Changes in adenoviral chromatin organization precede early gene activation upon infection.

Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60-70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.

TBK1 is part of a galectin 8 dependent membrane damage recognition complex and drives autophagy upon Adenovirus endosomal escape.

Intracellular pathogens cause membrane distortion and damage as they enter host cells. Cells perceive these membrane alterations as danger signals and respond by activating autophagy. This response has primarily been studied during bacterial invasion, and only rarely in viral infections. Here, we investigate the cellular response to membrane damage during adenoviral entry. Adenoviruses and their vector derivatives, that are an important vaccine platform against SARS-CoV-2, enter the host cell by endocytosis followed by lysis of the endosomal membrane. We previously showed that cells mount a locally confined autophagy response at the site of endosomal membrane lysis. Here we describe the mechanism of autophagy induction: endosomal membrane damage activates the kinase TBK1 that accumulates in its phosphorylated form at the penetration site. Activation and recruitment of TBK1 require detection of membrane damage by galectin 8 but occur independently of classical autophagy receptors or functional autophagy. Instead, TBK1 itself promotes subsequent autophagy that adenoviruses need to take control of. Deletion of TBK1 reduces LC3 lipidation during adenovirus infection and restores the infectivity of an adenovirus mutant that is restricted by autophagy. By comparing adenovirus-induced membrane damage to sterile lysosomal damage, we implicate TBK1 in the response to a broader range of types of membrane damage. Our study thus highlights an important role for TBK1 in the cellular response to adenoviral endosome penetration and places TBK1 early in the pathway leading to autophagy in response to membrane damage.

Nup358 and Transportin 1 Cooperate in Adenoviral Genome Import.

Nuclear import of viral genomes is an important step during the life cycle of adenoviruses (AdV), requiring soluble cellular factors as well as proteins of the nuclear pore complex (NPC). We addressed the role of the cytoplasmic nucleoporin Nup358 during adenoviral genome delivery by performing depletion/reconstitution experiments and time-resolved quantification of adenoviral genome import. Nup358-depleted cells displayed reduced efficiencies of nuclear import of adenoviral genomes, and the nuclear import receptor transportin 1 became rate limiting under these conditions. Furthermore, we identified a minimal N-terminal region of Nup358 that was sufficient to compensate for the import defect. Our data support a model where Nup358 functions as an assembly platform that promotes the formation of transport complexes, allowing AdV to exploit a physiological protein import pathway for accelerated transport of its DNA.IMPORTANCE Nuclear import of viral genomes is an essential step to initiate productive infection for several nuclear replicating DNA viruses. On the other hand, DNA is not a physiological nuclear import substrate; consequently, viruses have to exploit existing physiological transport routes. Here, we show that adenoviruses use the nucleoporin Nup358 to increase the efficiency of adenoviral genome import. In its absence, genome import efficiency is reduced and the transport receptor transportin 1 becomes rate limiting. We show that the N-terminal half of Nup358 is sufficient to drive genome import and identify a transportin 1 binding region. In our model, adenovirus genome import exploits an existing protein import pathway and Nup358 serves as an assembly platform for transport complexes.

In Vivo Labelling of Adenovirus DNA Identifies Chromatin Anchoring and Biphasic Genome Replication.

Adenoviruses are DNA viruses with a lytic infection cycle. Following the fate of incoming as well as recently replicated genomes during infections is a challenge. In this study, we used the ANCHOR3 technology based on a bacterial partitioning system to establish a versatile in vivo imaging system for adenoviral genomes. The system allows the visualization of both individual incoming and newly replicated genomes in real time in living cells. We demonstrate that incoming adenoviral genomes are attached to condensed cellular chromatin during mitosis, facilitating the equal distribution of viral genomes in daughter cells after cell division. We show that the formation of replication centers occurs in conjunction with in vivo genome replication and determine replication rates. Visualization of adenoviral DNA revealed that adenoviruses exhibit two kinetically distinct phases of genome replication. Low-level replication occurred during early replication, while high-level replication was associated with late replication phases. The transition between these phases occurred concomitantly with morphological changes of viral replication compartments and with the appearance of virus-induced postreplication (ViPR) bodies, identified by the nucleolar protein Mybbp1A. Taken together, our real-time genome imaging system revealed hitherto uncharacterized features of adenoviral genomes in vivo The system is able to identify novel spatiotemporal aspects of the adenovirus life cycle and is potentially transferable to other viral systems with a double-stranded DNA phase.IMPORTANCE Viruses must deliver their genomes to host cells to ensure replication and propagation. Characterizing the fate of viral genomes is crucial to understand the viral life cycle and the fate of virus-derived vector tools. Here, we integrated the ANCHOR3 system, an in vivo DNA-tagging technology, into the adenoviral genome for real-time genome detection. ANCHOR3 tagging permitted the in vivo visualization of incoming genomes at the onset of infection and of replicated genomes at late phases of infection. Using this system, we show viral genome attachment to condensed host chromosomes during mitosis, identifying this mechanism as a mode of cell-to-cell transfer. We characterize the spatiotemporal organization of adenovirus replication and identify two kinetically distinct phases of viral genome replication. The ANCHOR3 system is the first technique that allows the continuous visualization of adenoviral genomes during the entire virus life cycle, opening the way for further in-depth study.

Rapid Adenovirus typing method for species identification.

Adenoviruses are characterized by a large variability, reflected by their classification in species A to G. Certain species, eg A and C, could be associated with increased clinical severity, both in immunocompetent and immunocompromised hosts suggesting that in some instances species identification provides clinically relevant information. Here we designed a novel "pVI rapid typing method" to obtain quick, simple and cost effective species assignment for Adenoviruses, thanks to combined fusion temperature (Tm) and amplicon size analysis. Rapid typing results were compared to Sanger sequencing in the hexon gene for 140 Adenovirus-positive clinical samples included in the Typadeno study. Species A and C could be identified with a 100% positive predictive value, thus confirming the value of this simple typing method.

Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid.

Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.

Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer.

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507-19. ©2016 AACR.

Using AlphaScreen(®) to Identify Small-Molecule Inhibitors Targeting a Conserved Host-Pathogen Interaction.

AlphaScreen(®) is a technology particularly suitable for bi-molecular inhibitor screening assays, e.g. using protein-protein interactions with purified recombinant proteins. Each binding partner of the bi-molecular interaction is coupled either to donor or to acceptor beads. The technology is based on the quantifiable transfer of oxygen singlets from donor to acceptor microbeads brought together by a specific interaction between the partners. We identified the conserved interaction between WW domains of cellular ubiquitin ligases of the Nedd4 family and a short peptide motif (PPxY) present in several structural and non-structural viral proteins as a potential drug target. Using an AlphaScreen assay recapitulating the interaction between Nedd4.2 and the PPxY motif of the adenoviral capsid protein VI, we screened a library of small molecules and identified specific inhibitors of this interaction.

Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot.

HIV-1 Tat is efficiently secreted by HIV-1-infected or Tat-transfected cells. Accordingly, Tat concentrations in the nanomolar range have been measured in the sera of HIV-1-infected patients, and this protein acts as a viral toxin on bystander cells. Nevertheless, assaying Tat concentration in media or sera is not that straightforward because extracellular Tat is unstable and particularly sensitive to oxidation. Moreover, most anti-Tat antibodies display limited affinity. Here, we describe methods to quantify extracellular Tat using a sandwich ELISA or Western blotting when Tat is secreted by suspension or adherent cells, respectively. In both cases it is important to capture exported Tat using antibodies before any Tat oxidation occurs; otherwise it will become denatured and unreactive toward antibodies.

The amphipathic helix of adenovirus capsid protein VI contributes to penton release and postentry sorting.

UNLABELLED: Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE: In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle.

Transcriptional activation of the adenoviral genome is mediated by capsid protein VI.

Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins.

The ins and outs of HIV-1 Tat.

HIV-1 encodes for the small basic protein Tat (86-101 residues) that drastically enhances the efficiency of viral transcription. The mechanism enabling Tat nuclear import is not yet clear, but studies using reporter proteins fused to the Tat basic domain indicate that Tat could reach the nucleus by passive diffusion. Tat also uses an unusual transcellular transport pathway. The first step of this pathway involves high-affinity binding of Tat to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P(2)), a phospholipid that is concentrated in the inner leaflet of the plasma membrane and enables Tat recruitment at this level. Tat then crosses the plasma membrane to reach the outside medium. Although unconventional, Tat secretion by infected cells is highly active, and export is the major destination for HIV-1 Tat. Secreted Tat can bind to a variety of cell types using several different receptors. Most of them will allow Tat endocytosis. Upon internalization, low endosomal pH triggers a conformational change in Tat that results in membrane insertion. Later steps of Tat translocation to the target-cell cytosol are assisted by Hsp90, a general cytosolic chaperone. Cytosolic Tat can trigger various cell responses. Indeed, accumulating evidence suggests that extracellular Tat acts as a viral toxin that affects the biological activity of different cell types and has a key role in acquired immune-deficiency syndrome development. This review focuses on some of the recently identified molecular details underlying the unusual transcellular transport pathway used by Tat, such as the role of the single Trp in Tat for its membrane insertion and translocation.

Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells.

Human immunodeficiency virus type 1 (HIV-1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV-1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV-1-infected primary CD4(+) T-cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P(2) molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane-embedded PI(4,5)P(2) only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P(2)-mediated recruitment of cellular proteins. Tat-PI(4,5)P(2) interaction is strictly required for Tat secretion, a process that is very efficient, as approximately 2/3 of Tat are exported by HIV-1-infected cells during their lifespan. The function of extracellular Tat in HIV-1 infection might thus be more significant than earlier thought.

HIV-1 Tat is unconventionally secreted through the plasma membrane.

The Tat protein is required for efficient HIV-1 (human immunodeficiency virus type 1) transcription. Moreover, Tat is secreted by infected cells, and circulating Tat can affect several cell types, thereby contributing to HIV-1 pathogenesis. We monitored Tat secretion by transfected CD4+ T-cells. A Tat chimaera carrying an N-glycosylation site did not become glycosylated when expressed in cells, while the chimaera was glycosylated when mechanically introduced into purified microsomes. These data indicate that secreted Tat does not transit through the endoplasmic reticulum. The use of pharmacological inhibitors indicated that the Tat secretion pathway is unusual compared with previously identified unconventional secretion routes and does not involve intracellular organelles. Moreover, cell incubation at 16 degrees C inhibited Tat secretion and caused its accumulation at the plasma membrane, suggesting that secretion takes place at this level.

Intracellular pathway of Onconase that enables its delivery to the cytosol.

Onconase is an RNase with a very specific property because it is selectively toxic to transformed cells. This toxin is thought to recognize cell surface receptors, and the protection conferred by metabolic poisons against Onconase toxicity indicated that this RNase relies on endocytic uptake to kill cells. Nevertheless, its internalization pathway has yet to be unraveled. We show here that Onconase enters cells using AP-2/clathrin-mediated endocytosis. It is then routed, together with transferrin, to the receptor recycling compartment. Increasing the Onconase concentration in this structure using tetanus toxin light chain expression enhanced Onconase toxicity, indicating that recycling endosomes are a key compartment for Onconase cytosolic delivery. This intracellular destination is specific to Onconase because other (and much less toxic) RNases follow the default pathway to late endosomes/lysosomes. Drugs neutralizing endosomal pH increased Onconase translocation efficiency from purified endosomes during cell-free translocation assays by preventing Onconase dissociation from its receptor at endosomal pH. Consistently, endosome neutralization enhanced Onconase toxicity up to 100-fold. Onconase translocation also required cytosolic ATP hydrolysis. This toxin therefore shows an unusual entry process that relies on clathrin-dependent endocytic uptake and then neutralization of low endosomal pH for efficient translocation from the endosomal lumen to the cytosol.

In vivo homodimerisation of HTLV-1 Gag and MA gives clues to the retroviral capsid and TM envelope protein arrangement.

During retroviral particle formation, the capsid precursors (Gag) associate with the cell membrane via their matrix (MA) domain to form viral assembling particles. After budding, Gag and its proteolytically matured MA, form a shell in the released immature and mature particles, respectively. Although the arrangement of Gag domains in vitro and their radial organisation in retroviral particles have been extensively studied, little is known concerning Gag inter-subunit interactions in authentic retroviruses. We report that human T-cell leukemia virus type 1 Gag homodimerises in the cell via a disulphide bonding at cysteine 61 in the MA domain. Most Gags are homodimeric after budding and MAs are also dimeric in mature authentic virions. Molecular modelling of the MA domain indicates that non-covalent interactions at the MA dimer interface may also be important for Gag (and MA) dimerisation. In addition, all amino acids previously reported to be involved in MA-transmembrane (TM) interactions are located on the MA face opposite to the dimer interface. The model reveals that homodimerisation is compatible with a hexameric network of Gag and MA dimers that look like the hexameric networks observed for other retroviruses. These data, together with previous studies, lead us to propose a supra-molecular arrangement model in which the transmembrane glycoproteins of the virion envelope are anchored in a hexameric cage hole formed by the MA.

The ability of chloroquine to prevent tat-induced cytokine secretion by monocytes is implicated in its in vivo anti-human immunodeficiency virus type 1 activity.

Hydroxychloroquine at 1 microM reduces the load of human immunodeficiency virus type 1 (HIV-1) in patients, whereas chloroquine (CQ) concentrations above 3 microM are required for inhibition of HIV-1 replication in peripheral blood mononuclear cells. Exogenous HIV-1 Tat reaches the cytosol of T cells by using low endosomal pH, and endosome neutralization by CQ prevents Tat from entering and affecting T cells. We show here that 0.6 microM CQ inhibits cytokine secretion induced by Tat in monocytes without affecting lipopolysaccharide-triggered cytokine release. This finding suggests that the in vivo anti-HIV-1 effect of CQ results not from a direct effect on the infected cell but rather from the capacity of CQ to prevent Tat from perturbing the cytokine balance.

HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses.

The HIV-1 Tat protein is secreted by infected cells. Extracellular Tat can affect bystander uninfected T cells and induce numerous biological responses such as apoptosis and cytokine secretion. Tat is likely involved in several immune disorders during AIDS. Nevertheless, it is not known whether Tat triggers cell responses directly upon binding to signaling receptors at the plasma membrane or after delivery to the cytosol. The pathway that enables Tat to reach the cytosol is also unclear. Here we visualized Tat within T-cell-coated pits and endosomes. Moreover, inhibitors of clathrin/AP-2-mediated uptake such as chlorpromazine, activated RhoA, or dominant-negative mutants of Eps15, intersectin, dynamin, or rab5 impaired Tat delivery to the cytosol by preventing its endocytosis. Molecules neutralizing low endosomal pH or Hsp90 inhibitors abolished Tat entry at a later stage by blocking its endosomal translocation, as directly shown using a cell-free translocation assay. Finally, endosomal pH neutralization prevented Tat from inducing T-cell responses such as NF-kappaB activation, apoptosis, and interleukin secretion, indicating that cytosolic delivery is required for Tat signaling. Hence, Tat enters T cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH-induced and Hsp90-assisted endosomal translocation. Cell responses are then induced from the cytosol.