Spatial resolution of HIV-1 post-entry steps in resting CD4 T cells.

Resting CD4 T cells resist productive HIV-1 infection. The HIV-2/simian immunodeficiency virus protein viral accessory protein X (Vpx) renders these cells permissive to infection, presumably by alleviating blocks at cytoplasmic reverse transcription and subsequent nuclear import of reverse-transcription/pre-integration complexes (RTC/PICs). Here, spatial analyses using quantitative virus imaging techniques reveal that HIV-1 capsids containing RTC/PICs are readily imported into the nucleus, recruit the host dependency factor CPSF6, and translocate to nuclear speckles in resting CD4 T cells. Reverse transcription, however, remains incomplete, impeding proviral integration and viral gene expression. Vpx or pharmacological inhibition of the deoxynucleotide triphosphohydrolase (dNTPase) activity of the restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) increases levels of nuclear reverse-transcribed cDNA and facilitates HIV-1 integration. Nuclear import and intranuclear transport of viral complexes therefore do not pose important blocks to HIV-1 in resting CD4 T cells, and the limitation to reverse transcription by SAMHD1's dNTPase activity constitutes the main pre-integration block to infection.

Direct Capsid Labeling of Infectious HIV-1 by Genetic Code Expansion Allows Detection of Largely Complete Nuclear Capsids and Suggests Nuclear Entry of HIV-1 Complexes via Common Routes.

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.

Nuclear Capsid Uncoating and Reverse Transcription of HIV-1.

After cell entry, human immunodeficiency virus type 1 (HIV-1) replication involves reverse transcription of the RNA genome, nuclear import of the subviral complex without nuclear envelope breakdown, and integration of the viral complementary DNA into the host genome. Here, we discuss recent evidence indicating that completion of reverse transcription and viral genome uncoating occur in the nucleus rather than in the cytoplasm, as previously thought, and suggest a testable model for nuclear import and uncoating. Multiple recent studies indicated that the cone-shaped capsid, which encases the genome and replication proteins, not only serves as a reaction container for reverse transcription and as a shield from innate immune sensors but also may constitute the elusive HIV-1 nuclear import factor. Rupture of the capsid may be triggered in the nucleus by completion of reverse transcription, by yet-unknown nuclear factors, or by physical damage, and it appears to occur in close temporal and spatial association with the integration process.

HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells.

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.

When viruses infect human cells, they hijack the cell's machinery to produce the proteins they need to replicate. Retroviruses like HIV-1 do this by entering the nucleus and inserting their genetic information into the genome of the infected cell. This requires HIV-1 to convert its genetic material into DNA, which is then released from the protective shell surrounding it (known as the capsid) via a process called uncoating. The nucleus is enclosed within an envelope containing pores that molecules up to a certain size can pass through. Until recently these pores were thought to be smaller than the viral capsid, which led scientists to believe that the HIV-1 genome must shed this coat before penetrating the nucleus. However, recent studies have found evidence for HIV-1 capsid proteins and capsid structures inside the nucleus of some infected cells. This suggests that the capsid may not be removed before nuclear entry or that it may even play a role in helping the virus get inside the nucleus. To investigate this further, Müller et al. attached fluorescent labels to the newly made DNA of HIV-1 and some viral and cellular proteins. Powerful microscopy tools were then used to monitor the uncoating process in various cells that had been infected with the virus. Müller et al. found large amounts of capsid protein inside the nuclei of all the infected cells studied. During the earlier stages of infection, the capsid proteins were mostly associated with viral DNA and the capsid structure appeared largely intact. At later time points, the capsid structure had been broken down and the viral DNA molecules were gradually separating themselves from these remnants. These findings suggest that the HIV-1 capsid helps the virus get inside the nucleus and may protect its genetic material during conversion into DNA until right before integration into the cell's genome. Further experiments studying this process could lead to new therapeutic approaches that target the capsid as a way to prevent or treat HIV-1.

Cone-shaped HIV-1 capsids are transported through intact nuclear pores.

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.

Photocaged Hoechst Enables Subnuclear Visualization and Cell Selective Staining of DNA in vivo.

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Chemogenetic Control of Nanobodies.

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.

Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells.

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.IMPORTANCE 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.

A Spotlight on Viruses-Application of Click Chemistry to Visualize Virus-Cell Interactions.

The replication of a virus within its host cell involves numerous interactions between viral and cellular factors, which have to be tightly controlled in space and time. The intricate interplay between viral exploitation of cellular pathways and the intrinsic host defense mechanisms is difficult to unravel by traditional bulk approaches. In recent years, novel fluorescence microscopy techniques and single virus tracking have transformed the investigation of dynamic virus-host interactions. A prerequisite for the application of these imaging-based methods is the attachment of a fluorescent label to the structure of interest. However, their small size, limited coding capacity and multifunctional proteins render viruses particularly challenging targets for fluorescent labeling approaches. Click chemistry in conjunction with genetic code expansion provides virologists with a novel toolbox for site-specific, minimally invasive labeling of virion components, whose potential has just recently begun to be exploited. Here, we summarize recent achievements, current developments and future challenges for the labeling of viral nucleic acids, proteins, glycoproteins or lipids using click chemistry in order to study dynamic processes in virus-cell interactions.

Proximity-Triggered Covalent Stabilization of Low-Affinity Protein Complexes In Vitro and In Vivo.

The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions in vitro and in vivo.

HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

Quantitative analyses of anaerobic wastewater treatment processes: identifiability and parameter estimation.

We investigated the problem of identifying the parameters of a nonlinear fifth order model describing the population dynamics of two main bacterial groups in an anaerobic wastewater treatment process. In addition to addressing problems concerning structural and practical identifiability, we also analyzed how mathematical descriptions of bacterial population dynamics can model real data. Using three data sets recorded under different experimental conditions, we estimated important biochemical parameters and demonstrated that our model could describe the data successfully. Parameters, which are simultaneously determined using information from all three experiments, have more reliable estimates. We conclude that, after appropriate estimation, this model can be used for optimization and the control of continuous processes.