Interferon-Inducible Protein 16 (IFI16) Has a Broad-Spectrum Binding Ability Against ssDNA Targets: An Evolutionary Hypothesis for Antiretroviral Checkpoint.

The interferon γ-inducible protein 16 (IFI16) has recently been linked to the detection of nuclear and cytosolic DNA during infection with herpes simplex virus-1 and HIV. IFI16 binds dsDNA via HIN200 domains and activates stimulator of interferon genes (STING), leading to TANK (TRAF family member-associated NF-κB activator)-binding kinase-1 (TBK1)-dependent phosphorylation of interferon regulatory factor (IRF) 3 and transcription of type I interferons (IFNs) and related genes. To better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as cyclic dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to DNA ligands and viruses. In contrast, expression of the NF-κB-regulated cytokines IL-6 and IL-1β was unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of IFN-α and the IFN-stimulated gene retinoic acid-inducible gene I (RIG-I) in response to cyclic GMP-AMP, a second messenger produced by cyclic GMP-AMP synthase (cGAS) as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in these cells, suggesting that transcription of IFN-stimulated genes is dependent on IFI16. These results indicate a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in antiviral immunity.

Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

Polyglutamine binding protein 1 (PQBP1) inhibits innate immune responses to cytosolic DNA

Herpes simplex virus (HSV) is a ubiquitous human pathogen that causes a wide spectrum of disease, ranging from asymptomatic viral shedding to lethal encephalitis and disseminated disease [1,2]. These viruses belong to the neurotropic subfamily of α-herpesviruses, and after initial replication in epithelial cells, HSV enters sensory neurons to establish latency in neural ganglia. HSV can also progress to active lytic replication in the central nervous system, resulting in devastating encephalitis. To successfully replicate in the host nervous system, HSV encodes several viral proteins to counter the host innate response to infection. Among these, the multifunctional viral protein γ34.5 is central to countering several effector pathways in the host type I interferon (IFN) response. HSV γ34.5 is present in two copies in the repeated regions of the viral genome, and although initially described as a late gene, its expression is actually “leaky late,” with γ34.5 functioning to counter the host response after late viral DNA synthesis but also in the first hours of infection. Within γ34.5 are domains that specifically target host shutoff of protein synthesis [3], type I IFN induction through TANK-binding kinase (TBK1) [4], and inhibition of autophagy through Beclin 1 binding (Fig 1) [5]. HSV γ34.5 is required for full virulence in the murine brain [6,7]; however, recent evidence suggests that γ34.5 may function differently in newborn models of HSV disease compared to the adult [8]. Furthermore, some functions of γ34.5 are required for pathogenesis in non-nervous system tissue [9]. Here, we provide a brief overview of the multiple host responses modulated by γ34.5 for successful HSV replication in the nervous system and also discuss recent evidence that expands the role of γ34.5 to promote pathogenesis in several different tissue-types and across different developmental ages of the host. Fig 1 The HSV-1 major neurovirulence factor γ34.5 targets multiple different host pathways. HSV-1 γ34.5 Mediates Reversal of Host Shutoff of Total Protein Synthesis One of the earliest responses to infection is the type I IFN response and the innate pathways modulated by the IFN-inducible, double-stranded RNA–dependent protein kinase R (PKR) system. An important function of activated PKR during HSV infection is phosphorylation of the translation initiation factor eIF2α, resulting in translational arrest and reduction in the global synthesis of viral and cellular proteins [10]. However, HSV has evolved an effective strategy through γ34.5 to reverse the eIF2α kinase-mediated translational arrest to allow for successful viral replication. The carboxyl terminus of HSV-1 γ34.5 binds and retargets the host phosphatase PP1α to eIF2α, thus targeting eIF2α for dephosphorylation and reversing the shutoff of protein synthesis (Fig 2) [11]. Mutant viruses engineered to specifically disrupt the interaction between γ34.5 and the host phosphatase PP1α demonstrate the requirement of HSV-1–mediated retargeting of PP1α for pathogenesis in several different models of disease, including HSV keratitis [12], encephalitis, and disseminated disease in the neonate [9]. Interestingly, the carboxyl terminus of HSV-1 γ34.5 shares sequence homology with the host protein GADD34 (growth arrest and DNA damage-inducible gene 34) [13], which acts as PP1α regulatory subunit to target PP1α to eIF2α during periods of endoplasmic reticulum (ER) stress and the unfolded protein response. Earlier studies have shown that this host sequence and γ34.5 are interchangeable in the HSV-1 genome to preclude the premature shutoff of total protein synthesis, suggesting that during herpesvirus evolution, the virus acquired the GADD34 host sequence to improve viral replication and fitness [14]. Fig 2 Reversal of the host shutoff of protein synthesis mediated by HSV γ34.5. γ34.5 Binds TBK1 to Prevent Activation of the Type I IFN Response Prior to the initiation of the type I IFN response, HSV is detected in the host cell through several different pattern recognition receptors. For example, Toll-like receptor 3 (TLR3) detects HSV dsRNA in endosomes to stimulate IFN expression. In the cytoplasm, intracellular RNA and DNA sensors, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), interferon γ-inducible protein 16 (IFI16), and cyclic GMP-AMP synthase (cGAS), also detect HSV in the host cell [15–17]. Although these receptors detect different pathogen-associated molecular patterns, downstream signals are relayed through TBK1, which in turn phosphorylates and activates the interferon regulatory factor 3/7 (IRF3/7) for production of type I IFNs. HSV-1 γ34.5 counters this induction of the type I IFN response through binding of TBK1 with its amino terminus (Fig 1) [4]. Targeting of TBK1 by γ34.5 competes for IRF3 binding and ultimately inhibits IRF3 phosphorylation by TBK1, preventing IRF3 nuclear localization for type I IFN expression. A mutant virus deleted for the amino terminus of γ34.5 to demolish TBK1 binding demonstrates significantly increased IFN-β and interferon-stimulated gene (ISG) production in the first three to six hours of infection. In an ocular model of HSV disease, a virus deleted for TBK1 binding replicated poorly in the corneal epithelium and trigeminal ganglion and was effectively controlled by the host response before it reached the brain [18]. These findings reveal an additional role for γ34.5 in inhibiting the host response prior to transcription of type I IFNs and PKR up-regulation and demonstrate a role for early expression of this “leaky-late” gene.

Animal cells use pattern-recognition receptors (PRRs) to detect specific pathogens. Pathogen detection mounts an appropriate immune response, including interferon and cytokine induction. The intracellular PRR-signaling pathways that detect DNA viruses have been characterized, particularly in myeloid cells. In these pathways, cGMP-AMP synthase (cGAS) and the pyrin and HIN domain family member (PYHIN) protein interferon-γ–inducible protein 16 (IFI16) detect DNA and signal via stimulator of interferon genes protein (STING). However, although airway epithelial cells are frontline sentinels in detecting pathogens, information on how they respond to DNA viruses is limited, and the roles of PYHIN proteins in these cells are unknown. Here, we examined expression and activities of cGAS, STING, and PYHINs in human lung epithelial cells. A549 epithelial cells, commonly used for RNA-sensing studies, failed to respond to DNA because they lacked STING expression, and ectopic STING expression restored a cGAS-dependent DNA response in these cells. In contrast, NuLi-1 immortalized human bronchial epithelial cells did express STING, which was activated after DNA stimulation and mediated DNA-dependent gene induction. PYHIN1, which like IFI16 has been proposed to be a viral DNA sensor, was the only PYHIN protein expressed in both airway epithelial cell types. However, rather than having a role in DNA sensing, PYHIN1 induced proinflammatory cytokines in response to interleukin-1 (IL-1) or tumor necrosis factor α (TNFα) stimulation. Of note, PYHIN1, via its HIN domain, directly induced IL-6 and TNFα transcription, revealing that PYHIN proteins play a role in proinflammatory gene induction in airway epithelial cells.

Skin Stem Cells in Silence, Action, and Cancer.

Transposable elements, including endogenous retroviruses (ERVs), comprise almost 45% of the human genome. This could represent a significant pathogenic burden but it is becoming more evident that many of these elements have a positive contribution to make to normal human physiology. In particular, the contributions of human ERVs (HERVs) to gene regulation and the expression of noncoding RNAs has been revealed with the help of new and emerging genomic technologies. HERVs have the common provirus structure of coding open reading frames (ORFs) flanked by two long-terminal repeats (LTRs). However, over the course of evolution and as a consequence of host defence mechanisms, most of the sequences contain INDELs, mutations or have been reduced to single LTRs by recombination. These INDELs and mutations reduce HERV activity. However, there is a trade-off for the host cells in that HERVs can provide beneficial sources of genetic variation but with this benefit comes the risk of pathogenic activity and spread within the genome. For example, the LTRs are of critical importance as they contain promoter sequences and can regulate not only HERV expression but that of human genes. This is true even when the LTRs are located in intergenic regions or are in antisense orientation to the rest of the gene. Uncontrolled, this promoter activity could disrupt normal gene expression or transcript processing (e.g., splicing). Thus, control of HERVs and particularly their LTRs is essential for the cell to manage these elements and this control is achieved at multiple levels, including epigenetic regulations that permit HERV expression in the germline but silence it in most somatic tissues. We will discuss some of the common epigenetic mechanisms and how they affect HERV expression, providing detailed discussions of HERVs in stem cell, placenta and cancer biology.

It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.

Human endogenous retroviruses (HERVs) are remnants of retroviral germ line infections of human ancestors and make up ~8% of the human genome. Under physiological conditions, these elements are frequently inactive or non-functional due to deactivating mutations and epigenetic control. However, they can be reactivated under certain pathological conditions and produce viral transcripts and proteins. Several disorders, like multiple sclerosis or amyotrophic lateral sclerosis are associated with increased HERV expression. Although their detailed contribution to individual diseases has yet to be elucidated, an increasing number of studies in vitro and in vivo suggest HERVs as potent modulators of the immune system. They are able to affect the transcription of other immune-related genes, interact with pattern recognition receptors, and influence the positive and negative selection of developing thymocytes. Interestingly, HERV envelope proteins can both stimulate and suppress immune responses based on different mechanisms. In the light of HERV proteins becoming an emerging drug target for autoimmune-related disorders and cancer, we will provide an overview on recent findings of the complex interactions between HERVs and the human immune system with a focus on autoimmunity.

Ancient retrovirus infections played a role in human evolution and are still involved in innate immunity and neural plasticity.

Umbilical Cord Blood Stem Cells Induced into Myeloid, Erythroid, or Megakaryocytic Pathways Are Susceptible to Infection by Porcine Endogenous Retroviruses (PERV).

Hematopoietic development from human embryonic stem cell lines

Wnt Signaling, Lgr5, and Stem Cells in the Intestine and Skin

Bone Marrow-Derived Cells Are Not the Origin of the Cancer Stem Cells in Ultraviolet-Induced Skin Cancer

Enchytraeus japonensis is a small oligochaete that reproduces mainly asexually by fragmentation (autotomy) and regeneration. As sexual reproduction can also be induced, it is a good animal model for the study of both somatic and germline stem cells. To clarify the features of stem cells in regeneration, we investigated the proliferation and lineage of stem cells in E.japonensis. Neoblasts, which have the morphological characteristics of undifferentiated cells, were found to firmly adhere to the posterior surface of septa in each trunk segment. Also, smaller neoblast-like cells, which are designated as N-cells in this study, were located dorsal to the neoblasts on the septa. By conducting 5-bromo-2'-deoxyuridine (BrdU)-labeling-experiments, we have shown that neoblasts are slow-cycling (or quiescent) in intact growing worms, but proliferate rapidly in response to fragmentation. N-cells proliferate more actively than do neoblasts in intact worms. The results of pulse-chase experiments indicated that neoblast and N-cell lineage mesodermal cells that incorporated BrdU early in regeneration migrated toward the autotomized site to form the mesodermal region of the blastema, while the epidermal and intestinal cells also contributed to the blastema locally near the autotomized site. We have also shown that neoblasts have stem cell characteristics by expressing Ej-vlg2 and by the activity of telomerase during regeneration. Telomerase activity was high in the early stage of regeneration and correlated with the proliferation activity in the neoblast lineage of mesodermal stem cells. Taken together, our results indicate that neoblasts are mesodermal stem cells involved in the regeneration of E.japonensis.