Anna Luganini

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.

Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers

ABSTRACT In response to the need for new antiviral agents, dendrimer-based molecules have been recognized as having a large number of potential therapeutic applications. They include peptide-derivatized dendrimers, which are hyperbranched synthetic well-defined molecules which consist of a peptidyl branching core and covalently attached surface functional peptides. However, few studies have addressed their applications as direct-acting antiviral agents. Here, we report on the ability of the peptide dendrimer SB105 and its derivative, SB105_A10, to directly inhibit herpes simplex virus 1 (HSV-1) and HSV-2 in vitro replication, with favorable selective indexes discerned for both compounds. An analysis of their mode of action revealed that SB105 and SB105_A10 prevent HSV-1 and HSV-2 attachment to target cells, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal antiviral activity, was ineffective in blocking HSV attachment. Moreover, both SB105 and SB105_A10 retained their ability to inhibit HSV adsorption at pH 3.0 and 4.0 and in the presence of 10% human serum proteins, conditions mimicking the physiological properties of the vagina, a potential therapeutic location for such compounds. The inhibition of HSV adsorption is likely to stem from the ability of SB105_A10 to bind to the glycosaminoglycan moiety of cell surface heparan sulfate proteoglycans, thereby blocking virion attachment to target cells. Finally, when combined with acyclovir in checkerboard experiments SB105_A10 exhibited highly synergistic activity. Taken together, these findings suggest that SB105 and SB105_A10 are promising candidates for the development of novel topical microbicides for the prevention of HSV infections.

Peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate

Dendrimers are hyperbranched synthetic well-defined molecules with a number of potential applications, especially in relation to the need for new antiviral agents. One subclass of dendrimers are peptide-derivatized dendrimers which consist of a peptidyl branching core and covalently attached surface peptide functional units. Few studies have addressed the potential uses of peptide dendrimers as direct-acting antiviral agents. Here, we report on the ability of two peptide dendrimers, SB105 and SB105_A10, to directly and almost completely inhibit human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells; the agents were also found to inhibit murine CMV replication, whereas they were not able to inhibit adenovirus or vesicular stomatitis virus. The peptide dendrimers prevented adsorption of the HCMV to cells at 4 degrees C, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal anticytomegaloviral activity, was ineffective in blocking HCMV attachment. In effect, SB105_A10 bound to human cells through an interaction with cell surface heparan sulfate and thereby blocked virion attachment to target cells. These results indicate that the SB105 and SB105_A10 dendrimers could provide a useful starting point for the development of novel molecules to block HCMV infection.

Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

BackgroundHuman monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients.ResultsThe purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency ~20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated.ConclusionThe technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses.

Phosphorothioate-Modified Oligodeoxynucleotides Inhibit Human Cytomegalovirus Replication by Blocking Virus Entry

ABSTRACT Studies in animal models have provided evidence that Toll-like receptor 9 (TLR9) agonists, such as synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory deoxycytidyl-deoxyguanosine (CpG) motifs (CpG ODNs), protect against a wide range of viral pathogens. This antiviral activity has been suggested to be indirect and secondary to CpG-induced cytokines and inflammatory responses triggered through TLR9 activation. However, few studies have addressed the potential of CpG ODNs as direct antiviral agents. Here, we report on the ability of some CpG ODNs to directly suppress, almost completely, human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells. Murine CMV replication was inhibited as well, whereas no inhibition was observed for herpes simplex virus type 1, adenovirus, or vesicular stomatitis virus. The antiviral activity of these ODNs was significantly reduced when they were added after virus adsorption, indicating that their action may be primarily targeted to the very early phases of the HCMV cycle. In fact, the B-class prototype CpG ODN 2006 effectively prevented the nuclear localization of pp65 and input viral DNA, which suggests that it inhibits HCMV entry. Moreover, a CpG 2006 control, ODN 2137 without CpG motifs, also showed a potent inhibitory activity on the HCMV entry phase, indicating that the anticytomegaloviral activity is independent of the CpG motif. In contrast, a phosphodiester version of CpG 2006 showed reduced antiviral activity, indicating that the inhibitory activity is dependent on the phosphorothioate backbone of the ODN. These results suggest that this yet-unrecognized activity of CpG ODNs may be of interest in the development of novel anticytomegaloviral molecules.

Activation of the virus‐induced IKK/NF‐κB signalling axis is critical for the replication of human cytomegalovirus in quiescent cells

Activation of the IKK/NF‐κB signalling pathway is a hallmark of human cytomegalovirus (HCMV) infection. However, its role in regulating major immediate‐early promoter (MIEP)‐dependent transcription and HCMV replication remains controversial. This study uses a combination of genetic approaches to investigate the effects of cell culture conditions on the importance of virus‐induced NF‐κB activation during the infection of endothelial cells or fibroblasts. Adenoviral‐mediated expression of a dominant‐negative mutant of IKK2 kinase (dnIKK2) in human umbilical vein endothelial cells resulted in a strong reduction of IκBα degradation and NF‐κB activation following infection with an HCMV clinical isolate. Viral replication was impaired in dnIKK2‐expressing cells that were growth‐arrested before infection, but not in replicating cells. The inhibitory effect of dnIKK2 was independent from the virus strain and the cell type used, because replication of the laboratory AD169 strain was impaired as well in dnIKK2‐expressing quiescent fibroblasts. Moreover, progressive disruption of NF‐κB response elements within the MIEP in recombinant HCMV viruses derived from the clinical isolate prevented their replication in quiescent cells but not in actively growing cells. These results demonstrate an essential role of virus‐induced IKK/NF‐κB activity to trigger both viral IE gene expression and productive replication in quiescent cells.

Human cytomegalovirus productively infects lymphatic endothelial cells and induces a secretome that promotes angiogenesis and lymphangiogenesis through interleukin-6 and granulocyte–macrophage colony-stimulating factor

Endothelial cells (ECs) are a site of human cytomegalovirus (HCMV) productive replication, haematogenous dissemination and persistence, and are assumed to play a critical role in the development of HCMV-associated vascular diseases. Although early reports have shown the presence of HCMV antigens and DNA in lymphoid tissues, the ability of HCMV to infect lymphatic ECs (LECs) has remained unaddressed due to the lack of a suitable in vitro system. This study provided evidence that a clinical isolate of HCMV (retaining its natural endotheliotropism) was able to productively infect purified lymph node-derived LECs and that it dysregulated the expression of several LEC genes involved in the inflammatory response to viral infection. Qualitative and quantitative analysis of virus-free supernatants from HCMV-infected LEC cultures revealed virus-induced secretion of several cytokines, chemokines and growth factors, many of which are involved in the regulation of EC physiological properties. Indeed, functional assays demonstrated that the secretome produced by HCMV-infected LECs stimulated angiogenesis in both LECs and blood ECs, and that neutralization of either interleukin (IL)-6 or granulocyte-macrophage colony-stimulating factor (GM-CSF) in the secretome caused the loss of its angiogenic properties. The involvement of IL-6 and GM-CSF in the HCMV-mediated angiogenesis was further supported by the finding that the recombinant cytokines reproduced the angiogenic effects of the HCMV secretome. These findings suggest that HCMV induces haemangiogenesis and lymphangiogenesis through an indirect mechanism that relies on the stimulation of IL-6 and GM-CSF secretion from infected cells.

The US16 Gene of Human Cytomegalovirus Is Required for Efficient Viral Infection of Endothelial and Epithelial Cells

ABSTRACT The human cytomegalovirus (HCMV) US12 gene family comprises a set of 10 contiguous genes (US12 to US21), each encoding a predicted seven-transmembrane protein and whose specific functions have yet to be ascertained. While inactivation of individual US12 family members in laboratory strains of HCMV has not been found to affect viral replication in fibroblasts, inactivation of US16 was reported to increase replication in microvascular endothelial cells. Here, we investigate the properties of US16 further by ascertaining the expression pattern of its product. A recombinant HCMV encoding a tagged version of the US16 protein expressed a 33-kDa polypeptide that accumulated with late kinetics in the cytoplasmic virion assembly compartment. To elucidate the function(s) of pUS16, we generated US16-deficient mutants in the TR clinical strain of HCMV. According to previous studies, inactivation of US16 had no effect on viral replication in fibroblasts. In contrast, the US16-deficient viruses exhibited a major growth defect in both microvascular endothelial cells and retinal pigment epithelial cells. The expression of representative IE, E, and L viral proteins was impaired in endothelial cells infected with a US16 mutant virus, suggesting a defect in the replication cycle that occurs prior to IE gene expression. This defect must be due to an inefficient entry and/or postentry event, since pp65 and viral DNA did not move to the nucleus in US16 mutant-infected cells. Taken together, these data indicate that the US16 gene encodes a novel virus tropism factor that regulates, in a cell-specific manner, a pre-immediate-early phase of the HCMV replication cycle.

The Elk-1 and Serum Response Factor Binding Sites in the Major Immediate-Early Promoter of Human Cytomegalovirus Are Required for Efficient Viral Replication in Quiescent Cells and Compensate for Inactivation of the NF-κB Sites in Proliferating Cells

ABSTRACT The major immediate-early promoter (MIEP) region of human cytomegalovirus (HCMV) plays a critical role in the regulation of lytic and latent infections by integrating multiple signals supplied by the infecting virus, the type and physiological state of the host cell, and its extracellular surroundings. The interaction of cellular transcription factors with their cognate binding sites, which are present at high densities within the enhancer upstream from the MIEP core promoter, regulate the rate of IE gene transcription and thus affect the outcome of HCMV infection. We have shown previously that the NF-κB binding sites within the MIEP enhancer and cellular NF-κB activity induced by HCMV infection are required for efficient MIEP activity and viral replication in quiescent cells (P. Caposio, A. Luganini, G. Hahn, S. Landolfo, and G. Gribaudo, Cell. Microbiol. 9:2040-2054, 2007). We now show that the inactivation of either the Elk-1 or serum response factor (SRF) binding site within the enhancer also reduces MIEP activation and viral replication of recombinant HCMV viruses in quiescent fibroblasts. In these cells, we show that the expression of either Elk-1 or SRF is required for optimal IE gene expression, and that the HCMV-stimulated activation of the MEK1/2-ERK1/2 signaling axis leads to Elk-1 transcriptional competency. Furthermore, the replication kinetics of recombinant viruses in which NF-κB, Elk-1, and SRF binding sites all are inactivated demonstrate that the higher levels of Elk-1 and SRF binding to MIEP in proliferating cells can compensate even for a lack of HCMV-induced NF-κB-mediated MIEP transactivation. These observations highlight the importance of the combination of different MIEP binding sites to optimize IE gene expression in cells in different physiological states.

New cell-based indicator assays for the detection of human cytomegalovirus infection and screening of inhibitors of viral immediate-early 2 protein activity

Aims:  Expression of early (E) genes of human cytomegalovirus (HCMV) is stimulated cooperatively by the activities of host cell transcription factors and the viral immediate‐early 2 (IE2) protein. Taking advantage of the IE2‐dependent inducibility of E gene promoters, in this study, we generated cell‐based assays in which the expression of the enhanced green fluorescence protein (EGFP) reporter gene was driven by the UL54 or UL112/113 E promoters.