PAD-mediated citrullination is a novel candidate diagnostic marker and druggable target for HPV-associated cervical cancer.

Citrullination is an emerging post-translational modification catalyzed by peptidyl-arginine deiminases (PADs) that convert peptidyl-arginine into peptidyl-citrulline. In humans, the PAD family consists of five isozymes (PADs 1-4, 6) involved in multiple diseases, including cancer. Given that high-risk (hr) human papillomaviruses (HPVs) are the etiological agents of cervical cancer, in this study, we sought to determine whether PAD-mediated protein citrullination would play a functional role in the HPV-driven transformation of epithelial cells. Here we show that both total protein citrullination and PAD4 expression levels are significantly associated with cervical cancer progression. Specifically, epithelial immunostaining for PAD4 revealed an increasingly higher histoscore from low-grade (CIN1) to high-grade (CIN2, CIN3) cervical intraepithelial neoplasia, and invasive squamous cell carcinoma (SCC) lesions, raising the attractive possibility that PAD4 may be used as tumor staging markers. Furthermore, taking advantage of the epidermoid cervical cancer cell line CaSki, which harbors multiple copies of the integrated HPV16 genome, we show that the expression of E6 and E7 HPV oncoproteins is impaired by treatment with the pharmacological pan-PAD inhibitor BB-Cl-amidine. Consistently, p53 and p21, two targets of HPV oncoproteins, are upregulated by the PAD inhibitor, which undergoes cell growth arrest and apoptosis. Altogether, these findings highlight a novel mechanism by which hrHPVs alter host regulatory pathways involved in cell cycle and survival to gain viral fitness, raising the possibility that PADs may represent an attractive target for developing novel host-targeting antivirals effective in preventing cervical cancer progression.

Antiherpetic Activity of a Root Exudate from Solanum lycopersicum.

The rise of drug resistance to antivirals poses a significant global concern for public health; therefore, there is a pressing need to identify novel compounds that can effectively counteract strains resistant to current antiviral treatments. In light of this, researchers have been exploring new approaches, including the investigation of natural compounds as alternative sources for developing potent antiviral therapies. Thus, this work aimed to evaluate the antiviral properties of the organic-soluble fraction of a root exudate derived from the tomato plant Solanum lycopersicum in the context of herpesvirus infections. Our findings demonstrated that a root exudate from Solanum lycopersicum exhibits remarkable efficacy against prominent members of the family Herpesviridae, specifically herpes simplex virus type 1 (HSV-1) (EC50 25.57 µg/mL, SI > 15.64) and human cytomegalovirus (HCMV) (EC50 9.17 µg/mL, SI 32.28) by inhibiting a molecular event during the herpesvirus replication phase. Moreover, the phytochemical fingerprint of the Solanum lycopersicum root exudate was characterized through mass spectrometry. Overall, these data have unveiled a novel natural product with antiherpetic activity, presenting a promising and valuable alternative to existing drugs.

Citrullination profile analysis reveals peptidylarginine deaminase 3 as an HSV-1 target to dampen the activity of candidate antiviral restriction factors.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that remains latent in neuronal cell bodies but reactivates throughout an individual's life, causing severe adverse reactions, such as herpes simplex encephalitis (HSE). Recently, it has also been implicated in the etiology of Alzheimer's disease (AD). The absence of an effective vaccine and the emergence of numerous drug-resistant variants have called for the development of new antiviral agents that can tackle HSV-1 infection. Host-targeting antivirals (HTAs) have recently emerged as promising antiviral compounds that act on host-cell factors essential for viral replication. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, exhibits a marked inhibitory activity against HSV-1. Furthermore, we show that HSV-1 infection leads to enhanced protein citrullination through transcriptional activation of three PAD isoforms: PAD2, PAD3, and PAD4. Interestingly, PAD3-depletion by specific drugs or siRNAs dramatically inhibits HSV-1 replication. Finally, an analysis of the citrullinome reveals significant changes in the deimination levels of both cellular and viral proteins, with the interferon (IFN)-inducible proteins IFIT1 and IFIT2 being among the most heavily deiminated ones. As genetic depletion of IFIT1 and IFIT2 strongly enhances HSV-1 growth, we propose that viral-induced citrullination of IFIT1 and 2 is a highly efficient HSV-1 evasion mechanism from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection and demonstrate that PAD inhibitors efficiently suppress HSV-1 infection in vitro, which may provide the rationale for their repurposing as HSV-1 antiviral drugs.

Strigolactones as Broad-Spectrum Antivirals against ß-Coronaviruses through Targeting the Main Protease Mpro.

The current SARS-CoV-2 pandemic and the likelihood that new coronavirus strains will emerge in the immediate future point out the urgent need to identify new pan-coronavirus inhibitors. Strigolactones (SLs) are a class of plant hormones with multifaceted activities whose roles in plant-related fields have been extensively explored. Recently, we proved that SLs also exert antiviral activity toward herpesviruses, such as human cytomegalovirus (HCMV). Here we show that the synthetic SLs TH-EGO and EDOT-EGO impair ß-coronavirus replication including SARS-CoV-2 and the common cold human coronavirus HCoV-OC43. Interestingly, in silico simulations suggest the binding of SLs in the SARS-CoV-2 main protease (Mpro) active site, and this was further confirmed by an in vitro activity assay. Overall, our results highlight the potential efficacy of SLs as broad-spectrum antivirals against ß-coronaviruses, which may provide the rationale for repurposing this class of hormones for the treatment of COVID-19 patients.

IFI16 Impacts Metabolic Reprogramming during Human Cytomegalovirus Infection.

Cellular lipid metabolism plays a pivotal role in human cytomegalovirus (HCMV) infection, as increased lipogenesis in HCMV-infected cells favors the envelopment of newly synthesized viral particles. As all cells are equipped with restriction factors (RFs) able to exert a protective effect against invading pathogens, we asked whether a similar defense mechanism would also be in place to preserve the metabolic compartment from HCMV infection. Here, we show that gamma interferon (IFN-γ)-inducible protein 16 (IFI16), an RF able to block HCMV DNA synthesis, can also counteract HCMV-mediated metabolic reprogramming in infected primary human foreskin fibroblasts (HFFs), thereby limiting virion infectivity. Specifically, we find that IFI16 downregulates the transcriptional activation of the glucose transporter 4 (GLUT4) through cooperation with the carbohydrate-response element-binding protein (ChREBP), thereby reducing HCMV-induced transcription of lipogenic enzymes. The resulting decrease in glucose uptake and consumption leads to diminished lipid synthesis, which ultimately curbs the de novo formation of enveloped viral particles in infected HFFs. Consistently, untargeted lipidomic analysis shows enhanced cholesteryl ester levels in IFI16 KO versus wild-type (WT) HFFs. Overall, our data unveil a new role of IFI16 in the regulation of glucose and lipid metabolism upon HCMV replication and uncover new potential targets for the development of novel antiviral therapies. IMPORTANCE Human cytomegalovirus (HCMV) gathers all the substrates and enzymes necessary for the assembly of new virions from its host cell. For instance, HCMV is known to induce cellular metabolism of infected cells to favor virion assembly. Cells are, however, equipped with a first-line defense represented by restriction factors (RFs), which after sensing viral DNA can trigger innate and adaptive responses, thereby blocking HCMV replication. One such RF is IFN-γ-inducible protein 16 (IFI16), which we have shown to downregulate viral replication in human fibroblasts. Thus, we asked whether IFI16 would also play a role in preserving cellular metabolism upon HCMV infection. Our findings highlight an unprecedented role of IFI16 in opposing the metabolic changes elicited by HCMV, thus revealing new promising targets for antiviral therapy.

Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2.

The current SARS-CoV-2 pandemic, along with the likelihood that new coronavirus strains will appear in the nearby future, highlights the urgent need to develop new effective antiviral agents. In this scenario, emerging host-targeting antivirals (HTAs), which act on host-cell factors essential for viral replication, are a promising class of antiviral compounds. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, is endowed with a potent inhibitory activity against human beta-coronaviruses (HCoVs). Specifically, we show that infection of human fetal lung fibroblasts with HCoV-OC43 leads to enhanced protein citrullination through transcriptional activation of PAD4, and that inhibition of PAD4-mediated citrullination with either of the two pan-PAD inhibitors Cl-A and BB-Cl or the PAD4-specific inhibitor GSK199 curbs HCoV-OC43 replication. Furthermore, we show that either Cl-A or BB-Cl treatment of African green monkey kidney Vero-E6 cells, a widely used cell system to study beta-CoV replication, potently suppresses HCoV-OC43 and SARS-CoV-2 replication. Overall, our results demonstrate the potential efficacy of PAD inhibitors, in suppressing HCoV infection, which may provide the rationale for the repurposing of this class of inhibitors for the treatment of COVID-19 patients.

Human cytomegalovirus-induced host protein citrullination is crucial for viral replication.

Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection.

Human Cytomegalovirus and Autoimmune Diseases: Where Are We?

Human cytomegalovirus (HCMV) is a ubiquitous double-stranded DNA virus belonging to the ß-subgroup of the herpesvirus family. After the initial infection, the virus establishes latency in poorly differentiated myeloid precursors from where it can reactivate at later times to cause recurrences. In immunocompetent subjects, primary HCMV infection is usually asymptomatic, while in immunocompromised patients, HCMV infection can lead to severe, life-threatening diseases, whose clinical severity parallels the degree of immunosuppression. The existence of a strict interplay between HCMV and the immune system has led many to hypothesize that HCMV could also be involved in autoimmune diseases (ADs). Indeed, signs of active viral infection were later found in a variety of different ADs, such as rheumatological, neurological, enteric disorders, and metabolic diseases. In addition, HCMV infection has been frequently linked to increased production of autoantibodies, which play a driving role in AD progression, as observed in systemic lupus erythematosus (SLE) patients. Documented mechanisms of HCMV-associated autoimmunity include molecular mimicry, inflammation, and nonspecific B-cell activation. In this review, we summarize the available literature on the various ADs arising from or exacerbating upon HCMV infection, focusing on the potential role of HCMV-mediated immune activation at disease onset.

HPV Meets APOBEC: New Players in Head and Neck Cancer.

Besides smoking and alcohol, human papillomavirus (HPV) is a factor promoting head and neck squamous cell carcinoma (HNSCC). In some human tumors, including HNSCC, a number of mutations are caused by aberrantly activated DNA-modifying enzymes, such as the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) family of cytidine deaminases. As the enzymatic activity of APOBEC proteins contributes to the innate immune response to viruses, including HPV, the role of APOBEC proteins in HPV-driven head and neck carcinogenesis has recently gained increasing attention. Ongoing research efforts take the cue from two key observations: (1) APOBEC expression depends on HPV infection status in HNSCC; and (2) APOBEC activity plays a major role in HPV-positive HNSCC mutagenesis. This review focuses on recent advances on the role of APOBEC proteins in HPV-positive vs. HPV-negative HNSCC.

Where do we Stand after Decades of Studying Human Cytomegalovirus?

Human cytomegalovirus (HCMV), a linear double-stranded DNA betaherpesvirus belonging to the family of Herpesviridae, is characterized by widespread seroprevalence, ranging between 56% and 94%, strictly dependent on the socioeconomic background of the country being considered. Typically, HCMV causes asymptomatic infection in the immunocompetent population, while in immunocompromised individuals or when transmitted vertically from the mother to the fetus it leads to systemic disease with severe complications and high mortality rate. Following primary infection, HCMV establishes a state of latency primarily in myeloid cells, from which it can be reactivated by various inflammatory stimuli. Several studies have shown that HCMV, despite being a DNA virus, is highly prone to genetic variability that strongly influences its replication and dissemination rates as well as cellular tropism. In this scenario, the few currently available drugs for the treatment of HCMV infections are characterized by high toxicity, poor oral bioavailability, and emerging resistance. Here, we review past and current literature that has greatly advanced our understanding of the biology and genetics of HCMV, stressing the urgent need for innovative and safe anti-HCMV therapies and effective vaccines to treat and prevent HCMV infections, particularly in vulnerable populations.

Tuning the Orchestra: HCMV vs. Innate Immunity.

Understanding how the innate immune system keeps human cytomegalovirus (HCMV) in check has recently become a critical issue in light of the global clinical burden of HCMV infection in newborns and immunodeficient patients. Innate immunity constitutes the first line of host defense against HCMV as it involves a complex array of cooperating effectors - e.g., inflammatory cytokines, type I interferon (IFN-I), natural killer (NK) cells, professional antigen-presenting cells (APCs) and phagocytes - all capable of disrupting HCMV replication. These factors are known to trigger a highly efficient adaptive immune response, where cellular restriction factors (RFs) play a major gatekeeping role. Unlike other innate immunity components, RFs are constitutively expressed in many cell types, ready to act before pathogen exposure. Nonetheless, the existence of a positive regulatory feedback loop between RFs and IFNs is clear evidence of an intimate cooperation between intrinsic and innate immunity. In the course of virus-host coevolution, HCMV has, however, learned how to manipulate the functions of multiple cellular players of the host innate immune response to achieve latency and persistence. Thus, HCMV acts like an orchestra conductor able to piece together and rearrange parts of a musical score (i.e., innate immunity) to obtain the best live performance (i.e., viral fitness). It is therefore unquestionable that innovative therapeutic solutions able to prevent HCMV immune evasion in congenitally infected infants and immunocompromised individuals are urgently needed. Here, we provide an up-to-date review of the mechanisms regulating the interplay between HCMV and innate immunity, focusing on the various strategies of immune escape evolved by this virus to gain a fitness advantage.

The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1ß by Human Cytomegalovirus through Inhibition of NF-kB Activity.

Interleukin-1ß (IL-1ß) is a key effector of the inflammasome complex in response to pathogens and danger signals. Although it is well known that assembly of the inflammasome triggers proteolytic cleavage of the biologically inactive precursor pro-IL-1ß into its mature secreted form, the mechanism by which human cytomegalovirus (HCMV) regulates IL-1ß production via the inflammasome is still poorly understood. Here, we show that the infection of human foreskin fibroblasts (HFFs) with a mutant HCMV lacking the tegument protein pp65 (v65Stop) results in higher expression levels of mature IL-1ß compared to its wild-type counterpart, suggesting that pp65 mediates HCMV immune evasion through downmodulation of IL-1ß. Furthermore, we show that enhanced IL-1ß production by the v65Stop mutant is due in part to induction of DNA binding and the transcriptional activity of NF-κB. Lastly, we demonstrate that HCMV infection of HFFs triggers a non-canonical IL-1ß activation pathway where caspase-8 promotes IL-1ß maturation independently of caspase-1. Altogether, our findings provide novel mechanistic insights into the interplay between HCMV and the inflammasome system and raise the possibility of targeting pp65 to treat HCMV infection.

Modulation of the innate immune response by human cytomegalovirus.

The interplay between human cytomegalovirus (HCMV) and the innate immune response is a critical process that has attracted the attention of many research groups. The emerging scenario is that the immune response of an HCMV-infected host is mediated by a plethora of viral DNA sensors acting as pattern recognition receptors (PRRs), which are capable of inhibiting indirectly viral infection through the activation of two distinct downstream signaling cascades. The first one triggers the production of cytokines, chemokines and interferons (IFNs), while the second one leads to inflammasome complex formation, which in turn promotes the maturation and secretion of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß). An additional first line of defense against HCMV is represented by a multiplicity of constitutively expressed restriction factors that inhibit viral replication by directly interfering with the activity of essential viral/cellular genes. Here, we take a closer look at some of the most representative intrinsic restriction factors involved in HCMV infection (e.g. IFI16, ND10 complex, viperin and APOBEC3) and review our current understanding of the mechanisms that HCMV has evolved to counteract both IFN and inflammasome responses.

Human Cytomegalovirus Tegument Protein pp65 (pUL83) Dampens Type I Interferon Production by Inactivating the DNA Sensor cGAS without Affecting STING.

The innate immune response plays a pivotal role during human cytomegalovirus (HCMV) primary infection. Indeed, HCMV infection of primary fibroblasts rapidly triggers strong induction of type I interferons (IFN-I), accompanied by proinflammatory cytokine release. Here, we show that primary human foreskin fibroblasts (HFFs) infected with a mutant HCMV TB40/E strain unable to express UL83-encoded pp65 (v65Stop) produce significantly higher IFN-ß levels than HFFs infected with the wild-type TB40/E strain or the pp65 revertant (v65Rev), suggesting that the tegument protein pp65 may dampen IFN-ß production. To clarify the mechanisms through which pp65 inhibits IFN-ß production, we analyzed the activation of the cGAS/STING/IRF3 axis in HFFs infected with either the wild type, the revertant v65Rev, or the pp65-deficient mutant v65Stop. We found that pp65 selectively binds to cGAS and prevents its interaction with STING, thus inactivating the signaling pathway through the cGAS/STING/IRF3 axis. Consistently, addition of exogenous cGAMP to v65Rev-infected cells triggered the production of IFN-ß levels similar to those observed with v65Stop-infected cells, confirming that pp65 inactivation of IFN-ß production occurs at the cGAS level. Notably, within the first 24 h of HCMV infection, STING undergoes proteasome degradation independently of the presence or absence of pp65. Collectively, our data provide mechanistic insights into the interplay between HCMV pp65 and cGAS, leading to subsequent immune evasion by this prominent DNA virus.IMPORTANCE Primary human foreskin fibroblasts (HFFs) produce type I IFN (IFN-I) when infected with HCMV. However, we observed significantly higher IFN-ß levels when HFFs were infected with HCMV that was unable to express UL83-encoded pp65 (v65Stop), suggesting that pp65 (pUL83) may constitute a viral evasion factor. This study demonstrates that the HCMV tegument protein pp65 inhibits IFN-ß production by binding and inactivating cGAS early during infection. In addition, this inhibitory activity specifically targets cGAS, since it can be bypassed via the addition of exogenous cGAMP, even in the presence of pp65. Notably, STING proteasome-mediated degradation was observed in both the presence and absence of pp65. Collectively, our data underscore the important role of the tegument protein pp65 as a critical molecular hub in HCMV's evasion strategy against the innate immune response.

Intrinsic host restriction factors of human cytomegalovirus replication and mechanisms of viral escape.

Before a pathogen even enters a cell, intrinsic immune defenses are active. This first-line defense is mediated by a variety of constitutively expressed cell proteins collectively termed "restriction factors" (RFs), and they form a vital element of the immune response to virus infections. Over time, however, viruses have evolved in a variety ways so that they are able to overcome these RF defenses via mechanisms that are specific for each virus. This review provides a summary of the universal characteristics of RFs, and goes on to focus on the strategies employed by some of the most important RFs in their attempt to control human cytomegalovirus (HCMV) infection. This is followed by a discussion of the counter-restriction mechanisms evolved by viruses to circumvent the host cell's intrinsic immune defenses. RFs include nuclear proteins IFN-γ inducible protein 16 (IFI16) (a Pyrin/HIN domain protein), Sp100, promyelocytic leukemia, and hDaxx; the latter three being the keys elements of nuclear domain 10 (ND10). IFI16 inhibits the synthesis of virus DNA by down-regulating UL54 transcription - a gene encoding a CMV DNA polymerase; in response, the virus antagonizes IFI16 via a process involving viral proteins UL97 and pp65 (pUL83), which results in the mislocalizing of IFI16 into the cytoplasm. In contrast, viral regulatory proteins, including pp71 and IE1, seek to modify or disrupt the ND10 proteins and thus block or reverse their inhibitory effects upon virus replication. All in all, detailed knowledge of these HCMV counter-restriction mechanisms will be fundamental for the future development of new strategies for combating HCMV infection and for identifying novel therapeutic agents.

Innate nuclear sensor IFI16 translocates into the cytoplasm during the early stage of in vitro human cytomegalovirus infection and is entrapped in the egressing virions during the late stage.

UNLABELLED: Intrinsic immune mechanisms mediated by constitutively expressed proteins termed "restriction factors" provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the in vitro infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to the cytoplasm of HCMV-infected cells. ESCRT (endosomal sorting complex required for transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established. IMPORTANCE: Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.

Nuclear DNA sensor IFI16 as circulating protein in autoimmune diseases is a signal of damage that impairs endothelial cells through high-affinity membrane binding.

IFI16, a nuclear pathogenic DNA sensor induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. It is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. In this study, using an in-house capture enzyme-linked immunsorbent assay we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that the rIFI16 protein, when added in-vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. It was further demonstrated that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells. Free recombinant IFI16 binds these sites on HUVEC with dissociation constant of 2.7 nM, radioiodinated and unlabeled IFI16 compete for binding sites, with inhibition constant (Ki) of 14.43 nM and half maximal inhibitory concentration (IC50) of 67.88 nM; these data allow us to estimate the presence of 250,000 to 450,000 specific binding sites per cell. Corroborating the results from functional assays, this binding could be completely inhibited using anti-IFI16 N-terminal antibody, but not with an antibody raised against the IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of autoimmune patients which binds endothelial cells causing damage, suggesting a new pathogenic and alarmin function through which this protein triggers the development of autoimmunity.

Tumor-derived endothelial cells evade apoptotic activity of the interferon-inducible IFI16 gene.

The human interferon (IFN)-inducible IFI16 protein is a member of the 200-amino acid repeat family encoded by the HIN-200 genes. Forced IFI16 expression in normal human endothelial cells (ECs) inhibits cell growth and tube morphogenesis of ECs through the triggering of apoptosis by caspase-2 and caspase-3 via nuclear factor-κB (NF-κB) complex activation. Accumulating evidence suggests that tumor-derived ECs (TECs) possess a distinct and unique phenotype compared with normal ECs, and they may be able to acquire resistance to antiangiogenic agents such as IFNs. However, few functional studies are available on cultured TEC. In the present study, we have demonstrated that TEC obtained from tumors of various histological origin, namely kidney (Eck25), breast (B-TEC), and head and neck (HN4), continued to proliferate and generate microtubules on Matrigel following IFI16 overexpression. In contrast to normal ECs, they were resistant to apoptosis triggered by caspase-2 and caspase-3 activation via the NF-κB complex. At the molecular level, when overexpressed in TEC, IFI16 appeared unable to regulate NF-κB activity and lead to caspase activation. Altogether, these results indicate that TECs display abnormal responses, in terms of survival and angiogenic properties, to an antiproliferative and antiangiogenic IFN-inducible gene such as IFI16.

The proapoptotic activity of the Interferon-inducible gene IFI16 provides new insights into its etiopathogenetic role in autoimmunity.

Several lines of evidence link Interferons (IFNs) with autoimmune disorders. Autoantibodies against the Interferon-inducible IFI16 protein, a member of the HIN-200 family constitutively expressed in endothelial cells and keratinocytes, have been identified in patients affected by autoimmune diseases including Systemic Lupus Erythematosus (SLE), Sjogren Syndrome (SjS), and Scleroderma (SSc). These findings point to a role for IFI16 in the etiopathogenesis of autoimmune diseases, but the exact mechanisms involved in the development of autoimmunity remain obscure. In this study, we report for the first time that endothelial cells overexpressing IFI16 undergo apoptosis via the activation of caspase 2 and caspase 3, and that a positive feedback loop appears to link these two caspases. The relevance of IFI16-mediated apoptosis is highlighted by the observation that IFI16 knock down by RNA interference in endothelial cells inhibits the activation of both caspase 2 and caspase 3 by IFN-beta priming and synthetic double-stranded RNA treatment. Expression of a dominant-negative mutant of IKK2 kinase or treatment with AS602868, an inhibitor of IKK2 activity, results in a strong reduction of NF-kB activation along with absence of caspase 2 and caspase 3 activation and apoptosis induction. Collectively, our findings provide new insights into the role of IFI16 in the pathogenesis of autoimmune diseases by demonstrating that in addition to the stimulation of pro-inflammatory molecules, IFI16 also leads to apoptosis in endothelial cells.

The interferon-inducible HIN-200 gene family in apoptosis and inflammation: implication for autoimmunity.

The Ifi-200/HIN-200 gene family encodes highly homologous human (IFI16, myeloid cell nuclear differentiation antigen, absent in melanoma 2, and IFIX) and murine proteins (Ifi202a, Ifi202b, Ifi203, Ifi204, Ifi205, and Ifi206), which are induced by type I and II interferons (IFN). These proteins have been described as regulators of cell proliferation and differentiation and, more recently, several reports have suggested their involvement in both apoptotic and inflammatory processes. The relevance of HIN-200 proteins in human disease is beginning to be clarified, and emerging experimental data indicate their role in autoimmunity. Autoimmune disorders are sustained by perpetual activation of inflammatory process and a link between autoimmunity and apoptosis has been clearly established. Moreover, the interferon system is now considered as a key player in autoimmune disorders such as systemic lupus erythemathosus, systemic sclerosis, and Sjögren's syndrome, and it is therefore conceivable to hypothesize that HIN-200 may be among the pivotal mediators of IFN activity in autoimmune disease. In particular, the participation of HIN-200 proteins in apoptosis and inflammation could support their potential role in autoimmunity.