Assessing the influence of small structural modifications in simple DNA-based nanostructures on their role as drug nanocarriers.

DNA nanotechnology leverages Watson-Crick-Franklin base-pairing interactions to build complex DNA-based nanostructures (DNS). Due to DNA specific self-assembly properties, DNS can be designed with a total control of their architecture, which has been demonstrated to have an impact on the overall DNS features. Indeed, structural properties such as the shape, size and flexibility of DNS can influence their biostability as well as their ability to internalise into cells. We present here two series of simple DNS with small and precise variations related to their length or flexibility and study the influence that these structural changes have on their overall properties as drug nanocarriers. Results indicate that shorter and more flexible DNS present higher stability towards nuclease degradation. These structural changes also have a certain effect on their cell internalisation ability and drug release rate. Consequently, drug-loaded DNS cytotoxicity varies according to the design, with lower cell viability values obtained in the DNS exhibiting faster drug release and larger cell interaction rates. In summary, small changes in the structure of simple DNS can have an influence on their overall capabilities as drug nanocarriers. The effects reported here could guide the design of simple DNS for future therapeutic uses.

Lipin-2 regulates the antiviral and anti-inflammatory responses to interferon.

Interferons (IFN) are crucial antiviral and immunomodulatory cytokines that exert their function through the regulation of a myriad of genes, many of which are not yet characterized. Here, we reveal that lipin-2, a phosphatidic acid phosphatase whose mutations produce an autoinflammatory syndrome known as Majeed syndrome in humans, is regulated by IFN in a STAT-1-dependent manner. Lipin-2 inhibits viral replication both in vitro and in vivo. Moreover, lipin-2 also acts as a regulator of inflammation in a viral context by reducing the signaling through TLR3 and the generation of ROS and release of mtDNA that ultimately activate the NLRP3 inflammasome. Inhibitors of mtDNA release from mitochondria restrict IL-1ß production in lipin-2-deficient animals in a model of viral infection. Finally, analyses of databases from COVID-19 patients show that LPIN2 expression levels negatively correlate with the severity of the disease. Overall, these results uncover novel regulatory mechanisms of the IFN response driven by lipin-2 and open new perspectives for the future management of patients with LPIN2 mutations.

Discovery of the first PD-1 ligand encoded by a pathogen.

Large double-stranded DNA viruses deploy multiple strategies to subvert host immune defenses. Some of these tactics are mediated by viral gene products acquired by horizontal gene transfer from the corresponding hosts and shaped throughout evolution. The programmed death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, play a pivotal role attenuating T-cell responses and regulating immune tolerance. In this study, we report the first functional PD-L1 homolog gene (De2) found in a pathogen. De2, captured by a γ-herpesvirus from its host during co-evolution around 50 million years ago, encodes a cell-surface glycoprotein that interacts with high affinity and stability with host PD-1. We also find that mutations evolved by the viral protein result in a significant loss of its ability to interact in cis with CD80, an interaction that for PD-L1:CD80 has been reported to block PD-1 inhibitory pathways. Furthermore, we demonstrate that the viral protein strongly inhibits T-cell signaling. Our observations suggest that PD-L1 homologs may enable viruses to evade T cell responses, favor their replication, and prevent excessive tissue damage. Altogether, our findings reveal a novel viral immunosuppressive strategy and highlight the importance of the modulation of the PD-1/PD-L1 axis during viral infections.

Magnetogenetics: remote activation of cellular functions triggered by magnetic switches.

During the last decade, the possibility to remotely control intracellular pathways using physical tools has opened the way to novel and exciting applications, both in basic research and clinical applications. Indeed, the use of physical and non-invasive stimuli such as light, electricity or magnetic fields offers the possibility of manipulating biological processes with spatial and temporal resolution in a remote fashion. The use of magnetic fields is especially appealing for in vivo applications because they can penetrate deep into tissues, as opposed to light. In combination with magnetic actuators they are emerging as a new instrument to precisely manipulate biological functions. This approach, coined as magnetogenetics, provides an exclusive tool to study how cells transform mechanical stimuli into biochemical signalling and offers the possibility of activating intracellular pathways connected to temperature-sensitive proteins. In this review we provide a critical overview of the recent developments in the field of magnetogenetics. We discuss general topics regarding the three main components for magnetic field-based actuation: the magnetic fields, the magnetic actuators and the cellular targets. We first introduce the main approaches in which the magnetic field can be used to manipulate the magnetic actuators, together with the most commonly used magnetic field configurations and the physicochemical parameters that can critically influence the magnetic properties of the actuators. Thereafter, we discuss relevant examples of magneto-mechanical and magneto-thermal stimulation, used to control stem cell fate, to activate neuronal functions, or to stimulate apoptotic pathways, among others. Finally, although magnetogenetics has raised high expectations from the research community, to date there are still many obstacles to be overcome in order for it to become a real alternative to optogenetics for instance. We discuss some controversial aspects related to the insufficient elucidation of the mechanisms of action of some magnetogenetics constructs and approaches, providing our opinion on important challenges in the field and possible directions for the upcoming years.

Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion.

Viral infections are controlled, and very often cleared, by activated T lymphocytes. The inducible co-stimulator (ICOS) mediates its functions by binding to its ligand ICOSL, enhancing T-cell activation and optimal germinal center (GC) formation. Here, we show that ICOSL is heavily downmodulated during infection of antigen-presenting cells by different herpesviruses. We found that, in murine cytomegalovirus (MCMV), the immunoevasin m138/fcr-1 physically interacts with ICOSL, impeding its maturation and promoting its lysosomal degradation. This viral protein counteracts T-cell responses, in an ICOS-dependent manner, and limits virus control during the acute MCMV infection. Additionally, we report that blockade of ICOSL in MCMV-infected mice critically regulates the production of MCMV-specific antibodies due to a reduction of T follicular helper and GC B cells. Altogether, these findings reveal a novel mechanism evolved by MCMV to counteract adaptive immune surveillance, and demonstrates a role of the ICOS:ICOSL axis in the host defense against herpesviruses.

Divergent Traits and Ligand-Binding Properties of the Cytomegalovirus CD48 Gene Family.

The genesis of gene families by the capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a cell surface molecule that interacts via its N-terminal immunoglobulin (Ig) domain with the cell surface receptor 2B4 (CD244), regulating leukocyte cytotoxicity. We previously reported the presence of five CD48 homologs (vCD48s) in two related cytomegaloviruses, and demonstrated that one of them, A43, binds 2B4 and acts as a soluble CD48 decoy receptor impairing NK cell function. Here, we have characterized the rest of these vCD48s. We show that they are highly glycosylated proteins that display remarkably distinct features: divergent biochemical properties, cellular locations, and temporal expression kinetics. In contrast to A43, none of them interacts with 2B4. Consistent with this, molecular modeling of the N-terminal Ig domains of these vCD48s evidences notable changes as compared to CD48, suggesting that they interact with alternative targets. Accordingly, we demonstrate that one of them, S30, tightly binds CD2, a crucial T- and NK-cell adhesion and costimulatory molecule. Thus, our findings show how a key host immune receptor gene captured by a virus can be subsequently remodeled to evolve new immunoevasins with altered binding properties.

Subversion of natural killer cell responses by a cytomegalovirus-encoded soluble CD48 decoy receptor.

Throughout evolution, cytomegaloviruses (CMVs) have been capturing genes from their hosts, employing the derived proteins to evade host immune defenses. We have recently reported the presence of a number of CD48 homologs (vCD48s) encoded by different pathogenic viruses, including several CMVs. However, their properties and biological relevance remain as yet unexplored. CD48, a cosignaling molecule expressed on the surface of most hematopoietic cells, modulates the function of natural killer (NK) and other cytotoxic cells by binding to its natural ligand 2B4 (CD244). Here, we have characterized A43, the vCD48 exhibiting the highest amino acid sequence identity with host CD48. A43, which is encoded by owl monkey CMV, is a soluble molecule released from the cell after being proteolytically processed through its membrane proximal region. A43 is expressed with immediate-early kinetics, yielding a protein that is rapidly detected in the supernatant of infected cells. Remarkably, surface plasmon resonance assays revealed that this viral protein binds to host 2B4 with high affinity and slow dissociation rates. We demonstrate that soluble A43 is capable to abrogate host CD48:2B4 interactions. Moreover, A43 strongly binds to human 2B4 and prevents 2B4-mediated NK-cell adhesion to target cells, therefore reducing the formation of conjugates and the establishment of immunological synapses between human NK cells and CD48-expressing target cells. Furthermore, in the presence of this viral protein, 2B4-mediated cytotoxicity and IFN-γ production by NK cells are severely impaired. In summary, we propose that A43 may serve as a functional soluble CD48 decoy receptor by binding and masking 2B4, thereby impeding effective NK cell immune control during viral infections. Thus, our findings provide a novel example of the immune evasion strategies developed by viruses.

Viral CD229 (Ly9) homologs as new manipulators of host immunity.

The signaling lymphocytic activation molecule family (SLAMF) of receptors plays crucial roles during innate and adaptive immune responses. The SLAMF member CD229 (Ly9, SLAMF3) is a homophilic receptor predominantly expressed on the surface of B and T cells. CD229 acts as a cosignaling molecule, regulating lymphocyte homoeostasis and activation. To promote viral replication and survival in their hosts, viruses have developed sophisticated mechanisms to combat and avoid immune surveillance. Many of these strategies rely on host defense genes captured during the process of virus-host coevolution. In particular, large DNA viruses devote a wide range of proteins to interfere with almost every host immune pathway. Given that CD229 is critically involved in regulating immune responses, it is not surprising that viruses have designed tactics to mimic or interfere with this receptor. The discovery, in recent years, that some viruses have hijacked CD229 genes from their hosts, incorporating them as an integral part of their genomes, or have evolved proteins to directly target CD229, indicates that this is the case. While it is still an emerging area of research, the present review discusses these viral molecules and their potential in immune modulation. A more detailed understanding of the mechanisms of action and the functional implications of these new viral CD229 mimics may not only provide seminal information on viral immune evasion mechanisms but also, unveil unrecognized aspects of CD229 immune functions.

A Prominent Role of the Human Cytomegalovirus UL8 Glycoprotein in Restraining Proinflammatory Cytokine Production by Myeloid Cells at Late Times during Infection.

Human cytomegalovirus (HCMV) persistence in infected individuals relies on a plethora of mechanisms to efficiently reduce host immune responses. To that end, HCMV uses a variety of gene products, some of which have not been identified yet. Here we characterized the UL8 gene, which consists of two exons, sharing the first with the HCMV RL11 family member UL7 UL8 is a transmembrane protein with an N-terminal immunoglobulin (Ig)-like domain in common with UL7 but with an extended stalk and a distinctive cytoplasmic tail. The UL8 open reading frame gives rise to a heavily glycosylated protein predominantly expressed on the cell surface, from where it can be partially endocytosed and subsequently degraded. Infections with UL8-tagged viruses indicated that UL8 was synthesized with late-phase kinetics. By virtue of its highly conserved Ig-like domain, this viral protein interacted with a surface molecule present on activated neutrophils. Notably, when ectopically expressed in THP-1 myeloid cells, UL8 was able to significantly reduce the production of a variety of proinflammatory cytokines. Mutations in UL8 indicated that this functional effect was mediated by the cell surface expression of its Ig-like domain. To investigate the impact of the viral protein in the infection context, we engineered HCMVs lacking the UL8 gene and demonstrated that UL8 decreases the release of a large number of proinflammatory factors at late times after infection of THP-1 cells. Our data indicate that UL8 may exert an immunosuppressive role key for HCMV survival in the host.IMPORTANCE HCMV is a major pathogen that causes life-threatening diseases and disabilities in infected newborns and immunocompromised individuals. Containing one of the largest genomes among all reported human viruses, HCMV encodes an impressive repertoire of gene products. However, the functions of a large proportion of them still remain unknown, a fact that complicates the design of new therapeutic approaches to prevent or treat HCMV-associated diseases. In this report, we have conducted an extensive study of UL8, one of the previously uncharacterized HCMV open reading frames. We found that the UL8 protein is expressed at late times postinfection and utilized by HCMV to reduce the production of proinflammatory factors by infected myeloid cells. Thus, the work presented here points to a key role of UL8 as a novel HCMV immune modulator capable of restraining host antiviral defenses.

Genomic Programming of Human Neonatal Dendritic Cells in Congenital Systemic and In Vitro Cytomegalovirus Infection Reveal Plastic and Robust Immune Pathway Biology Responses.

Neonates and especially premature infants are highly susceptible to infection but still can have a remarkable resilience that is poorly understood. The view that neonates have an incomplete or deficient immune system is changing. Human neonatal studies are challenging, and elucidating host protective responses and underlying cognate pathway biology, in the context of viral infection in early life, remains to be fully explored. In both resource rich and poor settings, human cytomegalovirus (HCMV) is the most common cause of congenital infection. By using unbiased systems analyses of transcriptomic resources for HCMV neonatal infection, we find the systemic response of a preterm congenital HCMV infection, involves a focused IFN regulatory response associated with dendritic cells. Further analysis of transcriptional-programming of neonatal dendritic cells in response to HCMV infection in culture revealed an early dominant IFN-chemokine regulatory subnetworks, and at later times the plasticity of pathways implicated in cell-cycle control and lipid metabolism. Further, we identify previously unknown suppressed networks associated with infection, including a select group of GPCRs. Functional siRNA viral growth screen targeting 516-GPCRs and subsequent validation identified novel GPCR-dependent antiviral (ADORA1) and proviral (GPR146, RGS16, PTAFR, SCTR, GPR84, GPR85, NMUR2, FZ10, RDS, CCL17, and SORT1) roles. By contrast a gene family cluster of protocadherins is significantly differentially induced in neonatal cells, suggestive of possible immunomodulatory roles. Unexpectedly, programming responses of adult and neonatal dendritic cells, upon HCMV infection, demonstrated comparable quantitative and qualitative responses showing that functionally, neonatal dendritic cell are not overly compromised. However, a delay in responses of neonatal cells for IFN subnetworks in comparison with adult-derived cells are notable, suggestive of subtle plasticity differences. These findings support a set-point control mechanism rather than immaturity for explaining not only neonatal susceptibility but also resilience to infection. In summary, our findings show that neonatal HCMV infection leads to a highly plastic and functional robust programming of dendritic cells in vivo and in vitro. In comparison with adults, a minimal number of subtle quantitative and temporal differences may contribute to variability in host susceptibility and resilience, in a context dependent manner.

Immunoglobulin superfamily members encoded by viruses and their multiple roles in immune evasion.

Pathogens have developed a plethora of strategies to undermine host immune defenses in order to guarantee their survival. For large DNA viruses, these immune evasion mechanisms frequently rely on the expression of genes acquired from host genomes. Horizontally transferred genes include members of the immunoglobulin superfamily, whose products constitute the most diverse group of proteins of vertebrate genomes. Their promiscuous immunoglobulin domains, which comprise the building blocks of these molecules, are involved in a large variety of functions mediated by ligand-binding interactions. The flexible structural nature of the immunoglobulin domains makes them appealing targets for viral capture due to their capacity to generate high functional diversity. Here, we present an up-to-date review of immunoglobulin superfamily gene homologs encoded by herpesviruses, poxviruses, and adenoviruses, that include CD200, CD47, Fc receptors, interleukin-1 receptor 2, interleukin-18 binding protein, CD80, carcinoembryonic antigen-related cell adhesion molecules, and signaling lymphocyte activation molecules. We discuss their distinct structural attributes, binding properties, and functions, shaped by evolutionary pressures to disarm specific immune pathways. We include several novel genes identified from extensive genome database surveys. An understanding of the properties and modes of action of these viral proteins may guide the development of novel immune-modulatory therapeutic tools.

Signaling Lymphocytic Activation Molecule Family Receptor Homologs in New World Monkey Cytomegaloviruses.

UNLABELLED: Throughout evolution, large DNA viruses have been usurping genes from their hosts to equip themselves with proteins that restrain host immune defenses. Signaling lymphocytic activation molecule (SLAM) family (SLAMF) receptors are involved in the regulation of both innate and adaptive immunity, which occurs upon engagement with their ligands via homotypic or heterotypic interactions. Here we report a total of seven SLAMF genes encoded by the genomes of two cytomegalovirus (CMV) species, squirrel monkey CMV (SMCMV) and owl monkey CMV (OMCMV), that infect New World monkeys. Our results indicate that host genes were captured by retrotranscription at different stages of the CMV-host coevolution. The most recent acquisition led to S1 in SMCMV. S1 is a SLAMF6 homolog with an amino acid sequence identity of 97% to SLAMF6 in its ligand-binding N-terminal Ig domain. We demonstrate that S1 is a cell surface glycoprotein capable of binding to host SLAMF6. Furthermore, the OMCMV genome encodes A33, an LY9 (SLAMF3) homolog, and A43, a CD48 (SLAMF2) homolog, two soluble glycoproteins which recognize their respective cellular counterreceptors and thus are likely to be viral SLAMF decoy receptors. In addition, distinct copies of further divergent CD48 homologs were found to be encoded by both CMV genomes. Remarkably, all these molecules display a number of unique features, including cytoplasmic tails lacking characteristic SLAMF signaling motifs. Taken together, our findings indicate a novel immune evasion mechanism in which incorporation of host SLAMF receptors that retain their ligand-binding properties enables viruses to interfere with SLAMF functions and to supply themselves with convenient structural molds for expanding their immunomodulatory repertoires. IMPORTANCE: The way in which viruses shape their genomes under the continual selective pressure exerted by the host immune system is central for their survival. Here, we report that New World monkey cytomegaloviruses have broadly captured and duplicated immune cell receptors of the signaling lymphocyte activation molecule (SLAM) family during host-virus coevolution. Notably, we demonstrate that several of these viral SLAMs exhibit exceptional preservation of their N-terminal immunoglobulin domains, which results in maintenance of their ligand-binding capacities. At the same time, these molecules present distinctive structural properties which include soluble forms and the absence of typical SLAM signaling motifs in their cytoplasmic domains, likely reflecting the evolutionary adaptation undergone to efficiently interfere with host SLAM family activities. The observation that the genomes of other large DNA viruses might bear SLAM family homologs further underscores the importance of these molecules as a novel class of immune regulators and as convenient scaffolds for viral evolution.