HIV-1 uncoating requires long double-stranded reverse transcription products.

HIV-1 cores, which contain the viral genome and replication machinery, must disassemble (uncoat) during viral replication. However, the viral and host factors that trigger uncoating remain unidentified. Recent studies show that infectious cores enter the nucleus and uncoat near the site of integration. Here, we show that efficient uncoating of nuclear cores requires synthesis of a double-stranded DNA (dsDNA) genome >3.5 kb and that the efficiency of uncoating correlates with genome size. Core disruption by capsid inhibitors releases viral DNA, some of which integrates. However, most of the viral DNA is degraded, indicating that the intact core safeguards viral DNA. Atomic force microscopy and core content estimation reveal that synthesis of full-length genomic dsDNA induces substantial internal strain on the core to promote uncoating. We conclude that HIV-1 cores protect viral DNA from degradation by host factors and that synthesis of long double-stranded reverse transcription products is required to trigger efficient HIV-1 uncoating.

A quantitative model for virus uncoating predicts influenza A infectivity.

For virus infection of new host cells, the disassembly of the protective outer protein shell (capsid) is a critical step, but the mechanisms and host-virus interactions underlying the dynamic, active, and regulated uncoating process are largely unknown. Here, we develop an experimentally supported, multiscale kinetics model that elucidates mechanisms of influenza A virus (IAV) uncoating in cells. Biophysical modeling demonstrates that interactions between capsid M1 proteins, host histone deacetylase 6 (HDAC6), and molecular motors can physically break the capsid in a tug-of-war mechanism. Biochemical analysis and biochemical-biophysical modeling identify unanchored ubiquitin chains as essential and allow robust prediction of uncoating efficiency in cells. Remarkably, the different infectivity of two clinical strains can be ascribed to a single amino acid variation in M1 that affects binding to HDAC6. By identifying crucial modules of viral infection kinetics, the mechanisms and models presented here could help formulate novel strategies for broad-range antiviral treatment.

Reovirus genomic diversity confers plasticity for protease utility during adaptation to intracellular uncoating.

IMPORTANCE: Reoviruses infect many mammals and are widely studied as a model system for enteric viruses. However, most of our reovirus knowledge comes from laboratory strains maintained on immortalized L929 cells. Herein, we asked whether naturally circulating reoviruses possess the same genetic and phenotypic characteristics as laboratory strains. Naturally circulating reoviruses obtained from sewage were extremely diverse genetically. Moreover, sewage reoviruses exhibited poor fitness on L929 cells and relied heavily on gut proteases for viral uncoating and productive infection compared to laboratory strains. We then examined how naturally circulating reoviruses might adapt to cell culture conditions. Within three passages, virus isolates from the parental sewage population were selected, displaying improved fitness and intracellular uncoating in L929 cells. Remarkably, selected progeny clones were present at 0.01% of the parental population. Altogether, using reovirus as a model, our study demonstrates how the high genetic diversity of naturally circulating viruses results in rapid adaptation to new environments.

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.

Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus.

Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.

Maternal embryonic leucine zipper kinase (MELK) optimally regulates the HIV-1 uncoating process.

Human immunodeficiency virus type-1 (HIV-1) attaches to target cells and releases the capsid, an essential component of the viral core that contains viral RNA, into the cytoplasm. After invading target cells, the core structure gradually collapses. The timing of the disassembly of the HIV-1 capsid is essential for efficient viral cDNA synthesis and transport into the nucleus. HIV-1 uncoating is controlled by the host factor maternal embryonic leucine zipper kinase (MELK); however, the quantitative and dynamic relationship between the HIV-1 uncoating process and HIV-1 infection remains unresolved. In this study, we quantified the uncoating process on HIV-1 cDNA synthesis and transport into the nucleus by combining a mathematical model with in vitro data. In addition, detailed in silico simulations demonstrated host factors, including MELK, optimize transport efficiency. Our experimental-mathematical approach revealed quantitative dynamics of the HIV-1 uncoating process, indicating that increasing the speed of uncoating always reduces the amount of HIV-1 cDNA in the nucleus.

Strain and rupture of HIV-1 capsids during uncoating.

SignificanceThe mature capsids of HIV-1 are transiently stable complexes that self-assemble around the viral genome during maturation, and uncoat to release preintegration complexes that archive a double-stranded DNA copy of the virus in the host cell genome. However, a detailed view of how HIV cores rupture remains lacking. Here, we elucidate the physical properties involved in capsid rupture using a combination of large-scale all-atom molecular dynamics simulations and cryo-electron tomography. We find that intrinsic strain on the capsid forms highly correlated patterns along the capsid surface, along which cracks propagate. Capsid rigidity also increases with high strain. Our findings provide fundamental insight into viral capsid uncoating.

HIV-1 Nucleocapsid Protein Binds Double-Stranded DNA in Multiple Modes to Regulate Compaction and Capsid Uncoating.

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral DNA genome is stably contained within a narrow capsid with diameter similar to the persistence length of double-stranded (ds) DNA, and the role of NC in this process, are not well understood. In this study, we use optical tweezers, fluorescence imaging, and atomic force microscopy to observe NC binding a single long DNA substrate in multiple modes. We find that NC binds and saturates the DNA substrate in a non-specific binding mode that triggers uniform DNA self-attraction, condensing the DNA into a tight globule at a constant force up to 10 pN. When NC is removed from solution, the globule dissipates over time, but specifically-bound NC maintains long-range DNA looping that is less compact but highly stable. Both binding modes are additionally observed using AFM imaging. These results suggest multiple binding modes of NC compact DNA into a conformation compatible with reverse transcription, regulating the genomic pressure on the capsid and preventing premature uncoating.

Single-Particle Tracking Reveals the Interplay between HIV-1 Reverse Transcription and Uncoating.

Reverse transcription uses the reverse transcriptase enzyme to synthesize deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template. This plays an essential role in viral replication. There are still, however, many unknown facts regarding the timing and dynamic processes involved in this life stage. Here, three types of dual-fluorescence human immunodeficiency virus type-1 (HIV-1) particles were constructed with high infectivity, and the sequential process of reverse transcription was observed by real-time imaging of a single HIV-1 particle. Viral uncoating occurred at 60-120 min post infection. Subsequently, at 120-180 min post infection, the viral genome was separated into two parts and reverse-transcribed to generate a DNA product. Nevirapine (NVP), a reverse transcriptase inhibitor, can delay the dynamic process. This study revealed a delicate, sequential, and complex relationship between uncoating and reverse transcription, which may facilitate the development of antiviral drugs.

Chemical Evolution of Rhinovirus Identifies Capsid-Destabilizing Mutations Driving Low-pH-Independent Genome Uncoating.

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein.

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication.

Human coronavirus (HCoV) similar to other viruses rely on host cell machinery for both replication and to spread. The p97/VCP ATPase is associated with diverse pathways that may favor HCoV replication. In this study, we assessed the role of p97 and associated host responses in human lung cell line H1299 after HCoV-229E or HCoV-OC43 infection. Inhibition of p97 function by small molecule inhibitors shows antiviral activity, particularly at early stages of the virus life cycle, during virus uncoating and viral RNA replication. Importantly, p97 activity inhibition protects human cells against HCoV-induced cytopathic effects. The p97 knockdown also inhibits viral production in infected cells. Unbiased quantitative proteomics analyses reveal that HCoV-OC43 infection resulted in proteome changes enriched in cellular senescence and DNA repair during virus replication. Further analysis of protein changes between infected cells with control and p97 shRNA identifies cell cycle pathways for both HCoV-229E and HCoV-OC43 infection. Together, our data indicate a role for the essential host protein p97 in supporting HCoV replication, suggesting that p97 is a therapeutic target to treat HCoV infection.

Disassembling the Nature of Capsid: Biochemical, Genetic, and Imaging Approaches to Assess HIV-1 Capsid Functions.

The human immunodeficiency virus type 1 (HIV-1) capsid and its disassembly, or capsid uncoating, has remained an active area of study over the past several decades. Our understanding of the HIV-1 capsid as solely a protective shell has since shifted with discoveries linking it to other complex replication events. The interplay of the HIV-1 capsid with reverse transcription, nuclear import, and integration has led to an expansion of knowledge of capsid functionality. Coincident with advances in microscopy, cell, and biochemistry assays, several models of capsid disassembly have been proposed, in which it occurs in either the cytoplasmic, nuclear envelope, or nuclear regions of the cell. Here, we discuss how the understanding of the HIV-1 capsid has evolved and the key methods that made these discoveries possible.

Nuclear Import of HIV-1.

The delivery of the HIV-1 genome into the nucleus is an indispensable step in retroviral infection of non-dividing cells, but the mechanism of HIV-1 nuclear import has been a longstanding debate due to controversial experimental evidence. It was commonly believed that the HIV-1 capsid would need to disassemble (uncoat) in the cytosol before nuclear import because the capsid is larger than the central channel of nuclear pore complexes (NPCs); however, increasing evidence demonstrates that intact, or nearly intact, HIV-1 capsid passes through the NPC to enter the nucleus. With the protection of the capsid, the HIV-1 core completes reverse transcription in the nucleus and is translocated to the integration site. Uncoating occurs while, or after, the viral genome is released near the integration site. These independent discoveries reveal a compelling new paradigm of this important step of the HIV-1 life cycle. In this review, we summarize the recent studies related to HIV-1 nuclear import, highlighting the spatial-temporal relationship between the nuclear entry of the virus core, reverse transcription, and capsid uncoating.

Arginyl-tRNA-protein transferase 1 contributes to governing optimal stability of the human immunodeficiency virus type 1 core.

BACKGROUND: The genome of human immunodeficiency virus type 1 (HIV-1) is encapsulated in a core consisting of viral capsid proteins (CA). After viral entry, the HIV-1 core dissociates and releases the viral genome into the target cell, this process is called uncoating. Uncoating of HIV-1 core is one of the critical events in viral replication and several studies show that host proteins positively or negatively regulate this process by interacting directly with the HIV-1 CA. RESULTS: Here, we show that arginyl-tRNA-protein transferase 1 (ATE1) plays an important role in the uncoating process by governing the optimal core stability. Yeast two-hybrid screening of a human cDNA library identified ATE1 as an HIV-1-CA-interacting protein and direct interaction of ATE1 with Pr55gag and p160gag - pol via HIV-1 CA was observed by cell-based pull-down assay. ATE1 knockdown in HIV-1 producer cells resulted in the production of less infectious viruses, which have normal amounts of the early products of the reverse transcription reaction but reduced amounts of the late products of the reverse transcription. Interestingly, ATE1 overexpression in HIV-1 producer cells also resulted in the production of poor infectious viruses. Cell-based fate-of-capsid assay, a commonly used method for evaluating uncoating by measuring core stability, showed that the amounts of pelletable cores in cells infected with the virus produced from ATE1-knockdown cells increased compared with those detected in the cells infected with the control virus. In contrast, the amounts of pelletable cores in cells infected with the virus produced from ATE1-overexpressing cells decreased compared with those detected in the cells infected with the control virus. CONCLUSIONS: These results indicate that ATE1 expression levels in HIV-1 producer cells contribute to the adequate formation of a stable HIV-1 core. These findings provide insights into a novel mechanism of HIV-1 uncoating and revealed ATE1 as a new host factor regulating HIV-1 replication.

The Role of Capsid in HIV-1 Nuclear Entry.

HIV-1 can infect non-dividing cells. The nuclear envelope therefore represents a barrier that HIV-1 must traverse in order to gain access to the host cell chromatin for integration. Hence, nuclear entry is a critical step in the early stages of HIV-1 replication. Following membrane fusion, the viral capsid (CA) lattice, which forms the outer face of the retroviral core, makes numerous interactions with cellular proteins that orchestrate the progress of HIV-1 through the replication cycle. The ability of CA to interact with nuclear pore proteins and other host factors around the nuclear pore determines whether nuclear entry occurs. Uncoating, the process by which the CA lattice opens and/or disassembles, is another critical step that must occur prior to integration. Both early and delayed uncoating have detrimental effects on viral infectivity. How uncoating relates to nuclear entry is currently hotly debated. Recent technological advances have led to intense discussions about the timing, location, and requirements for uncoating and have prompted the field to consider alternative uncoating scenarios that presently focus on uncoating at the nuclear pore and within the nuclear compartment. This review describes recent advances in the study of HIV-1 nuclear entry, outlines the interactions of the retroviral CA protein, and discusses the challenges of investigating HIV-1 uncoating.

Overlap Intensity: An ImageJ Macro for Analyzing the HIV-1 In Situ Uncoating Assay.

Capsid uncoating is at the crossroads of early steps in HIV-1 replication. In recent years, the development of novel assays has expanded how HIV-1 uncoating can be studied. In the in situ uncoating assay, dual fluorescently labelled virus allows for the identification of fused viral cores. Antibody staining then detects the amount of capsid associated with each viral core at different times post-infection. Following fixed cell imaging, manual counting can be used to assess the fusion state and capsid signal for each viral core, but this method can introduce bias with increased time of analysis. To address these limitations, we developed the Overlap Intensity macro in ImageJ. This macro automates the detection of viral cores and quantification of overlapping fusion and capsid signals. We demonstrated the high accuracy of the macro by comparing core detection to manual methods. Analysis of an in situ uncoating assay further verified the macro by detecting progressive uncoating as expected. Therefore, this macro improves the accessibility of the in situ uncoating assay by replacing time-consuming manual methods or the need for expensive data analysis software. Beyond the described assay, the Overlap Intensity macro includes adjustable settings for use in other methods requiring quantification of overlapping fluorescent signals.

How Influenza Virus Uses Host Cell Pathways during Uncoating.

Influenza is a zoonotic respiratory disease of major public health interest due to its pandemic potential, and a threat to animals and the human population. The influenza A virus genome consists of eight single-stranded RNA segments sequestered within a protein capsid and a lipid bilayer envelope. During host cell entry, cellular cues contribute to viral conformational changes that promote critical events such as fusion with late endosomes, capsid uncoating and viral genome release into the cytosol. In this focused review, we concisely describe the virus infection cycle and highlight the recent findings of host cell pathways and cytosolic proteins that assist influenza uncoating during host cell entry.

Quantum Dots: A Promising Fluorescent Label for Probing Virus Trafficking.

Recent research has highlighted the immense potential of the quantum dot (QD)-based single-virus tracking (SVT) technique in virology. In these experiments, the infection behaviors of single viruses or viral components, labeled with QDs, could be tracked on time scales of milliseconds to hours in host cells. The trajectories of individual viruses are reconstructed with nanometer accuracy, and the underlying dynamic information on virus infection can be extracted to uncover the infection mechanisms of viruses. Therefore, QD-based single-virus tracking (QSVT) is an exquisitely selective and powerful approach to investigating how viruses are internalized in host cells dynamically to release their genome for viral replication and assembly that ensure the completion of viral life cycles.QDs are better candidates than organic dyes and fluorescent proteins for virus labeling and subsequent SVT due to the following considerations: (i) the high brightness of QDs makes it possible to label a virus with sufficient brightness using very few QDs or even just one QD; (ii) the extraordinary photostability of QDs allows one to track the infection process long term and quantify low probability events; (iii) the color-tunable emission property of QDs ensures multicolor labeling of various components of a virus simultaneously; and (iv) the abundant surface ligands of QDs facilitate the conjugation of a virus with a variety of labeling strategies. Therefore, the photoproperties of QDs make it possible to perform multicolor long-term SVT experiments quantitatively. Nowadays, the QD-based SVT (QSVT) technique has made prodigious achievements in unraveling the entry, trafficking, and uncoating mechanisms of viruses. This fascinating technique can provide spatiotemporal dynamic information on the viral journey in unprecedented detail and has revolutionized our understanding of virus infection.In this Account, we first introduce the advantages and the limitations of conventional SVT in virological research and the unique features of QDs as labels in the SVT field. We subsequently focus on the principles and related methods of QSVT and the current state of QD chemistry and QD-based virus labeling that resolves many issues associated with the tracking of individual viruses in live cells. Then we emphasize some new findings by this technique in the study of infection mechanisms. Finally, we will provide our insights into future challenges on this topic. With this Account, we hope to further stimulate the development of QSVT with a combined effort from different disciplines and, more importantly, to accelerate the applications of QSVT in virological research.

You can keep your coat on.

High-resolution imaging techniques reveal new insights into the actions of the retrovirus HIV-1 inside host cells.