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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.

The aim of the present study is to determine the expression levels of PYHIN (IFI16 and AIM2) and APOBEC3 (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) gene family members in a cohort of patients with head and neck squamous cell carcinoma (HNSCC) and assess their potential correlation with human papillomavirus (HPV) infection status, clinical characteristics, and survival. For this purpose, 34 HNSCC tissue specimens along with healthy surrounding mucosa were collected from patients surgically treated for HNSCC. Nucleic acids were isolated to assess the presence of HPV and the expression levels of selected molecular markers. Survival analysis was carried out using the Kaplan-Meier method. In HPV-negative (HPV - ) HNSCCs, we detected low mRNA expression levels of IFI16, A3A, and A3B, whereas these genes were upregulated of 2-100 folds in HPV-positive (HPV + ) tumors (p < 0.05). Interestingly, AIM2 gene expression levels were predominantly unchanged in HPV + HNSCCs compared to their HPV - counterparts, in which AIM2 was predominantly upregulated (10% vs. 50% of patients). In HPV - tumors, upregulation of TP53, NOTCH1, PD-L1, and IFI16 correlated with lower occurrence of nodal metastases. On the other hand, the expression of APOBEC family members did not correlate with clinical characteristics. Regarding survival, patients with upregulated A3F gene expression had a worse prognosis, while patients without changes in A3H expression had a lower survival rate. In conclusion, our findings indicate that the innate immune sensors IFI16 and AIM2 and some APOBEC family members could be potentially used as biomarkers for disease outcome in HNSCC patients regardless of HPV presence.

Interleukin-1β (IL-1β) 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-1β into its mature secreted form, the mechanism by which human cytomegalovirus (HCMV) regulates IL-1β 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-1β compared to its wild-type counterpart, suggesting that pp65 mediates HCMV immune evasion through downmodulation of IL-1β. Furthermore, we show that enhanced IL-1β 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-1β activation pathway where caspase-8 promotes IL-1β 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.

The germline encoded proteins serving as "pattern recognition receptors" (PRRs) constitute the earliest step in the innate immune response by recognizing the "pathogen-associated molecular patterns" (PAMPs) that comprise microbe nucleic acids and proteins usually absent from healthy hosts. Upon detection of exogenous nucleic acid two different innate immunity signaling cascades are activated. The first culminates in the production of chemokines, cytokines, and type I interferons (IFN-I), while the second leads to inflammasome complex formation. Human cytomegalovirus (HCMV), a member of the b-herpesvirus subfamily, is a widespread pathogen that infects the vast majority of the world's population. The virion has an icosahedral capsid that contains a linear dsDNA genome of approximately 240 kb, surrounded by an outer lipid envelope and a proteinaceous tegument containing several viral proteins. Despite the numerous and multifaceted antiviral effects of IFNs and cytokines, HCMV is able to invade, multiply, and establish persistent infection in healthy human hosts. To achieve this goal the virus has developed different strategies to block the IFN-I response and to alter the physiological outcomes of the IFN-inducible genes. This article focuses on HCMV tegument pp65 by reviewing its mechanisms of action in favoring virus evasion from the host innate immune response.

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-β 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-β production. To clarify the mechanisms through which pp65 inhibits IFN-β 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-β levels similar to those observed with v65Stop-infected cells, confirming that pp65 inactivation of IFN-β 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-β 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-β 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., (Copyright © 2018 American Society for Microbiology.)

Unlabelled: A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication., Importance: The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

IFI16, a member of the IFN-inducible PYHIN-200 gene family, displays multifaceted activity due to its ability to bind to various target proteins and, in turn, modulate a variety cell functions including proliferation, differentiation, apoptosis/pyroptosis, senescence, and in? ammation. The last few year have seen major advances in our knowledge of IFI16 antiviral activity and its role in the immune response. Indeed, a wealth of evidence now supports a key role of IFI16 in the activation of innate immunity and viral restriction against Herpesviruses and Lentiviruses, such that the definition of IFI16 as a "restriction factor" is now widely accepted. However, most viruses have developed their own unique strategy to antagonize IFI16, leading to a modification or disruption of its function. This review summarizes our current understanding of how viral replication is sensed and then inhibited by IFI16 protein and the viral strategies employed to defeat this host defense mechanism. We will focus mainly on Herpesviruses, although recent discoveries on the role of IFI16 in lentiviral infection will also be considered.

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The aim of this study was to analyze protein and gene expression of HER2 in 224 head and neck precancerous and malignant lesions by immunohistochemistry and FISH analysis. In parallel, expression of pStat3, Sox2, IFI16 and p16, Ki67 was evaluated. Immunohistochemical analysis was assessed on formalin-fixed paraffin-embedded (FFPE) tissue specimens. A combined method for HPV detection consisting of p16 immunostaining and two PCR probes was applied. HER2 gene status was evaluated by FISH analysis. HPV DNA was detected in 24% of cases with predominant HPV16 genotype. HPV-positive lesions had higher HER2, pStat3 and within carcinoma group, and higher IFI16 expression compared to the HPV-negative group (Fig. 1A-B-C). A strong positive correlation between Sox2 and proliferative activity was observed, whereas IFI16 expression displayed a negative relationship with Sox2 and Ki67 activity. The most striking result was higher pStat3 expression in HPV-positive lesions and its strong positive correlation with IFI16 expression. The presence of HPV may induce upregulation of HER2/neu, pStat3 and IFI16. High levels and a strong positive correlation between pStat3 and IFI16 suggest their synergistic pro-apoptotic effects in HPV-positive lesions.

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.

Objective: Several studies have shown the presence of anti-IFI16 antibodies in systemic lupus erythematosus (SLE), Sjögren Syndrome (SjS), systemic sclerosis (SSc) and other autoimmune diseases. However, the significance of anti-IFI16 antibodies in SLE has not been fully characterized. The aim of this study was to investigate associations between anti-IFI16 antibodies and clinical and serologic parameters of SLE., Methods: An enzyme-linked immunosorbent assay (ELISA) kit was used to measure anti-IFI16 antibodies in the sera of 168 SLE patients, 46 patients with any type of primary glomerulonephritis (GN) and 182 healthy controls (HCs). Associations between anti-IFI16 antibodies and clinical and serologic parameters of SLE were statistically evaluated using both univariate and multivariate analysis., Results: Significantly higher anti-IFI16 titres were observed in SLE patients compared to both non-SLE GN and HCs (median levels: 270.1 U/ml vs 132.1 U/ml, p = 0.001, and 52.9 U/ml, p < 0.0001, respectively). With cut-off levels corresponding to the 95th percentile of the control population (113 U/ml), 63% of the SLE patients tested positive for anti-IFI16 autoantibodies, compared to just 24% of patients with primary non-SLE GN and 5% of HCs. The presence of anti-IFI16 antibodies inversely correlated with proteinuria (univariate analysis) and C3 hypocomplementaemia (univariate and multivariate analyses)., Conclusions: The inverse correlations observed between anti-IFI16 positivity, proteinuria and C3 hypocomplementaemia suggest that anti-IFI16 antibodies do not contribute to renal inflammation in SLE; indeed they may even prevent complement consumption. Anti-IFI16 antibodies hold the potential to serve as a new biomarker of disease activity in SLE.

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between -54 and -43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor.

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.