Essential functions of Inositol hexakisphosphate (IP6) in Murine Leukemia Virus replication.

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.

Electro-flocculation of aquaculture wastewater microalgal communities reduces nutrient loading.

Land-based aquaculture provides dietary protein to the world's population in a sustainable way, but issues related to release of nitrogen rich wastewater limits its expansion. Sedimentation of naturally occurring microalgae that assimilate excess nitrogen, is slow and land intensive. Electro-flocculation, used in wastewater treatment processes, is a potential alternative for aquaculture. Trials of different electro-flocculation configurations applied to three prawn farm pondwater samples containing varying microalgal assemblages are reported. In 64 % of trials, electro-flocculation reduced total nitrogen (TN) and dissolved inorganic nitrogen (DIN) loads within regulatory limits.TN was reduced up to 83.2 % (10.93 to 1.83 mg.L-1) within 20 mins in stationary water, and DIN to 90.6 % (3.19 to 0.30 mg.L-1) in 102 mins trials in flowing water. Bellerochea andGloeocapsa spp. were dominant in wastewater. The role of microalgal community composition on flocculation is discussed, including evidence Bellerocheapromotes flocculation. This study confirmed electro-flocculation quickly reduces TN and DIN.

The HIV-1 capsid serves as a nanoscale reaction vessel for reverse transcription.

The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 particles can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, indicating that gp41 permits the entry of nucleotides into virions. Protease treatment of virions markedly reduced NERT suggesting the presence of a proteinaceous membrane channel. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.

Elasticity of the HIV-1 Core Facilitates Nuclear Entry and Infection.

HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid 1,2 . Recent studies have shown that HIV- 1 cores enter the nucleus prior to capsid disassembly 3-5 . Interactions with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Suppressors of the mutants restored elasticity and rescued infectivity and nuclear entry. Elasticity was also reduced by treatment of cores with the capsid-targeting compound PF74 and the antiviral drug lenacapavir. Our results indicate that capsid elasticity is a fundamental property of the HIV-1 core that enables its passage through the nuclear pore complex, thereby facilitating infection. These results provide new insights into the mechanisms of HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.

HIV-1 binds dynein directly to hijack microtubule transport machinery.

Viruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet the molecular mechanism that describes how viruses connect to the motor machinery is unknown. Here, we demonstrate the first example of viral microtubule trafficking from purified components: HIV-1 hijacking microtubule transport machinery. We discover that HIV-1 directly binds to the retrograde microtubule-associated motor, dynein, and not via a cargo adaptor, as previously suggested. Moreover, we show that HIV-1 motility is supported by multiple, diverse dynein cargo adaptors as HIV-1 binds to dynein light and intermediate chains on dynein's tail. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, like HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results introduce a new model of viral trafficking wherein a pathogen opportunistically 'hijacks' the microtubule transport machinery for motility, enabling multiple transport pathways through the host cytoplasm.

Multidisciplinary studies with mutated HIV-1 capsid proteins reveal structural mechanisms of lattice stabilization.

HIV-1 capsid (CA) stability is important for viral replication. E45A and P38A mutations enhance and reduce core stability, thus impairing infectivity. Second-site mutations R132T and T216I rescue infectivity. Capsid lattice stability was studied by solving seven crystal structures (in native background), including P38A, P38A/T216I, E45A, E45A/R132T CA, using molecular dynamics simulations of lattices, cryo-electron microscopy of assemblies, time-resolved imaging of uncoating, biophysical and biochemical characterization of assembly and stability. We report pronounced and subtle, short- and long-range rearrangements: (1) A38 destabilized hexamers by loosening interactions between flanking CA protomers in P38A but not P38A/T216I structures. (2) Two E45A structures showed unexpected stabilizing CANTD-CANTD inter-hexamer interactions, variable R18-ring pore sizes, and flipped N-terminal ß-hairpin. (3) Altered conformations of E45Aa α9-helices compared to WT, E45A/R132T, WTPF74, WTNup153, and WTCPSF6 decreased PF74, CPSF6, and Nup153 binding, and was reversed in E45A/R132T. (4) An environmentally sensitive electrostatic repulsion between E45 and D51 affected lattice stability, flexibility, ion and water permeabilities, electrostatics, and recognition of host factors.

HIV-1 capsid stability enables inositol phosphate-independent infection of target cells and promotes integration into genes.

The mature HIV-1 capsid is stabilized by host and viral determinants. The capsid protein CA binds to the cellular metabolites inositol hexakisphosphate (IP6) and its precursor inositol (1, 3, 4, 5, 6) pentakisphosphate (IP5) to stabilize the mature capsid. In target cells, capsid destabilization by the antiviral compounds lenacapavir and PF74 reveals a HIV-1 infectivity defect due to IP5/IP6 (IP5/6) depletion. To test whether intrinsic HIV-1 capsid stability and/or host factor binding determines HIV-1 insensitivity to IP5/6 depletion, a panel of CA mutants was assayed for infection of IP5/6-depleted T cells and wildtype cells. Four CA mutants with unstable capsids exhibited dependence on host IP5/6 for infection and reverse transcription (RTN). Adaptation of one such mutant, Q219A, by spread in culture resulted in Vpu truncation and a capsid three-fold interface mutation, T200I. T200I increased intrinsic capsid stability as determined by in vitro uncoating of purified cores and partially reversed the IP5/6-dependence in target cells for each of the four CA mutants. T200I further rescued the changes to lenacapavir sensitivity associated with the parental mutation. The premature dissolution of the capsid caused by the IP5/6-dependent mutations imparted a unique defect in integration targeting that was rescued by T200I. Collectively, these results demonstrate that T200I restored other capsid functions after RTN for the panel of mutants. Thus, the hyperstable T200I mutation stabilized the instability defects imparted by the parental IP5/6-dependent CA mutation. The contribution of Vpu truncation to mutant adaptation was linked to BST-2 antagonization, suggesting that cell-to-cell transfer promoted replication of the mutants. We conclude that interactions at the three-fold interface are adaptable, key mediators of capsid stability in target cells and are able to antagonize even severe capsid instability to promote infection.

Aging reduces manual dexterity and force production asymmetries between the hands.

Age-related effects on motor asymmetry provide insight into changes in cortical activation during aging. To investigate potential changes in manual performance associated with aging, we conducted the Jamar hand function test and the Purdue Pegboard test on young and older adults. All tests indicated reduced motor asymmetry in the older group. Further analysis suggested that a significant decline in dominant (right) hand function resulted in less asymmetric performance in older adults. The finding is inconsistent with the application of the HAROLD model in the motor domain, which assumes improved performance in the non-dominant hand, leading to a reduction of motor asymmetry in older adults. Based on the manual performance in young and older adults, it is suggested that aging reduces manual asymmetry in both force production and manual dexterity due to the reduced performance of the dominant hand.

The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores.

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

Modeling HIV-1 nuclear entry with nucleoporin-gated DNA-origami channels.

Delivering the virus genome into the host nucleus through the nuclear pore complex (NPC) is pivotal in human immunodeficiency virus 1 (HIV-1) infection. The mechanism of this process remains mysterious owing to the NPC complexity and the labyrinth of molecular interactions involved. Here we built a suite of NPC mimics-DNA-origami-corralled nucleoporins with programmable arrangements-to model HIV-1 nuclear entry. Using this system, we determined that multiple cytoplasm-facing Nup358 molecules provide avid binding for capsid docking to the NPC. The nucleoplasm-facing Nup153 preferentially attaches to high-curvature regions of the capsid, positioning it for tip-leading NPC insertion. Differential capsid binding strengths of Nup358 and Nup153 constitute an affinity gradient that drives capsid penetration. Nup62 in the NPC central channel forms a barrier that viruses must overcome during nuclear import. Our study thus provides a wealth of mechanistic insight and a transformative toolset for elucidating how viruses like HIV-1 enter the nucleus.

Utilising an internal focus of attention during preparation and an external focus during execution may facilitate motor learning.

Research with athletes and coaches has found that attentional focus strategies are more complex than using an internal or external focus exclusively. Recently Becker et al. [2020, March 1. The effects of attentional focus in the preparation and execution of a standing long jump. Psychological Research, 84(2), 285-291] found that switching attentional focus from internal in movement preparation to external during movement execution provided a benefit over internal focus and control conditions. The purpose of this study was to investigate the effects of attentional switching on motor skill acquisition. 79 participants were randomly divided into an internal (INT), external (EXT), or switching group (IES). Individuals performed 80 acquisition trials of a golf chipping task with their prescribed attentional focus during preparation and execution. 24-hours later 10-trial retention and transfer tests were performed. The primary analysis revealed significant improvement during acquisition (p < .001) but no significant differences between attentional focus groups during acquisition, retention, or transfer. A secondary analysis was performed with individuals low in attentional focus adherence removed (<60%). Groups significantly improved during acquisition (p < .001). For retention IES significantly outperformed INT (p < .05). These results suggest that the detrimental effects of an internal focus are only found during skill execution and not movement preparation. It is possible for learners to benefit from both an internal and external focus so long as the cues are provided during the correct phase of skill performance.Highlights Attentional focus is much more complex than using either an internal or external focus of attention. This study investigated the effects of switching attention during skill acquisition from an internal to external focus.A preliminary analysis revealed no significant differences between attentional focus groups of internal, external, or switching. One reason for this finding was the poor adherence to the prescribed focus cue.A secondary analysis where low adherence individuals were removed revealed a significant difference between the switching group and the internal focus group during retention.These finding suggest that the detrimental effects associated with an internal focus of attention are exclusive to using the focus during task execution. It is possible that an internal focus can still facilitate learning so long as an external focus is used during movement execution.

Reactive persistence, spatial management, and conservation of metapopulations: An application to seagrass restoration.

Assessing the conditions for persistence of spatially structured populations, especially those that are exploited by humans or threatened by global change, is of critical importance to inform management and conservation efforts. Observations for entire metapopulations are usually incomplete and rarely, if ever, sufficiently long to deduce population persistence from spatial patterns of abundance. Instead, insights based on metapopulation theory are often used for interpreting the demographic trajectories of real populations and for informing management decisions. The classical theoretical tool used to assess conditions for metapopulation persistence is the "invasibility criterion," which characterizes the asymptotic, or long-term, stability of a small colonizing population. Essentially, when the linear operator governing the metapopulation dynamics of an invasion event has a positive eigenvalue, recovery and resistance to extinction (resilience) are implied. The converse, however, is not necessarily the case-an invasion may grow over multiple generations, even when the eigenvalues indicate that extinction will eventually occur, a situation referred to here as "reactive persistence." For the management, restoration, and conservation of real metapopulations subject to continual disturbance, this transient behavior is often more relevant than the asymptotic behavior over long time scales. We develop the theoretical tools for assessing reactive persistence, demonstrating how the conditions for asymptotic and reactive persistence differ in both the patch-occupancy models suited to many terrestrial populations and those where local patch extinctions can be disregarded in the dynamics, often suited to marine species. After presenting the mathematical basis for generalizing the invasibility criterion to include reactive persistence, we illustrate how these concepts and tools can be applied in practice, using as a case study the population ecology and restoration of the seagrass Zostera muelleri (Irmisch ex Ascherson, 1867) in the Port of Gladstone in the Great Barrier Reef World Heritage Area Australia. It is shown how the analysis of the transient dynamics of the Z. muelleri metapopulation can be used to guide restoration efforts. Moreover, it is demonstrated that these reactive persistence concepts provide a more appropriate basis for site prioritization for restoration interventions than traditional stability analysis.

Effects of attentional focus and cognitive load on novice dart throwing: Evidence from quiet eye duration and pupillary responses.

The purpose of the present study was to investigate the effects of attentional focus and cognitive-load on motor performance, quiet-eye-duration, and pupil dilation. 18 participants completed a dart throwing task under four conditions, internal or external focus with high or low cognitive-load. Cognitive-load was created by a secondary tone detection task. During each trial participants pupil size and eye movements were recorded along with accuracy data of the dart throw. Results revealed that decreased cognitive-load increased accuracy while high load increased pupil size (p's < 0.05). An external focus resulted in the greatest accuracy while an external focus with high cognitive-load resulted in the longest quiet-eye-durations (p's < 0.05). Based on these findings an increase in pupil size is related to greater cognitive-load but doesn't explain the improvement in task performance. Likewise, an external focus of attention improved performance but was not strongly related to quiet-eye-duration. Results are further discussed in the article.

Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles.

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components.

HIV-1 mutants that escape the cytotoxic T-lymphocytes are defective in viral DNA integration.

HIV-1 replication is durably controlled without antiretroviral therapy (ART) in certain infected individuals called elite controllers (ECs). These individuals express specific human leukocyte antigens (HLA) that tag HIV-infected cells for elimination by presenting viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). In HIV-infected individuals expressing HLA-B27, CTLs primarily target the viral capsid protein (CA)-derived KK10 epitope. While selection of CA mutation R264K helps HIV-1 escape this potent CTL response, the accompanying fitness cost severely diminishes virus infectivity. Interestingly, selection of a compensatory CA mutation S173A restores HIV-1 replication. However, the molecular mechanism(s) underlying HIV-1 escape from this ART-free virus control by CTLs is not fully understood. Here, we report that the R264K mutation-associated infectivity defect arises primarily from impaired HIV-1 DNA integration, which is restored by the S173A mutation. Unexpectedly, the integration defect of the R264K variant was also restored upon depletion of the host cyclophilin A. These findings reveal a nuclear crosstalk between CA and HIV-1 integration as well as identify a previously unknown role of cyclophilin A in viral DNA integration. Finally, our study identifies a novel immune escape mechanism of an HIV-1 variant escaping a CA-directed CTL response.

HIV-1 CA Inhibitors Are Antagonized by Inositol Phosphate Stabilization of the Viral Capsid in Cells.

The HIV-1 capsid, composed of the CA protein, is the target of the novel antiretroviral drug lenacapavir (LCV). CA inhibitors block host factor binding and alter capsid stability to prevent nuclear entry and reverse transcription (RTN), respectively. Capsid stability is mediated in vitro by binding to the host cell metabolite inositol hexakisphosphate (IP6). IP6 depletion in target cells has little effect on HIV-1 infection. We hypothesized that capsid-altering concentrations of CA inhibitors might reveal an effect of IP6 depletion on HIV-1 infection in target cells. To test this, we studied the effects of IP6 depletion on inhibition of infection by the CA inhibitors PF74 and LCV. At low doses of either compound that affect HIV-1 nuclear entry, no effect of IP6 depletion on antiviral activity was observed. Increased antiviral activity was observed in IP6-depleted cells at inhibitor concentrations that affect capsid stability, correlating with increased RTN inhibition. Assays of uncoating and endogenous RTN of purified cores in vitro provided additional support. Our results show that inositol phosphates stabilize the HIV-1 capsid in target cells, thereby dampening the antiviral effects of capsid-targeting antiviral compounds. We propose that targeting of the IP6-binding site in conjunction with CA inhibitors will lead to robust antiretroviral therapy (ART). IMPORTANCE HIV-1 infection and subsequent depletion of CD4+ T cells result in AIDS. Antiretroviral therapy treatment of infected individuals prevents progression to AIDS. The HIV-1 capsid has recently become an ART target. Capsid inhibitors block HIV-1 infection at multiple steps, offering advantages over current ART. The cellular metabolite inositol hexakisphosphate (IP6) binds the HIV-1 capsid, stabilizing it in vitro. However, the function of this interaction in target cells is unclear. Our results imply that IP6 stabilizes the incoming HIV-1 capsid in cells, thus limiting the antiviral efficiency of capsid-destabilizing antivirals. We present a model of capsid inhibitor function and propose that targeting of the IP6-binding site in conjunction with capsid inhibitors currently in development will lead to more robust ART.

The HIV-1 capsid and reverse transcription.

The viral capsid plays a key role in HIV-1 reverse transcription. Recent studies have demonstrated that the small molecule IP6 dramatically enhances reverse transcription in vitro by stabilizing the viral capsid. Reverse transcription results in marked changes in the biophysical properties of the capsid, ultimately resulting in its breakage and disassembly. Here we review the research leading to these advances and describe hypotheses for capsid-dependent HIV-1 reverse transcription and a model for reverse transcription-primed HIV-1 uncoating.

CryoET structures of immature HIV Gag reveal six-helix bundle.

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

Correction: Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells.

[This corrects the article DOI: 10.1371/journal.ppat.1009190.].

HIV-1 uncoating occurs via a series of rapid biomechanical changes in the core related to individual stages of reverse transcription.

The HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6 Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. These reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.ImportanceFor successful infection, the HIV-1 genome, which is enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism of HIV-1 uncoating is a pivotal question of long standing. Using atomic force microscopy to analyze individual HIV-1 cores during reverse transcription, we observe a reproducible pattern of stiffness spikes. These spikes were shown to be associated with distinct stages of the reverse transcription reaction. Our findings suggest that these reverse-transcription-induced alterations gradually prepared the core for uncoating at the right time and location in target cells.