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.

Machine learning for cross-scale microscopy of viruses.

Despite advances in virological sciences and antiviral research, viruses continue to emerge, circulate, and threaten public health. We still lack a comprehensive understanding of how cells and individuals remain susceptible to infectious agents. This deficiency is in part due to the complexity of viruses, including the cell states controlling virus-host interactions. Microscopy samples distinct cellular infection stages in a multi-parametric, time-resolved manner at molecular resolution and is increasingly enhanced by machine learning and deep learning. Here we discuss how state-of-the-art artificial intelligence (AI) augments light and electron microscopy and advances virological research of cells. We describe current procedures for image denoising, object segmentation, tracking, classification, and super-resolution and showcase examples of how AI has improved the acquisition and analyses of microscopy data. The power of AI-enhanced microscopy will continue to help unravel virus infection mechanisms, develop antiviral agents, and improve viral vectors.

Engineered Human Adenoviruses of Species B and C Report Early, Intermediate Early, and Late Viral Gene Expression.

Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3, and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene. The 2A peptide induced ribosomal skipping during translation of the messenger RNA (mRNA) and gave rise to GFP from the corresponding viral promoters, as shown by immunoblotting and flow cytometry analyses of human and rodent cells. In human cells, both species B and C AdV exhibited highest reporter expression for fiber, followed by protein IX and lowest for E1A. Inoculation with either HAdV-C5 or -B3/35 viruses encoding protein IX- or fiber-GFP gave rise to higher GFP levels in hamster than mouse cells. Remarkably, despite rather low 2A ribosomal skipping efficiency of ∼50% for E1A-2A-GFP, protein IX-2A-GFP, and fiber-2A-GFP, unprocessed protein IX-2A-GFP and fiber-2A-GFP fusion proteins were efficiently incorporated into HAdV-B3 virions, respectively. These data indicate that the B3 C-termini of protein IX and fiber can be considered for retargeting engineered oncolytic or vaccination vectors, or for antigen display. The variable expression levels of transgenes from different subviral promoters may be used to improve oncolytic AdV vectors expressing therapeutic genes.

Adenovirus core protein V reinforces the capsid and enhances genome release from disrupted particles.

Out of the three core proteins in human adenovirus, protein V is believed to connect the inner capsid surface to the outer genome layer. Here, we explored mechanical properties and in vitro disassembly of particles lacking protein V (Ad5-ΔV). Ad5-ΔV particles were softer and less brittle than the wild-type ones (Ad5-wt), but they were more prone to release pentons under mechanical fatigue. In Ad5-ΔV, core components did not readily diffuse out of partially disrupted capsids, and the core appeared more condensed than in Ad5-wt. These observations suggest that instead of condensing the genome, protein V antagonizes the condensing action of the other core proteins. Protein V provides mechanical reinforcement and facilitates genome release by keeping DNA connected to capsid fragments that detach during disruption. This scenario is in line with the location of protein V in the virion and its role in Ad5 cell entry.

Label-free microscopy for virus infections.

Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques.

Vascular dysfunction in COVID-19 patients: update on SARS-CoV-2 infection of endothelial cells and the role of long non-coding RNAs.

Although COVID-19 is primarily a respiratory disease, it may affect also the cardiovascular system. COVID-19 patients with cardiovascular disorder (CVD) develop a more severe disease course with a significantly higher mortality rate than non-CVD patients. A common denominator of CVD is the dysfunction of endothelial cells (ECs), increased vascular permeability, endothelial-to-mesenchymal transition, coagulation, and inflammation. It has been assumed that clinical complications in COVID-19 patients suffering from CVD are caused by SARS-CoV-2 infection of ECs through the angiotensin-converting enzyme 2 (ACE2) receptor and the cellular transmembrane protease serine 2 (TMPRSS2) and the consequent dysfunction of the infected vascular cells. Meanwhile, other factors associated with SARS-CoV-2 entry into the host cells have been described, including disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), the C-type lectin CD209L or heparan sulfate proteoglycans (HSPG). Here, we discuss the current data about the putative entry of SARS-CoV-2 into endothelial and smooth muscle cells. Furthermore, we highlight the potential role of long non-coding RNAs (lncRNAs) affecting vascular permeability in CVD, a process that might exacerbate disease in COVID-19 patients.

High-content, arrayed compound screens with rhinovirus, influenza A virus and herpes simplex virus infections.

Viruses are genetically and structurally diverse, and outnumber cells by orders of magnitude. They can cause acute and chronic infections, suppress, or exacerbate immunity, or dysregulate survival and growth of cells. To identify chemical agents with pro- or antiviral effects we conducted arrayed high-content image-based multi-cycle infection screens of 1,280 mainly FDA-approved compounds with three human viruses, rhinovirus (RV), influenza A virus (IAV), and herpes simplex virus (HSV) differing in genome organization, composition, presence of an envelope, and tropism. Based on Z'-factors assessing screening quality and Z-scores ranking individual compounds, we identified potent inhibitors and enhancers of infection: the RNA mutagen 5-Azacytidine against RV-A16; the broad-spectrum antimycotic drug Clotrimazole inhibiting IAV-WSN; the chemotherapeutic agent Raltitrexed blocking HSV-1; and Clobetasol enhancing HSV-1. Remarkably, the topical antiseptic compound Aminacrine, which is clinically used against bacterial and fungal agents, inhibited all three viruses. Our data underscore the versatility and potency of image-based, full cycle virus propagation assays in cell-based screenings for antiviral agents.

Methylene blue, Mycophenolic acid, Posaconazole, and Niclosamide inhibit SARS-CoV-2 Omicron variant BA.1 infection of human airway epithelial organoids.

Sublineages of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron variants continue to amass mutations in the spike (S) glycoprotein, which leads to immune evasion and rapid spread of the virus across the human population. Here we demonstrate the susceptibility of the Omicron variant BA.1 (B.1.1.529.1) to four repurposable drugs, Methylene blue (MB), Mycophenolic acid (MPA), Posaconazole (POS), and Niclosamide (Niclo) in post-exposure treatments of primary human airway cell cultures. MB, MPA, POS, and Niclo are known to block infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) with the Wuhan strain, and four variants of concern (VoC), Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2) (Weiss et al., 2021, Murer et al., 2022). Our results here not only reinforce the broad anti-coronavirus effects of MB, MPA, POS and Niclo, but also demonstrate that the Omicron variant BA.1 (B.1.1.529.1) sheds infectious virus from HAEEC over at least 15 d, and maintains both intracellular and extracellular viral genomic RNA without overt toxicity, suggesting viral persistence. The data emphasize the potential of repurposable drugs against COVID-19.

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.

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.

The RGD-binding integrins αvß6 and αvß8 are receptors for mouse adenovirus-1 and -3 infection.

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvß6 and αvß8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mß6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvß6-positive CMT-93 cells, whereas mß8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvß8-positive M000216 cells. Soluble integrin αvß6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvß6/αvß8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvß6/ß8, where the distal leucine residue dips into a hydrophobic pocket of ß6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvß6/ß8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvß6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.

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.

Two years into COVID-19 - Lessons in SARS-CoV-2 and a perspective from papers in FEBS Letters.

The 2019 outbreak of coronavirus disease (COVID-19) in Wuhan (Hubei province of China) has given rise to a pandemic spread of virus, more than 240 million incidences and a death toll larger than 5 million people. COVID-19 has set off large efforts in research, therapy and patient care, as well as public and private debates in every imaginable form. A number of scientists used the publication platforms provided by the Federation of the European Biochemical Societies (FEBS) to present their research data, reviews, opinions and other contributions relating to COVID-19 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, I highlight the recent COVID-19 papers which have been published and collected in a Virtual Issue in FEBS Letters, and discuss their implications towards understanding the molecular, biochemical and cellular mechanisms of SARS-CoV-2 infections, vaccine development and antiviral discovery strategies.

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.

Microscopy deep learning predicts virus infections and reveals mechanics of lytic-infected cells.

Imaging across scales reveals disease mechanisms in organisms, tissues, and cells. Yet, particular infection phenotypes, such as virus-induced cell lysis, have remained difficult to study. Here, we developed imaging modalities and deep learning procedures to identify herpesvirus and adenovirus (AdV) infected cells without virus-specific stainings. Fluorescence microscopy of vital DNA-dyes and live-cell imaging revealed learnable virus-specific nuclear patterns transferable to related viruses of the same family. Deep learning predicted two major AdV infection outcomes, non-lytic (nonspreading) and lytic (spreading) infections, up to about 20 hr prior to cell lysis. Using these predictive algorithms, lytic and non-lytic nuclei had the same levels of green fluorescent protein (GFP)-tagged virion proteins but lytic nuclei enriched the virion proteins faster, and collapsed more extensively upon laser-rupture than non-lytic nuclei, revealing impaired mechanical properties of lytic nuclei. Our algorithms may be used to infer infection phenotypes of emerging viruses, enhance single cell biology, and facilitate differential diagnosis of non-lytic and lytic infections.

The HSV-1 Transcription Factor ICP4 Confers Liquid-Like Properties to Viral Replication Compartments.

Herpes Simplex Virus Type-1 (HSV-1) forms progeny in the nucleus within distinct membrane-less inclusions, the viral replication compartments (VRCs), where viral gene expression, DNA replication, and packaging occur. The way in which the VRCs maintain spatial integrity remains unresolved. Here, we demonstrate that the essential viral transcription factor ICP4 is an intrinsically disordered protein (IDP) capable of driving protein condensation and liquid-liquid phase separation (LLPS) in transfected cells. Particularly, ICP4 forms nuclear liquid-like condensates in a dose- and time-dependent manner. Fluorescence recovery after photobleaching (FRAP) assays revealed rapid exchange rates of EYFP-ICP4 between phase-separated condensates and the surroundings, akin to other viral IDPs that drive LLPS. Likewise, HSV-1 VRCs revealed by EYFP-tagged ICP4 retained their liquid-like nature, suggesting that they are phase-separated condensates. Individual VRCs homotypically fused when reaching close proximity and grew over the course of infection. Together, the results of this study demonstrate that the HSV-1 transcription factor ICP4 has characteristics of a viral IDP, forms condensates in the cell nucleus by LLPS, and can be used as a proxy for HSV-1 VRCs with characteristics of liquid-liquid phase-separated condensates.

Adenovirus - a blueprint for gene delivery.

A central quest in gene therapy and vaccination is to achieve effective and long-lasting gene expression at minimal dosage. Adenovirus vectors are widely used therapeutics and safely deliver genes into many cell types. Adenoviruses evolved to use elaborate trafficking and particle deconstruction processes, and efficient gene expression and progeny formation. Here, we discuss recent insights into how human adenoviruses deliver their double-stranded DNA genome into cell nuclei, and effect lytic cell killing, non-lytic persistent infection or vector gene expression. The mechanisms underlying adenovirus entry, uncoating, nuclear transport and gene expression provide a blueprint for the emerging field of synthetic virology, where artificial virus-like particles are evolved to deliver therapeutic payload into human cells without viral proteins and genomes.