Gutless Helper-Dependent and First-Generation HAdV5 Vectors Have Similar Mechanical Properties and Common Transduction Mechanisms.

Delivering vectorized information into cells with the help of viruses has been of high interest to fundamental and applied science, and bears significant therapeutic promise. Human adenoviruses (HAdVs) have been at the forefront of gene delivery for many years, and the subject of intensive development resulting in several generations of agents, including replication-competent, -defective or retargeted vectors, and recently also helper-dependent (HD), so-called gutless vectors lacking any viral protein coding information. While it is possible to produce HD-AdVs in significant amounts, physical properties of these virus-like particles and their efficiency of transduction have not been addressed. Here, we used single-cell and single virus particle assays to probe the effect of genome length on HAdV-C5 vector transduction. Our results demonstrate that first-generation C5 vectors lacking the E1/E3 regions of the viral genome as well as HD-AdV-C5 particles with a wild type (wt) ∼36 kbp or an undersized double-strand DNA genome are similar to human adenovirus C5 (HAdV-C5) wt regarding attachment to human lung epithelial cells, endocytic uptake, endosome penetration and dependency on the E3 RING ubiquitin ligase Mind Bomb 1 for DNA uncoating at the nuclear pore complex. Atomic force microscopy measurements of single virus particles indicated that small changes in the genome length from 94% to 103% of HAdV-C5 have no major impact on physical and mechanical features of AdV vectors. In contrast, an HD-AdV-C5 with ∼30 kbp genome was slightly stiffer and less heat-resistant than the other particles, despite comparable entry and transduction efficiencies in tissue culture cell lines, including murine alveolar macrophage-like Max-Planck-Institute (MPI)-2 cells. Together, our in vitro studies reinforce the use of HD-AdV vectors for effective single round gene delivery. The results illustrate how physical properties and cell entry behavior of single virus particles can provide functional information for anticipated therapeutic vector applications.

Sequence-Specific Features of Short Double-Strand, Blunt-End RNAs Have RIG-I- and Type 1 Interferon-Dependent or -Independent Anti-Viral Effects.

Pathogen-associated molecular patterns, including cytoplasmic DNA and double-strand (ds)RNA trigger the induction of interferon (IFN) and antiviral states protecting cells and organisms from pathogens. Here we discovered that the transfection of human airway cell lines or non-transformed fibroblasts with 24mer dsRNA mimicking the cellular micro-RNA (miR)29b-1* gives strong anti-viral effects against human adenovirus type 5 (AdV-C5), influenza A virus X31 (H3N2), and SARS-CoV-2. These anti-viral effects required blunt-end complementary RNA strands and were not elicited by corresponding single-strand RNAs. dsRNA miR-29b-1* but not randomized miR-29b-1* mimics induced IFN-stimulated gene expression, and downregulated cell adhesion and cell cycle genes, as indicated by transcriptomics and IFN-I responsive Mx1-promoter activity assays. The inhibition of AdV-C5 infection with miR-29b-1* mimic depended on the IFN-alpha receptor 2 (IFNAR2) and the RNA-helicase retinoic acid-inducible gene I (RIG-I) but not cytoplasmic RNA sensors MDA5 and ZNFX1 or MyD88/TRIF adaptors. The antiviral effects of miR29b-1* were independent of a central AUAU-motif inducing dsRNA bending, as mimics with disrupted AUAU-motif were anti-viral in normal but not RIG-I knock-out (KO) or IFNAR2-KO cells. The screening of a library of scrambled short dsRNA sequences identified also anti-viral mimics functioning independently of RIG-I and IFNAR2, thus exemplifying the diverse anti-viral mechanisms of short blunt-end dsRNAs.

Adenovirus entry: Stability, uncoating, and nuclear import.

Adenoviruses (AdVs) are widespread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver, and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID-19 vaccination. AdV vectors are physically and genetically stable and generally safe in humans. The particles have an icosahedral coat and a nucleoprotein core with a DNA genome. We describe the concept of AdV cell entry and highlight recent advances in cytoplasmic transport, uncoating, and nuclear import of the viral DNA. We highlight a recently discovered "linchpin" function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3 ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover mechanisms controlling capsid stability and premature uncoating and provide insight into nuclear transport of nucleic acids.

Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern.

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the ß-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

A viral ubiquitination switch attenuates innate immunity and triggers nuclear import of virion DNA and infection.

Antiviral defense and virus exclusion from the cell nucleus restrict foreign nucleic acid influx and infection. How the genomes of DNA viruses evade cytosolic pattern recognition and cross the nuclear envelope is incompletely understood. Here, we show that the virion protein V of adenovirus functions as a linchpin between the genome and the capsid, thereby securing particle integrity. Absence of protein V destabilizes cytoplasmic particles and promotes premature genome release, raising cytokine levels through the DNA sensor cGAS. Non-ubiquitinable V yields stable virions, genome misdelivery to the cytoplasm, and increased cytokine levels. In contrast, normal protein V is ubiquitinated at the nuclear pore complex, dissociates from the virion depending on the E3 ubiquitin ligase Mib1 and the proteasome, and allows genome delivery into the nucleus for infection. Our data uncover previously unknown cellular and viral mechanisms of viral DNA nuclear import in pathogenesis, vaccination, gene therapy, and synthetic biology.

Virus Infection Variability by Single-Cell Profiling.

Cell-to-cell variability of infection has long been known, yet it has remained one of the least understood phenomena in infection research. It impacts on disease onset and development, yet only recently underlying mechanisms have been studied in clonal cell cultures by single-virion immunofluorescence microscopy and flow cytometry. In this review, we showcase how single-cell RNA sequencing (scRNA-seq), single-molecule RNA-fluorescence in situ hybridization (FISH), and copper(I)-catalyzed azide-alkyne cycloaddition (click) with alkynyl-tagged viral genomes dissect infection variability in human and mouse cells. We show how the combined use of scRNA-FISH and click-chemistry reveals highly variable onsets of adenoviral gene expression, and how single live cell plaques reveal lytic and nonlytic adenovirus transmissions. The review highlights how scRNA-seq profiling and scRNA-FISH of coxsackie, influenza, dengue, zika, and herpes simplex virus infections uncover transcriptional variability, and how the host interferon response tunes influenza and sendai virus infections. We introduce the concept of "cell state" in infection variability, and conclude with advances by single-cell simultaneous measurements of chromatin accessibility and mRNA counts at high-throughput. Such technology will further dissect the sequence of events in virus infection and pathology, and better characterize the genetic and genomic stability of viruses, cell autonomous innate immune responses, and mechanisms of tissue injury.

On-chip transporting arresting and characterizing individual nano-objects in biological ionic liquids.

Understanding and controlling the individual behavior of nanoscopic matter in liquids, the environment in which many such entities are functioning, is both inherently challenging and important to many natural and man-made applications. Here, we transport individual nano-objects, from an assembly in a biological ionic solution, through a nanochannel network and confine them in electrokinetic nanovalves, created by the collaborative effect of an applied ac electric field and a rationally engineered nanotopography, locally amplifying this field. The motion of so-confined fluorescent nano-objects is tracked, and its kinetics provides important information, enabling the determination of their particle diffusion coefficient, hydrodynamic radius, and electrical conductivity, which are elucidated for artificial polystyrene nanospheres and subsequently for sub-100-nm conjugated polymer nanoparticles and adenoviruses. The on-chip, individual nano-object resolution method presented here is a powerful approach to aid research and development in broad application areas such as medicine, chemistry, and biology.

Double-stranded RNA bending by AU-tract sequences.

Sequence-dependent structural deformations of the DNA double helix (dsDNA) have been extensively studied, where adenine tracts (A-tracts) provide a striking example for global bending in the molecule. However, in contrast to dsDNA, sequence-dependent structural features of dsRNA have received little attention. In this work, we demonstrate that the nucleotide sequence can induce a bend in a canonical Watson-Crick base-paired dsRNA helix. Using all-atom molecular dynamics simulations, we identified a sequence motif consisting of alternating adenines and uracils, or AU-tracts, that strongly bend the RNA double-helix. This finding was experimentally validated using atomic force microscopy imaging of dsRNA molecules designed to display macroscopic curvature via repetitions of phased AU-tract motifs. At the atomic level, this novel phenomenon originates from a localized compression of the dsRNA major groove and a large propeller twist at the position of the AU-tract. Moreover, the magnitude of the bending can be modulated by changing the length of the AU-tract. Altogether, our results demonstrate the possibility of modifying the dsRNA curvature by means of its nucleotide sequence, which may be exploited in the emerging field of RNA nanotechnology and might also constitute a natural mechanism for proteins to achieve recognition of specific dsRNA sequences.

Cell-to-cell and genome-to-genome variability of adenovirus transcription tuned by the cell cycle.

In clonal cultures, not all cells are equally susceptible to virus infection, and the mechanisms underlying this are poorly understood. Here, we developed image-based single-cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts using single-molecule RNA fluorescence in situ hybridization (FISH) and the incoming 5-ethynyl-2'-deoxycytidine (EdC)-tagged viral genomes using a copper(I)-catalyzed azide-alkyne cycloaddition (click) reaction. Surprisingly, expression of the immediate early gene E1A only moderately correlated with the number of viral genomes in the cell nucleus. Intranuclear genome-to-genome heterogeneity was found at the level of viral transcription and, in accordance, individual genomes exhibited heterogeneous replication activity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A gene expression and displayed increased correlation between E1A gene expression and viral genome copy numbers. The combined image-based single-molecule procedures described here are ideally suited to explore the cell-to-cell variability in viral gene expression in a range of different settings, including the innate immune response.

Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry.

Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating.

The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections.

Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon.

The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome Release at the Nuclear Pore Complex.

Adenoviruses (AdVs) cause respiratory, ocular, and gastrointestinal tract infection and inflammation in immunocompetent people and life-threatening disease upon immunosuppression. AdV vectors are widely used in gene therapy and vaccination. Incoming particles attach to nuclear pore complexes (NPCs) of post-mitotic cells, then rupture and deliver viral DNA (vDNA) to the nucleus or misdeliver to the cytosol. Our genome-wide RNAi screen in AdV-infected cells identified the RING-type E3 ubiquitin ligase Mind bomb 1 (Mib1) as a proviral host factor for AdV infection. Mib1 is implicated in Notch-Delta signaling, ciliary biogenesis, and RNA innate immunity. Mib1 depletion arrested incoming AdVs at NPCs. Induced expression of full-length but not ligase-defective Mib1 in knockout cells triggered vDNA uncoating from NPC-tethered virions, nuclear import, misdelivery of vDNA, and vDNA expression. Mib1 is an essential host factor for AdV uncoating in human cells, and it provides a new concept for licensing virion DNA delivery through the NPC.

Single entity resolution valving of nanoscopic species in liquids.

Investigating biological and synthetic nanoscopic species in liquids, at the ultimate resolution of single entity, is important in diverse fields1-5. Progress has been made6-10, but significant barriers need to be overcome such as the need for intense fields, the lack of versatility in operating conditions and the limited functionality in solutions of high ionic strength for biological applications. Here, we demonstrate switchable electrokinetic nanovalving able to confine and guide single nano-objects, including macromolecules, with sizes down to around 10 nanometres, in a lab-on-chip environment. The nanovalves are based on spatiotemporal tailoring of the potential energy landscape of nano-objects using an electric field, modulated collaboratively by wall nanotopography and by embedded electrodes in a nanochannel system. We combine nanovalves to isolate single entities from an ensemble, and demonstrate their guiding, confining, releasing and sorting. We show on-demand motion control of single immunoglobulin G molecules, quantum dots, adenoviruses, lipid vesicles, dielectric and metallic particles, suspended in electrolytes with a broad range of ionic strengths, up to biological levels. Such systems can enable nanofluidic, large-scale integration and individual handling of multiple entities in applications ranging from single species characterization and screening to in situ chemical or biochemical synthesis in continuous on-chip processes.

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control.

Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.

Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages.

The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors.IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors.

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection.

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein-protein VII. Two other Ad core proteins, V and X/µ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII-virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection.

Cell Cycle-Dependent Kinase Cdk9 Is a Postexposure Drug Target against Human Adenoviruses.

Human adenoviruses (HAdVs) infect respiratory, gastrointestinal, and urinary tracts and give rise to eye infections and epidemic keratoconjunctivitis (EKC). They persist in lymphoid tissue and cause morbidity and mortality in immunocompromised people. Treatments with significant postexposure efficacy are not available. Here, we report that inhibition of the cell cycle-dependent kinase 9 (Cdk9) by RNA interference, or the compound flavopiridol, blocked infections with HAdV-C2/5, EKC-causing HAdV-D8/37, and progeny formation in human corneal epithelial and cancer cells. Flavopiridol abrogated the production of the immediate early viral transactivating protein E1A without affecting nuclear import of viral DNA. In morphometric plaque assays, the compound exhibited antiviral efficacy in both pre- and postexposure regimens with therapeutic indexes exceeding 10. The study identifies Cdk9 as a postexposure drug target against adenovirus infections in vitro and suggests that the clinically tested anticancer drug flavopiridol is a candidate for treating adenoviral EKC or adenovirus emergence upon immune suppression.

Fluorescence Tracking of Genome Release during Mechanical Unpacking of Single Viruses.

Viruses package their genome in a robust protein coat to protect it during transmission between cells and organisms. In a reaction termed uncoating, the virus is progressively weakened during entry into cells. At the end of the uncoating process the genome separates, becomes transcriptionally active, and initiates the production of progeny. Here, we triggered the disruption of single human adenovirus capsids with atomic force microscopy and followed genome exposure by single-molecule fluorescence microscopy. This method allowed the comparison of immature (noninfectious) and mature (infectious) adenovirus particles. We observed two condensation states of the fluorescently labeled genome, a feature of the virus that may be related to infectivity. Beyond tracking the unpacking of virus genomes, this approach may find application in testing the cargo release of bioinspired delivery vehicles.