Marco De Andrea

The intracellular DNA Sensor IFI16 gene acts as restriction factor for human cytomegalovirus replication

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.

Melusin is a new muscle-specific interactor for beta(1) integrin cytoplasmic domain.

Here we describe the isolation and partial characterization of a new muscle-specific protein (Melusin) which interacts with the integrin cytoplasmic domain. The cDNA encoding Melusin was isolated in a two-hybrid screening of a rat neonatal heart library using β1A and β1D integrin cytoplasmic regions as baits. Melusin is a cysteine-rich cytoplasmic protein of 38 kDa, with a stretch of acidic amino acid residues at the extreme carboxyl-terminal end. In addition, putative binding sites for SH3 and SH2 domains are present in the amino-terminal half of the molecule. Chromosomic analysis showed that melusin gene maps at Xq12.1/13 in man and in the synthenic region X band D in mouse. Melusin is expressed in skeletal and cardiac muscles but not in smooth muscles or other tissues. Immunofluorescence analysis showed that Melusin is present in a costamere-like pattern consisting of two rows flanking α-actinin at Z line. Its expression is up-regulated duringin vitro differentiation of the C2C12 murine myogenic cell line, and it is regulated during in vivo skeletal muscle development. A fragment corresponding to the tail region of Melusin interacted strongly and specifically with β1 integrin cytoplasmic domain in a two-hybrid test, but the full-length protein did not. Because the tail region of Melusin contains an acidic amino acid stretch resembling high capacity and low affinity calcium binding domains, we tested the possibility that Ca2+ regulates Melusin-integrin association. In vitro binding experiments demonstrated that interaction of full-length Melusin with detergent-solubilized integrin heterodimers occurred only in absence of cations, suggesting that it can be regulated by intracellular signals affecting Ca2+ concentration.

The retinoblastoma protein is an essential mediator that links the interferon-inducible 204 gene to cell-cycle regulation.

We have previously demonstrated that overexpression of p204, a member of the Ifi 200 gene family, inhibits growth, delays G0/G1 progression into S phase, and impairs E2F-mediated transcriptional activity. In this study, we show that p204 directly binds the retinoblastoma protein (pRb) in vivo to exert its activity. Transient p204 overexpression in Rb+/+ mouse embryo fibroblasts (MEF) inhibits cell proliferation, but does not affect cell growth in MEF derived from Rb−/− mice. Two human cell lines, Saos2 and C33A, bearing an inactive pRb, but not primary human embryo fibroblasts, are resistant to the p204 antiproliferative activity. p204 contains two 200 amino acid motifs, designated as type a or b domains, each containing a canonical Rb binding motif (LXCXE). When dominant-negative mutants at the Rb binding motif were transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreover p204 overexpression in Rb+/+ MEF led to a significant decrease of both DHFR and PCNA proteins, two S phase markers. By contrast, this effect was not observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb binding sites. Finally, overexpression of the LXCXE p204 mutant rendered Rb+/+ MEF resistant to the IFN-α antiproliferative activity, in comparison to the untransfected Rb+/+ MEF. As expected, Rb−/− cells were unsensitive to the IFN-α induced growth inhibition. Taken as a whole, these results suggest that (i) p204 contributes to the IFN-α antiproliferative activity and (ii) the primary target of p204 leading to efficient G1 arrest as well as to blockade of DNA replication from G1 phase is the pRb regulatory system.

Role of the Interferon-Inducible Gene IFI16 in the Etiopathogenesis of Systemic Autoimmune Disorders

Abstract:  Interferons (IFNs) are now known to exert a multitude of immunological functions on both the innate and adaptive immunity. Given their pleiotropic effects on the immune system, it is conceivable that excess type I IFN or aberrant regulation of its signaling could contribute to autoimmunity. Several lines of evidence link IFNs to autoimmune disorders, in particular to systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). Expression of a spectrum of genes that constitutes an “IFN signature” is the most significant observation indicating that IFNs may be dominant among the pathogenic mediators involved in some autoimmune diseases. A family of IFN‐inducible genes, designated HIN‐200 in the human and IFI‐200 in the murine species, encodes evolutionary related human (IFI16, MNDA, AIM2, IFIX) and murine proteins (Ifi202 a, Ifi202b, Ifi203, Ifi204, Ifi205/D3). Physiological IFI16 expression was found in cells of the immune system, in endothelial cells, and in stratified squamous epithelia, such as skin. The presence of anti‐IFI16 antibodies was reported in SLE and primary/secondary Sjögrens syndrome. More recently, we reported that anti‐IFI16 autoantibodies differentiate limited cutaneous systemic sclerosis (lcSSc) from diffuse systemic sclerosis (dcSSc). Molecular studies performed in primary endothelial cells overexpressing IFI16 demonstrated that it may be involved in the early steps of inflammation by modulating endothelial cell function, such as expression of adhesion molecules and chemokine production, cell growth, and apoptosis. Moreover, here we report that IFI16 expression is induced by proinflammatory cytokines. In this article the role of the IFI16 protein and its corresponding autoantibodies in the etiopathogenesis of systemic autoimmune diseases, in which chronic inflammation is involved, are discussed.

Immunohistochemical Expression Analysis of the Human Interferon-Inducible Gene IFI16, a Member of the HIN200 Family, Not Restricted to Hematopoietic Cells

This is the first description of an extensive immunohistochemical analysis of interferon (IFN)-inducible gene IFI16 expression in normal tissues. Immunohistochemical detection of IFI16 in paraffin-embedded tissues is achieved by using a polyclonal antibody raised against its C-terminal fragment that recognizes its three closely migrating isoforms in Western blotting. The results clearly indicate that IFI16 expression is not restricted to the hematopoietic compartment. In normal adult human tissues, it is prominent in stratified squamous epithelia and particularly intense in parabasal cells in the proliferating compartments, but it gradually decreases in the more differentiated suprabasal layers. Understanding of IFI16 expression in vivo is essential for interpretation of the results obtained from in vitro studies and elucidation of its physiologic role. The constitutive expression and wider distribution of IFI16 in normal human tissues, not restricted to the hematopoietic compartment, strongly support the possibility of an important role in cell differentiation that can be further modulated by other stimuli, such as IFN.

High β-HPV DNA Loads and Strong Seroreactivity Are Present in Epidermodysplasia Verruciformis

Epidermodysplasia verruciformis (EV) is a rare disease, characterized by cutaneous warts and associated with a strong predisposition to beta-genus human papillomavirus (HPV). Earlier studies reported high copy numbers of HPV-DNA in nearly all skin tumors from EV patients, but neither HPV replication status in non-lesional skin nor anti-HPV seroreactivity in these patients have been reported yet. We therefore performed a comprehensive viral load analysis for the more common beta-HPV types on skin samples and plucked eyebrow hairs from four EV patients treated at our dermatology department. The results clearly demonstrate that they carry a multiplicity (up to eighteen types) of beta-HPV genotypes in both skin sites. Worthy of note, a high intrapatient concordance for specific types between hair bulbs and skin biopsies was observed and the same beta-PV profile was maintained over time. Viral load analysis revealed a load range between less than one HPV-DNA copy per 100 cells to more than 400 HPV-DNA copies per cell in both eyebrow hairs and skin proliferative lesions. Evaluation of seroreactivity to beta-HPV types in the four EV patients revealed that antibodies against the 16 beta-HPV were significantly more prevalent and showed higher titers than in the controls.

Up-regulation of the interferon-inducible IFI16 gene by oxidative stress triggers p53 transcriptional activity in endothelial cells

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), induces injury of endothelium in a variety of pathophysiological conditions, such as inflammation, aging, and cancer. In our study, we characterized the signaling pathway linking oxidative stress induced by sublethal concentrations of H2O2 to p53 in primary human endothelial cells through the interferon (IFN)‐inducible gene IFI16. Induction of IFI16 by H2O2 was concentration‐ and time‐dependent (maximum at 50 μM, 6 h after treatment) and down‐regulated by pretreatment with N‐acetyl‐L‐cysteine, which acts as an antioxidant. This pathway is a general response to ROS and not specific to H2O2 treatment, as two other ROS‐generating compounds, i.e., S‐nitroso‐N‐acetylpenicillamine and tert‐butyl hydroperoxide, were equally capable to induce IFI16. Moreover, IFI16 up‐regulation is a result of protein accumulation, as expression of corresponding mRNA, assessed by real‐time polymerase chain reaction, was not affected. To investigate the mechanism of IFI16 accumulation, cells were incubated for 6 h in the presence of H2O2 or IFN‐β, and then cycloheximide was added to inhibit further protein synthesis. The half‐life of IFI16 protein was found to be significantly increased in H2O2‐treated cells compared with IFN‐β‐treated cells (t1/2=120 min vs. >30 min in H2O2‐ vs. IFN‐β‐treated cells, respectively). An increase of IFI16 was accompanied by interaction with p53 phosphorylated at its N terminus, as shown by immunoprecipitation experiments. Moreover, binding to IFI16 resulted in its transcriptional activation as shown by an increase in the activity of a reporter gene driven by p53‐responsive sequences derived from the p21WAF1 promoter, along with an increase in the p21 mRNA and protein levels. Altogether, these results demonstrate a novel role of IFI16 in the signal transduction pathway that leads to p53 activation by oxidative stress in endothelial cells.

Cell cycle and viral and immunologic profiles of head and neck squamous cell carcinoma as predictable variables of tumor progression

The aim of this study was to determine whether the aberrant expression of cell‐cycle or immune‐response markers together with human papillomavirus (HPV) positivity impacts patient survival in different head and neck squamous cell carcinoma (HNSCC) subsets.

Murine Cytomegalovirus Stimulates Cellular Thymidylate Synthase Gene Expression in Quiescent Cells and Requires the Enzyme for Replication

ABSTRACT Herpesviruses accomplish DNA replication either by expressing their own deoxyribonucleotide biosynthetic genes or by stimulating the expression of the corresponding cellular genes. Cytomegalovirus (CMV) has adopted the latter strategy to allow efficient replication in quiescent cells. In the present report, we show that murine CMV (MCMV) infection of quiescent fibroblasts induces both mRNA and protein corresponding to the cellular thymidylate synthase (TS) gene, which encodes the enzyme that catalyzes the de novo synthesis of thymidylic acid. The increase in TS gene expression was due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse TS promoter was induced following MCMV infection. Mutagenesis of the potential E2F-responsive element immediately upstream from the TS essential promoter region abolished the virus-mediated stimulation of the TS promoter, suggesting that the transactivating activity of MCMV infection was E2F dependent. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in TS promoter activity. Finally, MCMV replication and viral DNA synthesis were found to be inhibited by ZD1694, a quinazoline-based folate analog that inhibits TS activity. These results demonstrate that upregulation of cellular TS expression is required for efficient MCMV replication in quiescent cells.

Cell Cycle Arrest by Human Cytomegalovirus 86-kDa IE2 Protein Resembles Premature Senescence

ABSTRACT Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display β-galactosidase (β-Gal) activity at neutral pH (senescence-associated β-Gal [SA-β-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16INK4a, a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21CIP1/WAF. Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (α) genes are sufficient for the induction of SA-β-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16INK4a (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-β-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16INK4a and p53 pathways.