaging with electron cryotomography revealed that many viral capsids remained largely intact during the ERT reaction. Capsid uncoating, when observed, did not occur in an all-or-none fashion, as might have been anticipated for a highly cooperative structure. Rather, uncoating proceeded through a continuum of disassembly intermediates in which portions of the capsid wall appeared lost in patches, as revealed through subunit-level lattice mapping. Largely intact capsids in which viral nucleic acid strands extruded through lattice openings were observed after 8 to 10 hours, which was coincident with the maximal accumulation of late ERT products and integration events.

CONCLUSIONS

We have reconstituted efficient reverse transcription and integration—the major early steps of the HIV-1 life cycle—in a cell-free system. Our data indicate that the viral capsid plays an active and indispensable role in supporting efficient reverse transcription. Thus, we consider the entire core particle, including the outer capsid shell, to be the true viral “replication complex.” We further found that complete capsid uncoating may be a prerequisite for integration to occur. Thus, the capsid plays essential roles in the reactions that duplicate and archive the viral genome, in addition to previously established roles in protecting the viral genome from innate immune sensor surveillance and in helping the core to traverse the cytoplasm, enter the nucleus, and traffic to integration sites. We anticipate that our cell-free system will enable systematic analyses of the key steps in viral replication and integration and thereby elucidate the transformations that occur as HIV-1 proceeds through the first half of the viral life cycle.

Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro.

Images summarize stepwise reconstitution of highly efficient ERT and concerted integration in a cell-free system. Cryo–electron tomographic imaging showed that many core particles retained nearly complete outer viral capsids during the ERT reaction and that some lost surface patches, through which loops of the growing viral double-stranded cDNA could extrude. IP6, inositol hexakisphosphate; rNTPs, ribonucleoside triphosphates.

Abstract

During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells. We therefore reconstituted and imaged the early stages of HIV-1 replication in a cell-free system. HIV-1 cores released from permeabilized virions supported efficient, capsid-dependent endogenous reverse transcription to produce double-stranded DNA genomes, which sometimes looped out from ruptured capsid walls. Concerted integration of both viral DNA ends into a target plasmid then proceeded in a cell extract–dependent reaction. This reconstituted system uncovers the role of the capsid in templating replication.

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Science

Deep abiotic weathering of pyrite

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Getting rid of fool’s gold

Pyrite, also called fool’s gold, is an iron sulfide mineral that is very commonly found in rock but is almost nonexistent in sediments today. Pyrite oxidizes quickly and is a major source of sulfur to the ocean, but it is also a proxy for the oxygen content historically in Earth’s atmosphere. Gu et al. conducted a set of detailed observations of the pyrite oxidation process in a shale unit. The authors found that erosion tied to fracturing is just as important as the oxygen content for the dissolution process. They developed a model that helps determine the conditions in Earth’s past for which pyrite might have been stable and the role of microorganisms in the oxidation process.

Science, this issue p. eabb8092

Structured Abstract

INTRODUCTION

Oxidative weathering of pyrite, the most abundant sulfide mineral in Earth’s crust, is coupled to the biogeochemical cycles of sulfur, oxygen, carbon, and iron. Pyrite oxidation is key to these cycles because of its high reactivity with oxygen. Before the Great Oxidation Event (GOE), atmospheric oxygen concentrations were low on early Earth and pyrite was exposed at Earth’s surface, allowing erosion into sediments that were preserved in river deposits. Today, it oxidizes at depth in most rocks and is often not exposed at the land surface. To understand pyrite weathering through geologic time, researchers extrapolate the reaction kinetics based on studies from the laboratory or in acid mine drainage. Such work has emphasized the important role of microorganisms in catalyzing pyrite oxidation. But to interpret the oxidation rates of pyrite on early Earth requires knowledge of the rate-limiting step of the oxidation as it occurs naturally in rocks.

RATIONALE

We investigated the oxidation of pyrite in micrometer-sized grains, in centimeter-sized rock fragments, and in meter-scale boreholes at a small, well-studied catchment in a critical-zone observatory. Our goal was to determine the reaction mechanism of pyrite weathering in rocks as it occurs today. The slow-eroding catchment is underlain by shale, the most common rock type exposed on Earth. We determined weathering profiles of pyrite through chemical and microscopic analysis.

RESULTS

At the ridgelines of the shale watershed, most pyrite oxidation occurs within a 1-m-thick reaction zone ∼16 m below land surface, just above the depth of water table fluctuation. This is the reaction front at the borehole scale. Only limited oxidation occurs in halos around a few fractures at deeper depths. Above the depth where pyrite is 100% oxidized in all boreholes, rock fracture density and porosity are generally higher than below. However, the narrow parts of pore openings called pore throats remain small enough in oxidizing shale to limit access of microorganisms to the pyrite surface. During oxidation, iron oxides pseudomorphically replace the pyrite grains. High-resolution transmission electron microscopy (TEM) reveals that the oxidation front at grain scale is defined by a sharp interface between pyrite and an iron (oxyhydr)oxide (Fh) that is either ferrihydrite or feroxyhyte. This Fh then transforms into a banded structure of iron oxides that ultimately alter to goethite in outer layers. This complex oxidative transformation progresses inward from fractures when observed at clast scale.

CONCLUSION

Under today’s atmosphere, pyrite oxidation, rate-limited by diffusion of oxygen at the grain scale, is regulated by fracturing at clast scale. As pyrite is oxidized at borehole scale before reaching the land surface in most landscapes today, the oxidation rate is controlled by the movement of pyrite upward, which is in turn limited by the rate of erosion. Comparisons of shale landscapes with different erosion rates reveal that fracture spacing varies with erosion rate, so this suggests that fracture spacing may couple the landscape-scale to grain-scale rates. Microbial acceleration of oxidation globally today is unlikely in low-porosity rocks because pyrite oxidation usually occurs at depth, where pore throats limit access, as observed here for shales. Before the GOE, the rate of pyrite oxidation was instead controlled by the slower reaction kinetics in the presence of lower atmospheric oxygen concentrations. At that time, therefore, pyrite was exposed at the land surface, where microbial interaction could have accelerated the oxidation and acidified the landscape, as suggested by others. Our work highlights the importance of fracturing and erosion in addition to atmospheric oxygen as a control on the reactivity of this ubiquitous iron sulfide.

Schematic depiction of oxidative weathering of pyrite in rocks buried at meters depth.

Pyrite oxidation was studied from the molecular (TEM) scale of the pyrite―Fe oxide interface through clast and borehole scales to extrapolate to landscapes. The rate of oxidation of pyrite, limited at grain scale by oxygen diffusion through the shale matrix, is regulated at larger scales by fracturing and erosion.

Abstract

Pyrite is a ubiquitous iron sulfide mineral that is oxidized by trace oxygen. The mineral has been largely absent from global sediments since the rise in oxygen concentration in Earth’s early atmosphere. We analyzed weathering in shale, the most common rock exposed at Earth’s surface, with chemical and microscopic analysis. By looking across scales from 10−9 to 102 meters, we determined the factors that control pyrite oxidation. Under the atmosphere today, pyrite oxidation is rate-limited by diffusion of oxygen to the grain surface and regulated by large-scale erosion and clast-scale fracturing. We determined that neither iron- nor sulfur-oxidizing microorganisms control global pyrite weathering fluxes despite their ability to catalyze the reaction. This multiscale picture emphasizes that fracturing and erosion are as important as atmospheric oxygen in limiting pyrite reactivity over Earth’s history.

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Moving heart elements and cells

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Transposable elements comprise a large percentage of the human genome, with the endogenous retrovirus (ERV) subclass representing more than 8%. Using human pluripotent stem cell–derived cardiomyocytes and bioengineered micropatterning to recapitulate cardiogenesis, Wilson et al. found evidence that the primate-specific ERV MER41 is involved in primate heart development. A MER41-derived long noncoding RNA called BANCR is exclusively expressed in the fetal heart. When BANCR is eliminated, cardiomyocyte migration is disrupted. The cardiogenic transcription factor TBX5 and Hippo signaling factors TEAD4/YAP1 bind to a BANCR enhancer during fetal development. A related analysis in mouse shows that heart size increases with embryo BANCR knock-in.

Dev. Cell 54, 694 (2020).

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Obesity and inflammation

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Accumulation of fat cells (shown in yellow in this micrograph) may be promoted by gene variants linked to inflammation.

IMAGE: DAVID M. PHILLIPS/SCIENCE SOURCE

Obesity is associated with chronic inflammation, which can trigger other diseases such as atherosclerosis, type 2 diabetes, and even cancer. There appears to be a genetic component to excess fat accumulation, and studies suggest that inflammatory gene variants may contribute. Karunakaran et al. found that single-nucleotide polymorphisms in the human receptor-interacting serine/threonine-protein kinase 1 gene (RIPK1) increase its expression and are causally associated with obesity. RIPK1 is a key regulator of inflammatory responses and cell death. Silencing of Ripk1 in mice on a high-fat diet reduced fat mass, body weight, and inflammatory responses in adipose tissue. This suggests that RIPK1-mediated inflammation (and possibly other functions) contribute to obesity and that RIPK1 could be a therapeutic target.

Nat. Metab. 10.1038/s42255-020-00279-2 (2020).

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