44 results on '"Tatiana Gianni"'
Search Results
2. Decrease in Heparan Sulphate Binding in Tropism-Retargeted Oncolytic Herpes Simplex Virus (ReHV) Delays Blood Clearance and Improves Systemic Anticancer Efficacy
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Andrea Vannini, Federico Parenti, Cristina Forghieri, Gaia Vannini, Catia Barboni, Anna Zaghini, Tatiana Gianni, and Gabriella Campadelli-Fiume
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oncolytic virus ,oncolytic herpes simplex virus ,retargeting ,HER2 ,heparan sulphate ,systemic oncolytic therapy ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
The role of the interaction with cell-surface glycosaminoglycans (GAGs) during in vivo HSV infection is currently unknown. The rationale of the current investigation was to improve the anticancer efficacy of systemically administered retargeted oHSVs (ReHVs) by decreasing their binding to GAGs, including those of endothelial cells, blood cells, and off-tumor tissues. As a proof-of-principle approach, we deleted seven amino acids critical for interacting with GAGs from the glycoprotein C (gC) of R-337 ReHV. The modification in the resulting R-399 recombinant prolonged the half-life in the blood of systemically administered R-399 and enhanced its biodistribution to tumor-positive lungs and to the tumor-negative liver. Ultimately, it greatly increased the R-399 efficacy against metastatic-like lung tumors upon IV administration but not against subcutaneous tumors upon IT administration. These results provide evidence that the increased efficacy seen upon R-399 systemic administration correlated with the slower clearance from the circulation. To our knowledge, this is the first in vivo evidence that the partial impairment of the gC interaction with GAGs resulted in a prolonged half-life of circulating ReHV, an increase in the amount of ReHV taken up by tissues and tumors, and, ultimately, an enhanced anticancer efficacy of systemically administered ReHV.
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- 2024
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3. Innovative retargeted oncolytic herpesvirus against nectin4-positive cancers
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Andrea Vannini, Federico Parenti, Cristina Forghieri, Catia Barboni, Anna Zaghini, Gabriella Campadelli-Fiume, and Tatiana Gianni
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oncolytic virus ,oncolytic herpes simplex virus ,retargeting ,nectin4 ,anti-cancer vaccine ,immunotherapy ,Biology (General) ,QH301-705.5 - Abstract
Nectin4 is a recently discovered tumor associated antigen expressed in cancers that constitute relevant unmet clinical needs, including the undruggable triple negative breast cancer, pancreatic ductal carcinoma, bladder/urothelial cancer, cervical cancer, lung carcinoma and melanoma. So far, only one nectin4-specific drug—Enfortumab Vedotin—has been approved and the clinical trials that test novel therapeutics are only five. Here we engineered R-421, an innovative retargeted onco-immunotherapeutic herpesvirus highly specific for nectin4 and unable to infect through the natural herpes receptors, nectin1 or herpesvirus entry mediator. In vitro, R-421 infected and killed human nectin4-positive malignant cells and spared normal cells, e.g., human fibroblasts. Importantly from a safety viewpoint, R-421 failed to infect malignant cells that do not harbor nectin4 gene amplification/overexpression, whose expression level was moderate-to-low. In essence, there was a net threshold value below which cells were spared from infection, irrespective of whether they were malignant or normal; the only cells that R-421 targeted were the malignant overexpressing ones. In vivo, R-421 decreased or abolished the growth of murine tumors made transgenic for human nectin4 and conferred sensitivity to immune checkpoint inhibitors in combination therapies. Its efficacy was augmented by the cyclophosphamide immunomodulator and decreased by depletion of CD8-positive lymphocytes, arguing that it was in part T cell-mediated. R-421 elicited in-situ vaccination that protected from distant challenge tumors. This study provides proof-of-principle specificity and efficacy data justifying nectin4-retargeted onco-immunotherapeutic herpesvirus as an innovative approach against a number of difficult-to-drug clinical indications.
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- 2023
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4. Efficacy of Systemically Administered Retargeted Oncolytic Herpes Simplex Viruses—Clearance and Biodistribution in Naïve and HSV-Preimmune Mice
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Andrea Vannini, Federico Parenti, Catia Barboni, Cristina Forghieri, Valerio Leoni, Mara Sanapo, Daniela Bressanin, Anna Zaghini, Gabriella Campadelli-Fiume, and Tatiana Gianni
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oncolytic virus ,oncolytic herpes simplex virus ,retargeting ,HER2 ,PSMA ,anti-cancer vaccine ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
We investigated the anticancer efficacy, blood clearance, and tissue biodistribution of systemically administered retargeted oncolytic herpes simplex viruses (ReHVs) in HSV-naïve and HSV-preimmunized (HSV-IMM) mice. Efficacy was tested against lung tumors formed upon intravenous administration of cancer cells, a model of metastatic disease, and against subcutaneous distant tumors. In naïve mice, HER2- and hPSMA-retargeted viruses, both armed with mIL-12, were highly effective, even when administered to mice with well-developed tumors. Efficacy was higher for combination regimens with immune checkpoint inhibitors. A significant amount of infectious virus persisted in the blood for at least 1 h. Viral genomes, or fragments thereof, persisted in the blood and tissues for days. Remarkably, the only sites of viral replication were the lungs of tumor-positive mice and the subcutaneous tumors. No replication was detected in other tissues, strengthening the evidence of the high cancer specificity of ReHVs, a property that renders ReHVs suitable for systemic administration. In HSV-IMM mice, ReHVs administered at late times failed to exert anticancer efficacy, and the circulating virus was rapidly inactivated. Serum stability and in vivo whole blood stability assays highlighted neutralizing antibodies as the main factor in virus inactivation. Efforts to deplete mice of the neutralizing antibodies are ongoing.
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- 2023
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5. Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV
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Andrea Vannini, Federico Parenti, Daniela Bressanin, Catia Barboni, Anna Zaghini, Gabriella Campadelli-Fiume, and Tatiana Gianni
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oncolytic virus ,oncolytic herpes simplex virus ,retargeting ,PSMA ,prostate cancer ,vaccination ,Microbiology ,QR1-502 - Abstract
Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.
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- 2021
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6. Genotype of Immunologically Hot or Cold Tumors Determines the Antitumor Immune Response and Efficacy by Fully Virulent Retargeted oHSV
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Tatiana Gianni, Valerio Leoni, Mara Sanapo, Federico Parenti, Daniela Bressanin, Catia Barboni, Anna Zaghini, Gabriella Campadelli-Fiume, and Andrea Vannini
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oncolytic virus ,oncolytic herpes simplex virus ,retargeting ,HER2 ,tumor genotype ,vaccination ,Microbiology ,QR1-502 - Abstract
We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased; Cd40 and Cd27 co-immunostimulatory markers were increased; the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely.
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- 2021
- Full Text
- View/download PDF
7. Immunotherapeutic Efficacy of Retargeted oHSVs Designed for Propagation in an Ad Hoc Cell Line
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Andrea Vannini, Valerio Leoni, Mara Sanapo, Tatiana Gianni, Giorgia Giordani, Valentina Gatta, Catia Barboni, Anna Zaghini, and Gabriella Campadelli-Fiume
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oncolytic virus ,herpes simplex virus ,retargeted virus ,tropism retargeting ,tumor ,immunotherapy ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Our laboratory has pursued the generation of cancer-specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor-Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off-target and off-tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R-335 and R-337 prototypes were armed with murine IL-12. Intratumorally-administered R-337 conferred almost complete protection from LLC-1-HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long-term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen-agnostic vaccines.
- Published
- 2021
- Full Text
- View/download PDF
8. Insertion of a ligand to HER2 in gB retargets HSV tropism and obviates the need for activation of the other entry glycoproteins.
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Biljana Petrovic, Tatiana Gianni, Valentina Gatta, and Gabriella Campadelli-Fiume
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Herpes simplex virus (HSV) entry into the cells requires glycoproteins gD, gH/gL and gB, activated in a cascade fashion by conformational modifications induced by cognate receptors and intermolecular signaling. The receptors are nectin1 and HVEM (Herpes virus entry mediator) for gD, and αvβ6 or αvβ8 integrin for gH. In earlier work, insertion of a single chain antibody (scFv) to the cancer receptor HER2 (human epidermal growth factor receptor 2) in gD, or in gH, resulted in HSVs specifically retargeted to the HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Here, the scFv to HER2 was inserted in gB (gBHER2). The insertion re-targeted the virus tropism to the HER2-positive cancer cells. This was unexpected since gB is known to be a fusogenic glycoprotein, not a tropism determinant. The gB-retargeted recombinant offered the possibility to investigate how HER2 mediated entry. In contrast to wt-gB, the activation of the chimeric gBHER2 did not require the activation of the gD and of gH/gL by their respective receptors. Furthermore, a soluble form of HER2 could replace the membrane-bound HER2 in mediating virus entry, hinting that HER2 acted by inducing conformational changes to the chimeric gB. This study shows that (i) gB can be modified and become the major determinant of HSV tropism; (ii) the chimeric gBHER2 bypasses the requirement for receptor-mediated activation of other essential entry glycoproteins.
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- 2017
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9. The epithelial αvβ3-integrin boosts the MYD88-dependent TLR2 signaling in response to viral and bacterial components.
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Tatiana Gianni and Gabriella Campadelli-Fiume
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
TLR2 is a cell surface receptor which elicits an immediate response to a wide repertoire of bacteria and viruses. Its response is usually thought to be proinflammatory rather than an antiviral. In monocytic cells TLR2 cooperates with coreceptors, e.g. CD14, CD36 and αMβ2-integrin. In an earlier work we showed that αvβ3-integrin acts in concert with TLR2 to elicit an innate response to HSV, and to lipopolysaccharide. This response is characterized by production of IFN-α and -β, a specific set of cytokines, and NF-κB activation. We investigated the basis of the cooperation between αvβ3-integrin and TLR2. We report that β3-integrin participates by signaling through Y residues located in the C-tail, known to be involved in signaling activity. αvβ3-integrin boosts the MYD88-dependent TLR2 signaling and IRAK4 phosphorylation in 293T and in epithelial, keratinocytic and neuronal cell lines. The replication of ICP0minus HSV is greatly enhanced by DN versions of MYD88, of Akt - a hub of this pathway, or by β3integrin-silencing. αvβ3-integrin enables the recruitment of TLR2, MAL, MYD88 at lipid rafts, the platforms from where the signaling starts. The PAMP of the HSV-induced innate response is the gH/gL virion glycoprotein, which interacts with αvβ3-integrin and TLR2 independently one of the other, and cross-links the two receptors. Given the preferential distribution of αvβ3-integrin to epithelial cells, we propose that αvβ3-integrin serves as coreceptor of TLR2 in these cells. The results open the possibility that TLR2 makes use of coreceptors in a variety of cells to broaden its spectrum of activity and tissue specificity.
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- 2014
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10. αvβ6- and αvβ8-integrins serve as interchangeable receptors for HSV gH/gL to promote endocytosis and activation of membrane fusion.
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Tatiana Gianni, Stefano Salvioli, Liudmila S Chesnokova, Lindsey M Hutt-Fletcher, and Gabriella Campadelli-Fiume
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Herpes simplex virus (HSV)--and herpesviruses in general--encode for a multipartite entry/fusion apparatus. In HSV it consists of the HSV-specific glycoprotein D (gD), and three additional glycoproteins, gH/gL and gB, conserved across the Herpesviridae family and responsible for the execution of fusion. According to the current model, upon receptor binding, gD propagates the activation to gH/gL and to gB in a cascade fashion. Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis, to avoid premature activation and exhaustion of the glycoproteins. We considered the possibility that such control might be carried out by as yet unknown receptors. Indeed, receptors for HSV gB, but not for gH/gL, have been described. In other members of the Herpesviridae family, such as Epstein-Barr virus, integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins. We report that αvβ6- and αvβ8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells. Evidence rests on loss of function experiments, in which integrins were blocked by antibodies or silenced, and gain of function experiments in which αvβ6-integrin was expressed in integrin-negative cells. αvβ6- and αvβ8-integrins acted independently and are thus interchangeable. Both bind gH/gL with high affinity. The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes. In the case of αvβ8, but not αvβ6-integrin, the portal of entry is located at lipid microdomains and requires dynamin 2. Thus, a major role of αvβ6- or αvβ8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis. We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion.
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- 2013
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11. Halting the Spread of Herpes Simplex Virus-1: The Discovery of an Effective Dual αvβ6/αvβ8 Integrin Ligand
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Stefano Tomassi, Francesco Saverio Di Leva, Giovanna Musco, Salvatore Di Maro, Horst Kessler, Michael Weinmüller, Tatiana Gianni, Barbara Romano, Jussara Amato, Giacomo Quilici, Ettore Novellino, Angelo A. Izzo, Andrea Vannini, Florian Reichart, Vincenzo Maria D'Amore, Luciana Marinelli, Tomassi, S., D'Amore, V. M., Di Leva, F. S., Vannini, A., Quilici, G., Weinmuller, M., Reichart, F., Amato, J., Romano, B., Izzo, A. A., Di Maro, S., Novellino, E., Musco, G., Gianni, T., Kessler, H., Marinelli, L., Tomassi S., D'Amore V.M., Di Leva F.S., Vannini A., Quilici G., Weinmuller M., Reichart F., Amato J., Romano B., Izzo A.A., Di Maro S., Novellino E., Musco G., Gianni T., Kessler H., and Marinelli L.
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Integrins ,Small interfering RNA ,Molecular model ,Integrin ,Peptide ,Herpesvirus 1, Human ,Integrin ligand ,Ligands ,medicine.disease_cause ,Peptides, Cyclic ,01 natural sciences ,Article ,03 medical and health sciences ,HEK293 Cell ,Antigens, Neoplasm ,Drug Discovery ,medicine ,Humans ,030304 developmental biology ,chemistry.chemical_classification ,Infectivity ,0303 health sciences ,Binding Sites ,biology ,Binding Site ,Virus Internalization ,Combined approach ,ddc ,0104 chemical sciences ,Cell biology ,Molecular Docking Simulation ,010404 medicinal & biomolecular chemistry ,HEK293 Cells ,Herpes simplex virus ,chemistry ,biology.protein ,Oligopeptide ,Molecular Medicine ,Oligopeptides ,Human ,Protein Binding - Abstract
Over recent years, αvβ6 and αvβ8 Arg-Gly-Asp (RGD) integrins have risen to prominence as interchangeable co-receptors for the cellular entry of herpes simplex virus-1 (HSV-1). In fact, the employment of subtype-specific integrin-neutralizing antibodies or gene-silencing siRNAs has emerged as a valuable strategy for impairing HSV infectivity. Here, we shift the focus to a more affordable pharmaceutical approach based on small RGD-containing cyclic pentapeptides. Starting from our recently developed αvβ6-preferential peptide [RGD-Chg-E]-CONH2 (1), a small library of N-methylated derivatives (2–6) was indeed synthesized in the attempt to increase its affinity toward αvβ8. Among the novel compounds, [RGD-Chg-(NMe)E]-CONH2 (6) turned out to be a potent αvβ6/αvβ8 binder and a promising inhibitor of HSV entry through an integrin-dependent mechanism. Furthermore, the renewed selectivity profile of 6 was fully rationalized by a NMR/molecular modeling combined approach, providing novel valuable hints for the design of RGD integrin ligands with the desired specificity profile.
- Published
- 2021
12. Genotype of Immunologically Hot or Cold Tumors Determines the Antitumor Immune Response and Efficacy by Fully Virulent Retargeted oHSV
- Author
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Gabriella Campadelli-Fiume, Anna Zaghini, Valerio Leoni, Tatiana Gianni, Mara Sanapo, Federico Parenti, Andrea Vannini, Daniela Bressanin, Catia Barboni, Gianni T., Leoni V., Sanapo M., Parenti F., Bressanin D., Barboni C., Zaghini A., Campadelli-Fiume G., and Vannini A.
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Receptor, ErbB-2 ,medicine.medical_treatment ,Cell ,Herpesvirus 1, Human ,Immune checkpoint inhibitor ,Virus Replication ,immune checkpoint inhibitors ,Mice ,0302 clinical medicine ,Neoplasms ,skin and connective tissue diseases ,0303 health sciences ,Mice, Inbred BALB C ,Immunosuppression ,oncolytic herpes simplex virus ,Primary tumor ,QR1-502 ,3. Good health ,Oncolytic Viruses ,Infectious Diseases ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Immunotherapy ,Oncolytic herpes simplex viru ,Genotype ,T cell ,Mice, Transgenic ,Biology ,tumor genotype ,Microbiology ,Article ,03 medical and health sciences ,Immune system ,Virology ,HER2 ,medicine ,Animals ,Humans ,030304 developmental biology ,oncolytic virus ,retargeting ,Immunity ,Oncolytic viru ,medicine.disease ,vaccination ,Oncolytic virus ,Mice, Inbred C57BL ,Viral replication ,Cancer research - Abstract
We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased, Cd40 and Cd27 co-immunostimulatory markers were increased, the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely.
- Published
- 2021
13. αvβ3-integrin regulates PD-L1 expression and is involved in cancer immune evasion
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Catia Barboni, Anna Zaghini, Valerio Leoni, Tatiana Gianni, Gabriella Campadelli-Fiume, Andrea Vannini, Paolo Malatesta, Mara Sanapo, Vannini A., Leoni V., Barboni C., Sanapo M., Zaghini A., Malatesta P., Campadelli-Fiume G., and Gianni T.
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medicine.medical_treatment ,Integrin ,Cell ,αvβ3-integrin ,PD-L1 expression ,Corrections ,combination therapy ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Antigen ,Cancer immunotherapy ,medicine ,Splenocyte ,immune evasion ,030304 developmental biology ,0303 health sciences ,cancer immunotherapy ,Multidisciplinary ,biology ,Chemistry ,Immunotherapy ,3. Good health ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,CD8 - Abstract
Tumors utilize a number of effective strategies, including the programmed death 1/PD ligand 1 (PD-1/PD-L1) axis, to evade immune-mediated control of their growth. PD-L1 expression is mainly induced by IFN receptor signaling or constitutively induced. Integrins are an abundantly expressed class of proteins which play multiple deleterious roles in cancer and exert proangiogenic and prosurvival activities. We asked whether αvβ3-integrin positively regulates PD-L1 expression and the anticancer immune response. We report that αvβ3-integrin regulated constitutive and IFN-induced PD-L1 expression in human and murine cancerous and noncancerous cells. αvβ3-integrin targeted STAT1 through its signaling C tail. The implantation of β3-integrin-depleted tumor cells led to a dramatic decrease in the growth of primary tumors, which exhibited reduced PD-L1 expression and became immunologically hot, with increased IFNγ content and CD8+ cell infiltration. In addition, the implantation of β3-integrin-depleted tumors elicited an abscopal immunotherapeutic effect measured as protection from the challenge tumor and durable splenocyte and serum reactivity to B16 cell antigens. These modifications to the immunosuppressive microenvironment primed cells for checkpoint (CP) blockade. When combined with anti-PD-1, β3-integrin depletion led to durable therapy and elicited an abscopal immunotherapeutic effect. We conclude that in addition to its previously known roles, αvβ3-integrin serves as a critical component of the cancer immune evasion strategy and can be an effective immunotherapy target.
- Published
- 2019
14. Immunotherapeutic Efficacy of Retargeted oHSVs Designed for Propagation in an Ad Hoc Cell Line
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Catia Barboni, Anna Zaghini, Tatiana Gianni, Valentina Gatta, Andrea Vannini, Giorgia Giordani, Mara Sanapo, Gabriella Campadelli-Fiume, Valerio Leoni, Vannini A., Leoni V., Sanapo M., Gianni T., Giordani G., Gatta V., Barboni C., Zaghini A., and Campadelli-fiume G.
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0301 basic medicine ,Cancer Research ,tumor ,medicine.medical_treatment ,antigen-agnostic vaccination ,Review ,Biology ,medicine.disease_cause ,Retargeted viru ,lcsh:RC254-282 ,tropism retargeting ,Antigen‐agnostic vaccination ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,HER2 ,medicine ,Herpes simplex viru ,oncolytic virus ,Tumor microenvironment ,retargeted virus ,Oncolytic viru ,Immunotherapy ,vaccination ,herpes simplex virus ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,3. Good health ,Oncolytic virus ,checkpoint inhibitor ,030104 developmental biology ,Herpes simplex virus ,Oncology ,030220 oncology & carcinogenesis ,Cancer cell ,Retargeting ,Cancer research ,Vero cell ,immunotherapy - Abstract
Simple Summary The onco-immunotherapeutic viruses, among which stand the onco-immunotherapeutic herpes simplex viruses, have gained renewed interest due to their ability to unlock the potential of checkpoint inhibitors in preclinical and clinical settings. In prior decades, safety concerns led to the generation of overall safe, partially or highly attenuated oncolytic viruses. Current focus is on more efficacious onco-immunotherapeutic viruses with limited ability to cause off-tumor and off-target infections and the capability to subvert the tumor microenvironment immunosuppression—hence to potentiate checkpoint inhibitors. These viruses might serve as potential partners of T-cell therapies. Abstract Our laboratory has pursued the generation of cancer-specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor-Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off-target and off-tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R-335 and R-337 prototypes were armed with murine IL-12. Intratumorally-administered R-337 conferred almost complete protection from LLC-1-HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long-term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen-agnostic vaccines.
- Published
- 2021
15. Rescue, Purification, and Characterization of a Recombinant HSV Expressing a Transgenic Protein
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Tatiana Gianni, Laura Menotti, Valentina Gatta, Simona Pepe, Biljana Petrovic, Gabriella Campadelli-Fiume, Andrea Vannini, Valerio Leoni, Diefenbach R., Fraefel C. (eds), and Andrea Vannini, Biljana Petrovic, Valentina Gatta, Valerio Leoni, Simona Pepe, Laura Menotti, Gabriella Campadelli-Fiume, Tatiana Gianni
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0301 basic medicine ,Virus quantification ,Transgene ,Biology ,Recombinant virus ,Genome ,Recombineering ,law.invention ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,mIL12 transgene expression ,In vivo ,law ,Plaque purification ,030220 oncology & carcinogenesis ,Recombinant DNA ,Interleukin 12 ,HSV rescue ,Virion purification ,Plaque assay - Abstract
In the previous chapter, we describe the engineering of a HSV-BAC genome by galK recombineering. Here we describe the procedures to reconstitute, or regenerate, the replicating recombinant virus, and the methods to purify it and characterize it for the correct expression of the transgene. We present the example of R-115, a recombinant expressing murine interleukin 12 (mIL12) from the US1-US2 intergenic region. A specific method for the production of highly purified virions by iodixanol gradient, suitable for in vivo applications, is also detailed.
- Published
- 2019
16. oHSV Genome Editing by Means of galK Recombineering
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Andrea Vannini, Tatiana Gianni, Laura Menotti, Valerio Leoni, Biljana Petrovic, Simona Pepe, Gabriella Campadelli-Fiume, Valentina Gatta, Russell J. Diefenbach, Cornel Fraefel, and Laura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-Fiume
- Subjects
0301 basic medicine ,Bacterial artificial chromosome ,Virus arming ,viruses ,Virus engineering ,Computational biology ,Amplicon ,Biology ,Recombinant virus ,medicine.disease_cause ,Genome ,Recombineering ,galK recombineering ,Interleukin 12 ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Herpes simplex virus ,Genome editing ,030220 oncology & carcinogenesis ,medicine ,Herpes simplex viru ,Transgene expression ,Oncolytic virotherapy ,Gene - Abstract
Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.
- Published
- 2019
17. Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV
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Tatiana Gianni, Gabriella Campadelli-Fiume, Catia Barboni, Federico Parenti, Daniela Bressanin, Anna Zaghini, Andrea Vannini, Vannini A., Parenti F., Bressanin D., Barboni C., Zaghini A., Campadelli-Fiume G., and Gianni T.
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Male ,medicine.medical_treatment ,Immune checkpoint inhibitor ,urologic and male genital diseases ,immune checkpoint inhibitors ,Prostate cancer ,0302 clinical medicine ,Prostate ,Tumor Microenvironment ,Precision Medicine ,In Situ Hybridization ,Oncolytic Virotherapy ,0303 health sciences ,oncolytic herpes simplex virus ,prostate cancer ,Primary tumor ,QR1-502 ,3. Good health ,Oncolytic Viruses ,Infectious Diseases ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Kallikreins ,immunotherapy ,Oncolytic herpes simplex viru ,Antineoplastic Agents ,Microbiology ,Article ,03 medical and health sciences ,Immune system ,Antigen ,Cell Line, Tumor ,Virology ,LNCaP ,PSMA ,medicine ,Humans ,oncolytic virus ,retargeting ,030304 developmental biology ,business.industry ,Oncolytic viru ,Prostatic Neoplasms ,Cancer ,Immunotherapy ,Prostate-Specific Antigen ,vaccination ,medicine.disease ,in situ vaccine ,Cancer research ,business ,Biomarkers - Abstract
Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.
- Published
- 2021
18. oHSV Genome Editing by Means of galK Recombineering
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Laura, Menotti, Valerio, Leoni, Valentina, Gatta, Biljana, Petrovic, Andrea, Vannini, Simona, Pepe, Tatiana, Gianni, and Gabriella, Campadelli-Fiume
- Subjects
Gene Editing ,Chromosomes, Artificial, Bacterial ,Galactokinase ,Mice ,Viral Proteins ,Escherichia coli Proteins ,Escherichia coli ,Animals ,Herpesvirus 1, Human - Abstract
Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.
- Published
- 2019
19. Rescue, Purification, and Characterization of a Recombinant HSV Expressing a Transgenic Protein
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Andrea, Vannini, Biljana, Petrovic, Valentina, Gatta, Valerio, Leoni, Simona, Pepe, Laura, Menotti, Gabriella, Campadelli-Fiume, and Tatiana, Gianni
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Recombination, Genetic ,Chromosomes, Artificial, Bacterial ,Mice ,Cell Line, Tumor ,Animals ,Gene Expression ,Herpesvirus 1, Human ,Interleukin-12 ,Recombinant Proteins - Abstract
In the previous chapter, we describe the engineering of a HSV-BAC genome by galK recombineering. Here we describe the procedures to reconstitute, or regenerate, the replicating recombinant virus, and the methods to purify it and characterize it for the correct expression of the transgene. We present the example of R-115, a recombinant expressing murine interleukin 12 (mIL12) from the US1-US2 intergenic region. A specific method for the production of highly purified virions by iodixanol gradient, suitable for in vivo applications, is also detailed.
- Published
- 2019
20. Correction for Vannini et al., αvβ3-integrin regulates PD-L1 expression and is involved in cancer immune evasion
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Paolo Malatesta, Valerio Leoni, Andrea Vannini, Gabriella Campadelli-Fiume, Mara Sanapo, Catia Barboni, Tatiana Gianni, and Anna Zaghini
- Subjects
αvβ3 integrin ,Multidisciplinary ,cancer immunotherapy ,business.industry ,Cancer ,αvβ3-integrin ,Biological Sciences ,PD-L1 expression ,Evasion (ethics) ,medicine.disease ,Microbiology ,combination therapy ,Immune system ,PNAS Plus ,Cancer research ,Medicine ,Pd l1 expression ,business ,immune evasion - Abstract
Significance The PD-1/PD-L1 axis is a master player in the tumor immune evasion strategy. Checkpoint inhibitors, including anti–PD-1/PD-L1, are revolutionizing cancer immunotherapy. There is intense interest in dissecting their regulation and improving their application, mainly by combination therapies. The significance of the current findings is 2-fold. Our results suggest αvβ3-integrin as a critical regulator of PD-L1 expression and a key component of the tumor immune evasion machinery. Indeed, αvβ3-integrin depletion impairs tumor growth and elicits immunotherapeutic protection. Second, αvβ3-integrin blockade primes tumors for anti–PD-1 therapy and induces durable anticancer immune protection when combined with anti–PD-1 therapy. αvβ3-integrin is a readily druggable target that adds to the list of molecules suitable for combinatorial cancer immunotherapy., Tumors utilize a number of effective strategies, including the programmed death 1/PD ligand 1 (PD-1/PD-L1) axis, to evade immune-mediated control of their growth. PD-L1 expression is mainly induced by IFN receptor signaling or constitutively induced. Integrins are an abundantly expressed class of proteins which play multiple deleterious roles in cancer and exert proangiogenic and prosurvival activities. We asked whether αvβ3-integrin positively regulates PD-L1 expression and the anticancer immune response. We report that αvβ3-integrin regulated constitutive and IFN-induced PD-L1 expression in human and murine cancerous and noncancerous cells. αvβ3-integrin targeted STAT1 through its signaling C tail. The implantation of β3-integrin–depleted tumor cells led to a dramatic decrease in the growth of primary tumors, which exhibited reduced PD-L1 expression and became immunologically hot, with increased IFNγ content and CD8+ cell infiltration. In addition, the implantation of β3-integrin–depleted tumors elicited an abscopal immunotherapeutic effect measured as protection from the challenge tumor and durable splenocyte and serum reactivity to B16 cell antigens. These modifications to the immunosuppressive microenvironment primed cells for checkpoint (CP) blockade. When combined with anti–PD-1, β3-integrin depletion led to durable therapy and elicited an abscopal immunotherapeutic effect. We conclude that in addition to its previously known roles, αvβ3-integrin serves as a critical component of the cancer immune evasion strategy and can be an effective immunotherapy target.
- Published
- 2019
21. Simultaneous Insertion of Two Ligands in gD for Cultivation of Oncolytic Herpes Simplex Viruses in Noncancer Cells and Retargeting to Cancer Receptors
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Valentina Gatta, Valerio Leoni, Tatiana Gianni, Gabriella Campadelli-Fiume, and Biljana Petrovic
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0301 basic medicine ,gD ,Immunology ,Herpesvirus 1, Human ,Biology ,Microbiology ,Virus ,Gene Delivery ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Viral Envelope Proteins ,HER2 ,Virology ,Chlorocebus aethiops ,Animals ,Humans ,Receptor ,Vero Cells ,retargeting ,oncolytic virus ,HSV ,Vero ,3. Good health ,Oncolytic virus ,Oncolytic Viruses ,Viral Tropism ,030104 developmental biology ,030220 oncology & carcinogenesis ,Insect Science ,Cancer cell ,Retargeting ,Vero cell ,biology.protein ,Antibody ,Peptides - Abstract
Insertion of a single-chain variable-fragment antibody (scFv) to HER2 (human epidermal growth factor receptor 2) in gD, gH, or gB gives rise to herpes simplex viruses (HSVs) specifically retargeted to HER2-positive cancer cells, hence to highly specific nonattenuated oncolytic agents. Clinical-grade virus production cannot rely on cancer cells. Recently, we developed a double-retargeting strategy whereby gH carries the GCN4 peptide for retargeting to the noncancer producer Vero-GCN4R cell line and gD carries the scFv to HER2 for cancer retargeting. Here, we engineered double-retargeted recombinants, which carry both the GCN4 peptide and the scFv to HER2 in gD. Novel, more-advantageous detargeting strategies were devised so as to optimize the cultivation of the double-retargeted recombinants. Nectin1 detargeting was achieved by deletion of amino acids (aa) 35 to 39, 214 to 223, or 219 to 223 and replacement of the deleted sequences with one of the two ligands. The last two deletions were not attempted before. All recombinants exhibited the double retargeting to HER2 and to the Vero-GCN4R cells, as well as detargeting from the natural receptors HVEM and nectin1. Of note, some recombinants grew to higher yields than others. The best-performing recombinants carried a gD deletion as small as 5 amino acids and grew to titers similar to those exhibited by the singly retargeted R-LM113 and by the nonretargeted R-LM5. This study shows that double retargeting through insertion of two ligands in gD is feasible and, when combined with appropriate detargeting modifications, can result in recombinants highly effective in vitro and in vivo . IMPORTANCE There is increasing interest in oncolytic viruses following the FDA and European Medicines Agency (EMA) approval of the oncolytic HSV Oncovex GM-CSF and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly immune checkpoint inhibitors. A strategy to gain high cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. However, cultivation of retargeted oncolytics in cells expressing the cancer receptor may not be approvable by regulatory agencies. We devised a strategy for their cultivation in noncancer cells. Here, we describe a double-retargeting strategy, based on the simultaneous insertion of two ligands in gD, one for retargeting to a producer, universal Vero cell derivative and one for retargeting to the HER2 cancer receptor. These insertions were combined with novel, minimally disadvantageous detargeting modifications. The current and accompanying studies indicate how to best achieve the clinical-grade cultivation of retargeted oncolytics.
- Published
- 2018
22. Type I Interferon and NF-κB Activation Elicited by Herpes Simplex Virus gH/gL via αvβ3 Integrin in Epithelial and Neuronal Cell Lines
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Tatiana Gianni, Gabriella Campadelli-Fiume, Valerio Leoni, T. Gianni, V. Leoni, and G. Campadelli-Fiume
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viruses ,Immunology ,Cellular Response to Infection ,Herpesvirus 1, Human ,Integrin alpha5 ,Biology ,medicine.disease_cause ,DENDRITIC CELLS ,Microbiology ,Cell Line ,Viral Proteins ,Viral Envelope Proteins ,Interferon ,Viral entry ,Virology ,medicine ,TLR2 ,Humans ,Receptor ,Neurons ,Membrane Glycoproteins ,αvβ3 integrin in epithelial and neuronal cell line ,TOLL-LIKE RECEPTOR-2 ,Contraindications ,Integrin beta3 ,NF-kappa B ,Epithelial Cells ,Herpes Simplex ,Herpesvirus glycoprotein B ,Molecular biology ,infection ,POLIOVIRUS RECEPTOR ,LIPID RAFTS ,Herpes simplex virus ,Cell culture ,Insect Science ,ENTRY ,REPLICATION ,Interferon Type I ,recognition ,protein ,Interferon type I ,Molecular Chaperones ,medicine.drug - Abstract
αvβ3 integrin represents a novel sensing system which detects herpes simplex virus (HSV) and bacterial constituents. In cooperation with Toll-like receptor 2 (TLR2), it elicits an innate response that leads to activation of type I interferon (IFN), NF-κB, and a specific set of cytokines. We report that this defensive branch is functional in cells which represent experimental models of epithelial, including keratinocytic, and neuronal cells. These are the major targets of HSV in vivo . HSV entered the three cell lines via distinct routes. Hence, the defensive response was independent of the route of virus entry. Soluble gH/gL sufficed to elicit type I IFN and NF-κB activation and represents the viral pathogen-associated molecular pattern (PAMP) of this defense system.
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- 2013
23. αvβ3-integrin is a major sensor and activator of innate immunity to herpes simplex virus-1
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Liudmila S. Chesnokova, Tatiana Gianni, Gabriella Campadelli-Fiume, Lindsey M. Hutt-Fletcher, Valerio Leoni, Gianni T., Leoni V., Chesnokova L.S., Hutt-Fletcher L.M., and Campadelli-Fiume G.
- Subjects
Gene Expression ,Syk ,Enzyme-Linked Immunosorbent Assay ,Herpesvirus 1, Human ,Biology ,Virus Replication ,medicine.disease_cause ,Polymerase Chain Reaction ,Cell Line ,Innate/physiology ,medicine ,Gene Silencing ,Receptor ,Human/immunology ,Multidisciplinary ,Innate immune system ,Herpesvirus 1 ,NF-kappa B ,Cytokines/metabolism ,Immunity ,Pattern recognition receptor ,HUMANS ,Biological Sciences ,Integrin alphaVbeta3/physiology ,Integrin alphaVbeta3 ,Virology ,Immunity, Innate ,Cell biology ,TLR2 ,Herpes simplex virus ,NF-kappa B/metabolism ,Human/physiology ,Cytokines ,Proto-oncogene tyrosine-protein kinase Src ,Interferon regulatory factors - Abstract
Pathogens are sensed by Toll-like receptors (TLRs) and a growing number of non-TLR receptors. Integrins constitute a family of signaling receptors exploited by viruses and bacteria to access cells. By gain- and loss-of-function approaches we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against herpes simplex virus (HSV). αvβ3-integrin signaled through two pathways. One concurred with TLR2, affected activation/induction of interferons type 1 (IFNs-1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and a polarized set of cytokines and receptors. The virion glycoproteins gH/gL sufficed to induce IFN1 and NF-κB via this pathway. The other pathway was TLR2-independent, involved sarcoma (SRC)-spleen tyrosine kinase (SYK)-Caspase recruitment domain-containing protein 9 (CARD9)-TRIF (TIR-domain-containing adapter-inducing interferon-β), and affected interferon regulatory factor 3 and 7 (IRF3-IRF7). The importance of αvβ3-integrin-mediated defense is reflected in the observation that HSV evolved the immediate-early infected cellular protein 0 (ICP0) protein to counteract it. We propose that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors, a role likely exerted toward viruses and bacteria that interact with integrins and mount an innate response.
- Published
- 2012
24. Integrins as Herpesvirus Receptors and Mediators of the Host Signalosome
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Donna Collins-McMillen, Tatiana Gianni, Andrew D. Yurochko, Gabriella Campadelli-Fiume, and DIPARTIMENTO DI MEDICINA SPECIALISTICA, DIAGNOSTICA E SPERIMENTALE
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0301 basic medicine ,Herpesvirus 4, Human ,Integrins ,viruses ,Integrin ,Cytomegalovirus ,Virus Attachment ,KSHV ,medicine.disease_cause ,Herpesviridae ,Receptor tyrosine kinase ,03 medical and health sciences ,Species Specificity ,Viral entry ,EBV ,Virology ,medicine ,Humans ,Simplexvirus ,HCMV ,HSV ,Signal transduction ,Receptor ,biology ,Virus Internalization ,Herpesvirus glycoprotein B ,Endocytosis ,3. Good health ,Cell biology ,030104 developmental biology ,Herpes simplex virus ,Herpesvirus 8, Human ,biology.protein ,Receptors, Virus ,Signal Transduction - Abstract
none 4 si NIH National Institute of General Medical Sciences (NIGMS), NIH National Institute of Allergy & Infectious Diseases (NIAID), NIH National Institute of General Medical Sciences (NIGMS), NIH National Institute of Allergy & Infectious Diseases (NIAID) The repertoire of herpesvirus receptors consists of nonintegrin and integrin molecules. Integrins interact with the conserved glycoproteins gH/gL or gB. This interaction is a conserved biology across the Herpesviridae family, likely directed to promote virus entry and endocytosis. Herpesviruses exploit this interaction to execute a range of critical functions that include (a) relocation of nonintegrin receptors (e.g., herpes simplex virus nectin1 and Kaposi's sarcoma-associated herpesvirus EphA2), or association with nonintegrin receptors (i.e., human cytomegalovirus EGFR), to dictate species-specific entry pathways; (b) activation of multiple signaling pathways (e.g., Ca2+ release, c-Src, FAK, MAPK, and PI3K); and (c) association with Rho GTPases, tyrosine kinase receptors, Toll-like receptors, which result in cytoskeletal remodeling, differential cell type targeting, and innate responses. In turn, integrins can be modulated by viral proteins (e.g., Epstein-Barr virus LMPs) to favor spread of transformed cells. We propose that herpesviruses evolved a multipartite entry system to allow interaction with multiple receptors, including integrins, required for their sophisticated life cycle. mixed Campadelli, MARIA GABRIELLA; Collins Mcmillen, Donna; Gianni, Tatiana; Yurochko, Andrew D. Campadelli, MARIA GABRIELLA; Collins Mcmillen, Donna; Gianni, Tatiana; Yurochko, Andrew D.
- Published
- 2016
25. Retargeting Strategies for Oncolytic Herpes Simplex Viruses
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Tatiana Gianni, Elisa Avitabile, Costanza Casiraghi, Gabriella Campadelli-Fiume, Valerio Leoni, Biljana Petrovic, Valentina Gatta, Campadelli, MARIA GABRIELLA, Petrovic, Biljana, Leoni, Valerio, Gianni, Tatiana, Avitabile, Elisa, Casiraghi, Costanza, and Gatta, Valentina
- Subjects
0301 basic medicine ,Receptor, ErbB-2 ,Infectious Disease ,Review ,Biology ,Virus ,03 medical and health sciences ,Virology ,Simplexvirus ,Receptor ,Tropism ,retargeting ,Viral Structural Proteins ,oncolytic HSV ,Virus Internalization ,Recombinant Proteins ,3. Good health ,Oncolytic virus ,Oncolytic Viruses ,Viral Tropism ,030104 developmental biology ,Infectious Diseases ,Apoptosis ,HER-2 ,Retargeting ,Cancer cell ,Cancer research ,Tissue tropism ,Receptors, Virus ,Single-Chain Antibodies - Abstract
Most of the oncolytic herpes simplex viruses (HSVs) exhibit a high safety profile achieved through attenuation. They carry defects in virulence proteins that antagonize host cell response to the virus, including innate response, apoptosis, authophagy, and depend on tumor cell proliferation. They grow robustly in cancer cells, provided that these are deficient in host cell responses, which is often the case. To overcome the attenuation limits, a strategy is to render the virus highly cancer-specific, e.g., by retargeting their tropism to cancer-specific receptors, and detargeting from natural receptors. The target we selected is HER-2, overexpressed in breast, ovarian and other cancers. Entry of wt-HSV requires the essential glycoproteins gD, gH/gL and gB. Here, we reviewed that oncolytic HSV retargeting was achieved through modifications in gD: the addition of a single-chain antibody (scFv) to HER-2 coupled with appropriate deletions to remove part of the natural receptors' binding sites. Recently, we showed that also gH/gL can be a retargeting tool. The insertion of an scFv to HER-2 at the gH N-terminus, coupled with deletions in gD, led to a recombinant capable to use HER-2 as the sole receptor. The retargeted oncolytic HSVs can be administered systemically by means of carrier cells-forcedly-infected mesenchymal stem cells. Altogether, the retargeted oncolytic HSVs are highly cancer-specific and their replication is not dependent on intrinsic defects of the tumor cells. They might be further modified to express immunomodulatory molecules.
- Published
- 2016
26. αvβ3 Integrin Boosts the Innate Immune Response Elicited in Epithelial Cells through Plasma Membrane and Endosomal Toll-Like Receptors
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Tatiana Gianni, Costanza Casiraghi, Gabriella Campadelli-Fiume, Casiraghi, Costanza, Gianni, Tatiana, and Campadelli-Fiume, Gabriella
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0301 basic medicine ,Integrin ,Immunology ,Cellular Response to Infection ,Endosomes ,Microbiology ,Collagen receptor ,03 medical and health sciences ,HEK293 Cell ,Virology ,Endosome ,Humans ,Simplexvirus ,Gene Silencing ,Simplexviru ,Integrin alphaVbeta3 ,Epithelial Cell ,Innate immune system ,030102 biochemistry & molecular biology ,biology ,Cell Membrane ,NF-kappa B ,Epithelial Cells ,Immunity, Innate ,Cell biology ,Toll-Like Receptor 3 ,030104 developmental biology ,HEK293 Cells ,Integrin alpha M ,Insect Science ,TLR3 ,biology.protein ,Integrin, beta 6 ,Signal transduction ,Human ,Signal Transduction - Abstract
We report that αvβ3 integrin strongly affects the innate immune response in epithelial cells. αvβ3 integrin greatly increased the response elicited via plasma membrane Toll-like receptors (TLRs) by herpes simplex virus or bacterial ligands. The endosomal TLR3, not the cytosolic sensor interferon gamma-inducible protein 16 (IFI16), was also boosted by αvβ3 integrin. The boosting was exerted specifically by αvβ3 integrin but not by αvβ6 or αvβ8 integrin. Current and previous work indicates that integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered an integrin-TLR response.
- Published
- 2015
27. Helicity propensity and interaction of synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H: A circular dichroism study
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Carlo Bertucci, Barbara Sanavio, Angela Piccoli, Tatiana Gianni, B.Sanavio, A.Piccoli, T.Gianni, and C.Bertucci
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Protein Folding ,Conformational change ,Circular dichroism ,Protein Conformation ,Amino Acid Motifs ,Biophysics ,medicine.disease_cause ,Biochemistry ,Article ,Thermal denaturation ,Protein Structure, Secondary ,Analytical Chemistry ,Protein structure ,Viral Envelope Proteins ,Peptide folding ,Peptide-peptide complexes ,medicine ,Molecular Biology ,Protein secondary structure ,chemistry.chemical_classification ,SECONDARY STRUCTURE ,Chemistry ,Circular Dichroism ,Temperature ,Protein Structure, Tertiary ,THERMAL STABILITY ,Heptad repeat ,Crystallography ,Herpes simplex virus ,Solvents ,Secondary structure estimation ,Protein folding ,Peptides ,Glycoprotein ,PEPTIDE DIMER ,Software - Abstract
The secondary structure of two synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H was determined by circular dichroism. In particular, the propensity of these peptides to assume an ordered structure was investigated upon by changing the solvent's polarity and the temperature. A reduction of solvent polarity led to a significant increase in the alpha-helix content in the case of HR1, whereas only a slight change in the secondary structure was observed in the case of HR2. In both cases the conformational change followed a two-state transition model. The interaction of the peptides was monitored by the conformational change in the mixture with respect to the single peptides. However, formation of the complex did not significantly enhance thermal stability. A reliable estimation of the secondary structure was obtained by optimising the experimental conditions to collect CD data down to 180 nm, and by comparing the structure data yielded by different software packages.
- Published
- 2007
28. αvβ6- and αvβ8-integrins serve as interchangeable receptors for HSV gH/gL to promote endocytosis and activation of membrane fusion
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Gabriella Campadelli-Fiume, Tatiana Gianni, Liudmila S. Chesnokova, Lindsey M. Hutt-Fletcher, Stefano Salvioli, Tatiana Gianni, Stefano Salvioli, Liudmila S. Chesnokova, Lindsey M. Hutt-Fletcher, and Gabriella Campadelli-Fiume
- Subjects
lcsh:Immunologic diseases. Allergy ,Integrins ,Endosome ,viruses ,membrane fusion ,Immunology ,Integrin ,Spodoptera ,Endocytosis ,medicine.disease_cause ,Microbiology ,Viral Envelope Proteins ,Antigens, Neoplasm ,Virology ,Genetics ,medicine ,Sf9 Cells ,Animals ,Humans ,Simplexvirus ,Receptor ,lcsh:QH301-705.5 ,Molecular Biology ,Cells, Cultured ,Dynamin ,biology ,Lipid bilayer fusion ,Virus Internalization ,Molecular biology ,Herpesvirus glycoprotein B ,Herpes simplex virus ,HEK293 Cells ,lcsh:Biology (General) ,biology.protein ,Receptors, Virus ,αvβ6- and αvβ8-integrin ,Parasitology ,lcsh:RC581-607 ,K562 Cells ,receptors for HSV gH/gL ,HERPES SIMPLEX VIRUS ,HeLa Cells ,Research Article - Abstract
Herpes simplex virus (HSV) - and herpesviruses in general - encode for a multipartite entry/fusion apparatus. In HSV it consists of the HSV-specific glycoprotein D (gD), and three additional glycoproteins, gH/gL and gB, conserved across the Herpesviridae family and responsible for the execution of fusion. According to the current model, upon receptor binding, gD propagates the activation to gH/gL and to gB in a cascade fashion. Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis, to avoid premature activation and exhaustion of the glycoproteins. We considered the possibility that such control might be carried out by as yet unknown receptors. Indeed, receptors for HSV gB, but not for gH/gL, have been described. In other members of the Herpesviridae family, such as Epstein-Barr virus, integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins. We report that αvβ6- and αvβ8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells. Evidence rests on loss of function experiments, in which integrins were blocked by antibodies or silenced, and gain of function experiments in which αvβ6-integrin was expressed in integrin-negative cells. αvβ6- and αvβ8-integrins acted independently and are thus interchangeable. Both bind gH/gL with high affinity. The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes. In the case of αvβ8, but not αvβ6-integrin, the portal of entry is located at lipid microdomains and requires dynamin 2. Thus, a major role of αvβ6- or αvβ8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis. We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion., Author Summary In order to infect their hosts and cause disease, viruses must enter their host cells. The human pathogen herpes simplex virus (HSV) - and herpesviruses in general - are equipped with a complex, multipartite entry apparatus, made of four glycoproteins – gD, gH/gL, gB. These glycoproteins must be activated in a timely, coordinated manner. According to the current model, the flux of activation goes from receptor-bound gD, to gH/gL and gB. The premature activation, and hence exhaustion of the glycoproteins must also be prevented. We report on a checkpoint at the gH/gL level. Specifically, αvβ6- and αvβ8-integrins serve as receptors for HSV entry into keratinocytes and other epithelial and neuronal cells. Both bind gH/gL with high affinity. The interaction profoundly affects the pathway of HSV entry, promoting HSV endocytosis into acidic endosomes. For αvβ8-integrin, the portal of entry is at lipid microdomains and requires dynamin 2. We propose that, by placing the activation of gH/gL under control of an integrin trigger point, HSV can synchronize virion endocytosis with the cascade of activation that culminates in the execution of fusion between the virion envelope and cellular membranes.
- Published
- 2013
29. Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry
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Tatiana Gianni, Gabriella Campadelli-Fiume, Raffaele Massaro, Gianni, T., Massaro, R., and Campadelli-Fiume, G.
- Subjects
glycoprotein ,Integrins ,viruses ,Amino Acid Motifs ,Nectins ,Integrin ,Herpesvirus 1, Human ,gL ,virus entry ,Biology ,medicine.disease_cause ,Models, Biological ,Herpesviridae ,gH ,Viral Envelope Proteins ,Antigens, Neoplasm ,Viral entry ,Cell Line, Tumor ,medicine ,Humans ,Receptor ,chemistry.chemical_classification ,Brefeldin A ,Multidisciplinary ,Cell adhesion molecule ,Cell Membrane ,Virion ,Antibodies, Monoclonal ,Epithelial Cells ,Herpes Simplex ,Virus Internalization ,herpes simplex viru ,Antibodies, Neutralizing ,Virology ,Molecular biology ,Herpesvirus glycoprotein B ,Endocytosis ,Protein Structure, Tertiary ,Herpes simplex virus ,Microscopy, Fluorescence ,Solubility ,PNAS Plus ,chemistry ,Mutation ,Proteolysis ,biology.protein ,Glycoprotein ,Cell Adhesion Molecules - Abstract
Significance Entry of enveloped viruses into the cell requires the activation of viral glycoproteins, often mediated by cellular receptors. Herpesviruses infect cells via a multipartite system, which includes species-specific glycoproteins plus conserved apparatus gH/gL and gB. HSV makes use of αvβ6- or αvβ8-integrins as gH/gL receptors. The interaction of HSV gH/gL with integrins resulted in the dissociation of gL. The dissociation took place if all the actors of the entry apparatus were present, i.e., under conditions that lead to glycoprotein activation and virus entry. We propose that ( i ) gL is a regulator of gH and prevents its activation until integrins promote gL dissociation from gH/gL. ( ii ) Dissociation from an inhibitory regulator represents a previously unidentified mechanism of activation of viral fusion glycoproteins.
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- 2015
30. The herpesvirus glycoproteins B and H·L are sequentially recruited to the receptor-bound gD to effect membrane fusion at virus entry
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Gabriella Campadelli-Fiume, Cristina Forghieri, and Tatiana Gianni
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Multidisciplinary ,Viral protein ,viruses ,Lipid bilayer fusion ,Transfection ,Biological Sciences ,Biology ,medicine.disease_cause ,Virology ,Herpesvirus glycoprotein B ,Virus ,Cell biology ,Herpes simplex virus ,Viral envelope ,Viral entry ,medicine - Abstract
Four glycoproteins (gD, gB, gH, and gL) are required for herpes simplex virus entry into the cell or for cell–cell fusion in transfected cells. gD serves as the receptor-binding glycoprotein and as the trigger of fusion; the other three execute fusion between the viral envelope and the plasma and endocytic membranes or the membranes of adjacent cells and are highly conserved among members of the herpesvirus family. Details of the interaction of gD with gB, gH, and gL were not known. Here, we report that the four glycoproteins assemble into a complex initiated by the interaction of gD with its cellular receptor. gB is recruited to the gD–receptor complex next, even in the absence of gH·gL. gH·gL is recruited next, but only to the receptor–gD–gB ensemble. A complex with the composition receptor–gD–gB–gH·gL is assembled transiently with a life span of 15–30 min in cells exposed to virus but can also be found in infected cells and in cells committed to form polykaryocytes after transfection of the glycoprotein quartet. The results indicate that the complex assembly is a critical step in the process of virus entry and fusion, and that no viral protein other than those that participate in the complex itself is required for complex assembly. These findings imply critical protein–protein interactions among the quartet as herpes simplex virions enter the cells and at cell–cell fusion, define a specific order of recruitment, and place gH·gL as the last link in the process of glycoprotein recruitment to the complex.
- Published
- 2006
31. A Heptad Repeat in Herpes Simplex Virus 1 gH, Located Downstream of the α-Helix with Attributes of a Fusion Peptide, Is Critical for Virus Entry and Fusion
- Author
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Laura Menotti, Tatiana Gianni, and Gabriella Campadelli-Fiume
- Subjects
Author's Correction ,Repetitive Sequences, Amino Acid ,viruses ,Molecular Sequence Data ,Immunology ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,Membrane Fusion ,Microbiology ,Protein Structure, Secondary ,Cell Line ,Viral Envelope Proteins ,Viral entry ,Cricetinae ,Virology ,Chlorocebus aethiops ,medicine ,Animals ,Amino Acid Sequence ,Peptide sequence ,Coiled coil ,Base Sequence ,Virulence ,Genetic Complementation Test ,Lipid bilayer fusion ,Herpesvirus glycoprotein B ,Virus-Cell Interactions ,Protein Structure, Tertiary ,Cell biology ,Heptad repeat ,Herpes simplex virus ,Ectodomain ,Insect Science ,COS Cells ,DNA, Viral - Abstract
Entry of herpes simplex virus 1 (HSV-1) into cells occurs by fusion with cell membranes; it requires gD as the receptor binding glycoprotein and the trigger of fusion, and the trio of the conserved glycoproteins gB, gH, and gL to execute fusion. Recently, we reported that the ectodomain of HSV-1 gH carries a hydrophobic α-helix (residues 377 to 397) with attributes of an internal fusion peptide (T. Gianni, P. L. Martelli, R. Casadio, and G. Campadelli-Fiume, J. Virol. 79: 2931-2940, 2005). Downstream of this α-helix, a heptad repeat (HR) with a high propensity to form a coiled coil was predicted between residues 443 and 471 and was designated HR-1. The simultaneous substitution of two amino acids in HR-1 (E450G and L453A), predicted to abolish the coiled coil, abolished the ability of gH to complement the infectivity of a gH-null HSV mutant. When coexpressed with gB, gD, and gL, the mutant gH was unable to promote cell-cell fusion. These defects were not attributed to a defect in heterodimer formation with gL, the gH chaperone, or in trafficking to the plasma membrane. A 25-amino-acid synthetic peptide with the sequence of HR-1 (pep-gH wt25 ) inhibited HSV replication if present at the time of virus entry into the cell. A scrambled peptide had no effect. The effect was specific, as pep-gH wt25 did not reduce HSV-2 and pseudorabies virus infection. The presence of a functional HR in the HSV-1 gH ectodomain strengthens the view that gH has attributes typical of a viral fusion glycoprotein.
- Published
- 2005
32. Entry of Herpes Simplex Virus Mediated by Chimeric Forms of Nectin1 Retargeted to Endosomes or to Lipid Rafts Occurs through Acidic Endosomes
- Author
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Gabriella Campadelli-Fiume, Laura Menotti, Tatiana Gianni, GIANNI T., CAMPADELLI-FIUME G., and MENOTTI L.
- Subjects
Herpesvirus entry mediator ,Endosome ,Nectins ,Immunology ,Endocytic cycle ,VIRAL ENTRY ,Biology ,Endocytosis ,Membrane Fusion ,Microbiology ,Ammonium Chloride ,Membrane Microdomains ,NECTIN1 RECEPTOR RETARGETING ,Viral entry ,Cricetinae ,Virology ,Animals ,Simplexvirus ,Lipid raft ,Tyrphostins ,ENDOSOMES ,Virus-Cell Interactions ,Cell biology ,Androstadienes ,ErbB Receptors ,LIPID RAFTS ,Ectodomain ,Biochemistry ,Insect Science ,Quinazolines ,Receptors, Virus ,Macrolides ,Signal transduction ,Wortmannin ,Cell Adhesion Molecules ,HERPES SIMPLEX VIRUS - Abstract
Herpes simplex virus (HSV) enters cells by fusion with target membranes, commonly the plasma membrane. In some cells, including CHO cells expressing the nectin1 or herpesvirus entry mediator receptors, entry occurs through an endocytic route. We report the following results. (i) When expressed in J cells, nectin1 and HVEM mediated a pathway of entry insensitive to endosome acidification inhibitors. (ii) A chimeric nectin1 receptor competent for endosomal uptake by fusion of the nectin1 ectodomain with the transmembrane sequence and cytoplasmic tail of the epidermal growth factor receptor (EGFR1) (nectin1-EGFR1) and chimeric nectin1 sorted to lipid rafts by a glycosylphosphatidylinositol anchor mediated endocytic entry blocked by the early endosome inhibitor wortmannin and by the endosome acidification inhibitors bafilomycin and NH 4 Cl. (iii) Entry mediated by nectin1-EGFR1 was selectively inhibited by AG1478, a tyrosine phosphorylation inhibitor that targets the EGFR1 cytoplasmic tail and blocks the signaling pathway that culminates in clathrin-dependent uptake of the receptor into endosomes. We draw the following conclusions. (i) The same receptor may initiate different routes of infection, depending on the cell in which it is expressed. Hence, the cell is a determinant that controls whether a given receptor initiates a plasma membrane or an endocytic route of entry. (ii) Receptors whose physiology involves uptake into endosomes or sorting to lipid rafts are suitable to serve as HSV receptors. (iii) Structural features of the receptors are additional determinants that control whether HSV entry occurs at the plasma membrane or at endosomes. These findings are relevant to studies of HSV retargeting to specific receptors.
- Published
- 2004
33. Coexpression of UL20p and gK Inhibits Cell-Cell Fusion Mediated by Herpes Simplex Virus Glycoproteins gD, gH-gL, and Wild-Type gB or an Endocytosis-Defective gB Mutant and Downmodulates Their Cell Surface Expression
- Author
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Gabriella Campadelli-Fiume, Elisa Avitabile, Giulia Lombardi, Miriam Capri, Tatiana Gianni, AVITABILE E., LOMBARDI G., GIANNI T., CAPRI M., and CAMPADELLI-FIUME G.
- Subjects
DOWN-REGULATION ,Syncytium ,Cell fusion ,HERPES SIMPLEX ,MEMBRANE FUSION ,ENDOCYTOSIS ,Endoplasmic reticulum ,Immunology ,Lipid bilayer fusion ,Biology ,Endocytosis ,Microbiology ,Herpesvirus glycoprotein B ,Molecular biology ,Virus-Cell Interactions ,Cell Line ,VIRAL GLYCOPROTEINS ,Viral Proteins ,Viral Envelope Proteins ,Cytoplasm ,Cell culture ,Virology ,Insect Science ,Animals - Abstract
Syncytium formation in cells that express herpes simplex virus glycoprotein B (gB), gD, gH, and gL is blocked by gK (E. Avitabile, G. Lombardi, and G. Campadelli-Fiume, J. Virol. 77: 6836-6844, 2003). Here, we report the results of two series of experiments. First, UL20 protein (UL20p) expression weakly inhibited cell-cell fusion. Coexpression of UL20p and gK drastically reduced fusion in a cell-line-dependent manner, with the highest inhibition in BHK cells. Singly expressed UL20p and gK localized at the endoplasmic reticulum and nuclear membranes. When they were coexpressed, both proteins relocalized to the Golgi apparatus. Remarkably, in cells that coexpressed UL20p and gK, the antifusion activity correlated with a downmodulation of gD, gB, gH, and gL cell surface expression. Second, gB Δ867 has a partial deletion in the cytoplasmic tail that removed endocytosis motifs. Whereas wild-type (wt) gB was internalized in vesicles lined with the endosomal marker Rab5, gB Δ867 was not internalized, exhibited enhanced cell surface expression, and was more efficient in mediating cell-cell fusion than wt gB. The antifusion activity of UL20p and gK was also exerted when gB Δ867 replaced wt gB in the cell fusion assay. These studies show that the gB C tail carries a functional endocytosis motif(s) and that the removal of the motif correlated with increased gB surface expression and increased fusion activity. We conclude that cell-cell fusion in wt-virus-infected cells is negatively controlled by at least two mechanisms. The novel mechanism described here involves the concerted action of UL20p and gK and correlates with a moderate but consistent reduction in the cell surface expression of the fusion glycoproteins. This mechanism is independent of the one exerted through endocytosis-mediated downmodulation of gB from the plasma membrane.
- Published
- 2004
34. Microglial cells protect cerebellar granule neurons from apoptosis: Evidence for reciprocal signaling
- Author
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Antonio Contestabile, Tatiana Gianni, and Elisabetta Polazzi
- Subjects
Nervous system ,Cerebellum ,Programmed cell death ,Cell Survival ,Population ,Tetrazolium Salts ,Apoptosis ,Cell Count ,Cell Communication ,Biology ,Neuroprotection ,Cerebellar Cortex ,Cellular and Molecular Neuroscience ,medicine ,Animals ,Rats, Wistar ,education ,Potassium Deficiency ,Cells, Cultured ,Neurons ,education.field_of_study ,Microglia ,Depolarization ,Coculture Techniques ,Rats ,Thiazoles ,medicine.anatomical_structure ,Animals, Newborn ,nervous system ,Neurology ,Culture Media, Conditioned ,Neuroglia ,Neuroscience - Abstract
The microglia are the immune cell population of the nervous system and play important roles both in normal function and in disease. Reciprocal neuron–microglia interactions are not well understood, in particular those concerning the crosstalk between the two cell populations when neuronal damage does occur. We have used a well-established model of apoptosis in cerebellar granule neurons to test the effect of co-culturing microglial cells with them or of exposing them to microglia-conditioned medium. Microglial cells, derived from cortical or cerebellar mixed glial cultures and plated over cerebellar granule neurons, protected these neurons from apoptosis induced by shifting them, at 7 days in vitro, for 24 h from a depolarizing (high-potassium) to a nondepolarizing (low-potassium) medium. The same result was achieved when microglial cells obtained from mixed glial cortical cultures were plated over a membrane well insert in the culture chamber, permitting medium exchange without physical contact with granule neurons. A similar result was obtained when the low-potassium, apoptosis-inducing medium was conditioned by 48-h exposure to microglial cells; 24-h exposure to microglial cells was not enough to confer neuroprotective capability to the conditioned medium. However in double-conditioned medium experiments, in which the medium was first exposed to apoptotic neurons and then to microglial cells, unknown signal(s) released by apoptotic neurons, conferred to the 24-h conditioned medium a strong neuroprotective action, similar to that observed in the co-cultures experiments. This finding, together with the results from co-culture experiments, is explained by admitting that molecules released in the medium by apoptotic neurons potentiate the anti-apoptotic activity of microglia. Our results, therefore, demonstrate not only that normally microglial cells release in the medium molecule(s) able to rescue neurons from apoptotic death, but that unknown diffusible signal(s) from apoptotic neurons enhance(s) microglial neuroprotective properties as well. GLIA 36:271–280, 2001. © 2001 Wiley-Liss, Inc.
- Published
- 2001
35. alphaV-beta3-Integrin Relocalizes nectin1 and Routes Herpes Simplex Virus to Lipid Rafts
- Author
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Gabriella Campadelli-Fiume, Tatiana Gianni, Gianni T., and Campadelli-Fiume G
- Subjects
GLYCOPROTEIN H ,Endosome ,viruses ,ENDOCYTOSIS ,Immunology ,Nectins ,Herpesvirus 1, Human ,Biology ,Endocytosis ,medicine.disease_cause ,Microbiology ,Virus ,Cell Line ,Membrane Microdomains ,Nectin ,Virology ,medicine ,Humans ,LIPID RAFT ,Lipid raft ,Integrin alphaVbeta3 ,AVB3INTEGRIN ,Herpes Simplex ,Virus Internalization ,Cell biology ,Transport protein ,Virus-Cell Interactions ,Protein Transport ,Herpes simplex virus ,Insect Science ,lipids (amino acids, peptides, and proteins) ,Cell Adhesion Molecules ,HERPES SIMPLEX VIRUS - Abstract
Herpes simplex virus (HSV) enters cells by fusion at plasma membranes or endosomes. Cellular factors route the virus to different pathways. αVβ3-integrin directs HSV to a lipid raft and acidic endosome pathway. We report that infection mediated by nectin1 plus αVβ3-integrin exhibits the same characteristics as entry mediated by raft-located forms of nectin. αVβ3-integrin relocalizes nectin1 to lipid rafts, independently of virus. Thus, HSV routing to the lipid raft-dependent pathway is consequent to the integrin-induced relocalization of nectin1. Inhibition by the Na+/H+ exchanger 5-( N -ethyl- N -isopropyl)amirolide suggests that αVβ3-integrin overexpression favors HSV macropinocytic uptake in some cells but not in others.
- Published
- 2012
36. Viral and cellular contributions to herpes simplex virus entry into the cell
- Author
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Elisa Avitabile, Gabriella Campadelli-Fiume, Tatiana Gianni, Laura Menotti, Campadelli-Fiume G., Menotti L., Avitabile E., and Gianni T.
- Subjects
Simplexvirus/genetic ,VIRAL ENVELOPE PROTEINS/GENETICS ,viruses ,Biology ,medicine.disease_cause ,Virus ,Herpes Simplex/metabolism ,Viral Envelope Proteins ,Viral entry ,Herpes Simplex/genetic ,Virology ,medicine ,Animals ,Humans ,Simplexvirus ,chemistry.chemical_classification ,Animal ,Herpes Simplex ,Viral tegument ,Virus Internalization ,Herpesvirus glycoprotein B ,Herpes simplex virus ,Capsid ,chemistry ,Cytoplasm ,Herpes Simplex/virology ,Viral Envelope Proteins/metabolism ,Glycoprotein ,SIMPLEXVIRUS/PHYSIOLOGY ,Human - Abstract
Herpes simplex virus (HSV) entry into the cell involves the fusion of the virion envelope with a cellular membrane and delivery of capsid and tegument proteins to the cytoplasm. Our understanding of this phenomenon has greatly increased in recent years. On the virus side, the multipartite nature of the entry-fusion machinery (made of the glycoproteins gD, the heterodimer gH/gL and gB) entails a mechanism of gD activation promoted by the gD encounter with one of its receptor; and cross-talk among the entry-fusion glycoproteins, which culminates in gB activation and fusion execution. On the cell side, machineries and signalling activities are put in place. The number of known receptors and sentinels is increasing. The cell routes the virus through alternative entry pathways by means of routing factors, exemplified by αVβ3-integrin and paired immunoglobulin-like type 2 receptor alpha. Of the signalling events, a key one is the immediate host response to incoming virions. Unexpectedly, this is in part triggered by the same virion components and some cellular factors that also promote virus entry. Hence, a link is emerging between two phenomena so far considered as distinct.
- Published
- 2011
37. Herpes simplex virus glycoproteins H/L bind to cells independently of alpha V- beta 3 integrin and inhibit virus entry, and their constitutive expression restricts infection
- Author
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Rebecca M. DuBois, Gabriella Campadelli-Fiume, Stefano Salvioli, Scott S. Blystone, Arianna Cerretani, Tatiana Gianni, Félix A. Rey, Gianni T., Cerretani A., Dubois R., Salvioli S., Blystone S.S., Rey F., and Campadelli-Fiume G.
- Subjects
Immunology ,Integrin ,medicine.disease_cause ,Microbiology ,Cell Line ,Viral Envelope Proteins ,Viral entry ,Virology ,medicine ,Animals ,Humans ,Simplexvirus ,RGD motif ,chemistry.chemical_classification ,biology ,Integrin beta3 ,Transfection ,Virus Internalization ,Herpesvirus glycoprotein B ,Molecular biology ,Virus-Cell Interactions ,Herpes simplex virus ,chemistry ,Cell culture ,Insect Science ,biology.protein ,Glycoprotein ,Protein Binding - Abstract
Herpes simplex virus (HSV) fusion with cells requires the gD, gB, and gH/gL glycoprotein quartet. gD serves as a receptor binding glycoprotein. gB and gH/gL execute fusion in an as-yet-unclear manner. To better understand the role of gH/gL in HSV entry, we produced a soluble version of gH/gL carrying a One-STrEP tag (gH t.st /gL). Previous findings implicated integrins as possible ligands to gH/gL (C. Parry et al., J. Gen. Virol. 86:7-10, 2005). We report that (i) gH t.st /gL bound a number of cells in a dose-dependent manner at concentrations similar to those required for the binding of soluble gB or gD. (ii) gH t.st /gL inhibited HSV entry at the same concentrations required for binding. It also inhibited cell-cell fusion in transfected cells. (iii) The absence of β3 integrin did not prevent the binding of gH t.st /gL to CHO cells and infection inhibition. Conversely, integrin-negative K562 cells did not acquire the ability to bind gH t.st /gL when hyperexpressing αVβ3 integrin. (iv) Constitutive expression of wild-type gH/gL (wt-gH/gL) restricted infection in all of the cell lines tested, a behavior typical of glycoproteins which bind cellular receptors. The extent of restriction broadly paralleled the efficiency of gH/gL transfection. RGD motif mutant gH/gL could not be differentiated from wt-gH with respect to restriction of infection. Cumulatively, the present results provide several lines of evidence that HSV gH/gL interacts with a cell surface cognate protein(s), that this protein is not necessarily an αVβ3 integrin, and that this interaction is required for the process of virus entry/fusion.
- Published
- 2010
38. {alpha}V{beta}3-integrin routes herpes simplex virus to an entry pathway dependent on cholesterol-rich lipid rafts and dynamin2
- Author
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VALENTINA GATTA, Gabriella Campadelli-Fiume, Valentina GATTA, TATIANA GIANNI, Gianni T., Gatta V., and Campadelli-Fiume G.
- Subjects
Alpha-v beta-3 ,Integrin ,Nectins ,CHO Cells ,Biology ,Endocytosis ,Antibodies ,chemistry.chemical_compound ,Dynamin II ,Cricetulus ,Membrane Microdomains ,Cricetinae ,Animals ,Humans ,Simplexvirus ,Lipid raft ,Integrin alphaVbeta3 ,Multidisciplinary ,Innate immune system ,Pinocytosis ,HEK 293 cells ,Virus Internalization ,Biological Sciences ,Immunity, Innate ,Cell biology ,Cholesterol ,chemistry ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Cell Adhesion Molecules - Abstract
HSVs enter cells in a receptor-dependent [nectin1 or herpesviruses entry mediator (HVEM)] fashion by fusion of the viral envelope with plasma membrane (neutral pH compartment), by endocytosis into neutral or acidic compartments, or by macropinocytosis/phagocytosis. The cellular determinants of the route of entry are unknown. Here, we asked what cellular factors determine the pathway of HSV entry. CHO cells lack β 3 -integrin and the respective α-subunits’ heterodimers. We report that, in the absence of α V β 3 -integrin, HSV enters CHO-nectin1 cells through a pathway independent of cholesterol-rich rafts and dynamin2. In the presence of α V β 3 -integrin, HSV enters CHO-nectin1 cells through a pathway dependent on cholesterol-rich rafts and dynamin2. HSV enters J-nectin1 and 293T cells through a neutral compartment independent of cholesterol-rich rafts and dynamin2. α V β 3 -integrin overexpression in these cells modifies the route of entry to an acidic compartment dependent on cholesterol-rich rafts and dynamin2, hence similar to that in α V β 3 -integrin–positive CHO-nectin1 cells. In some cells, the diversion of entry from an integrin- and raft-independent pathway to an acidic compartment requiring cholesterol-rich lipids rafts and dynamin2 is irreversible. Indeed, HSV cannot infect CHO-nectin1-α V β 3 cells through any compartment when the αvβ3-integrin–dependent pathway is blocked by anti-integrin antibody, anti-dynamin2, or anti-acidification drugs. We conclude that the αvβ3-integrin is a determinant in the choice of HSV entry pathway into cells. Because the pathway dictated by αvβ3-integrin is through lipid rafts, the platforms for a number of Toll-like receptors, current findings raise the possibility that αvβ3-integrin acts as a sentinel of innate immunity.
- Published
- 2010
39. Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
- Author
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Gabriella Campadelli-Fiume, Michele Amasio, Tatiana Gianni, Gianni T, Amasio M, and Campadelli-Fiume G.
- Subjects
VIRAL ENVELOPE PROTEINS/GENETICS ,Blotting, Western ,Green Fluorescent Proteins ,Biomolecular Networks ,Biology ,medicine.disease_cause ,Biochemistry ,Membrane Fusion ,Protein structure ,Viral Envelope Proteins ,Viral entry ,Chlorocebus aethiops ,medicine ,Animals ,Immunoprecipitation ,Simplexvirus ,Binding site ,Molecular Biology ,Vero Cells ,chemistry.chemical_classification ,Lipid bilayer fusion ,Cell Biology ,Herpesvirus glycoprotein B ,Molecular biology ,MEMBRANE FUSION/PHYSIOLOGY ,Protein Structure, Tertiary ,Herpes simplex virus ,chemistry ,Ectodomain ,Glycoprotein ,Genetic Engineering ,SIMPLEXVIRUS/PHYSIOLOGY ,Plasmids - Abstract
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. gD serves as a receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in an N-terminal domain carrying the receptor-binding sites and a C-terminal domain carrying the profusion domain, required for fusion but not receptor binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other, we searched for biochemical defined complexes in infected and transfected cells and in virions. Previously, interactions were detected in transfected whole cells by split green fluorescent protein complementation (Atanasiu, D., Whitbeck, J. C., Cairns, T. M., Reilly, B., Cohen, G. H., and Eisenberg, R. J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 18718-18723; Avitabile, E., Forghieri, C., and Campadelli-Fiume, G. (2007) J. Virol. 81, 11532-11537); it was not determined whether they led to biochemical complexes. Infected cells harbor a gD-gH complex (Perez-Romero, P., Perez, A., Capul, A., Montgomery, R., and Fuller, A. O. (2005) J. Virol. 79, 4540-4544). We report that gD formed complexes with gB in the absence of gH/gL and with gH/gL in the absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry and did not increase at virus entry into the cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the amino acids 240-260 portion and downstream with Thr304-Pro305 as critical residues and of the binding site to gH/gL at the amino acids 260-310 portion with Pro291-Pro292 as critical residues. The results indicate that gD carries composite-independent binding sites for gB and gH/gL, both of which are partly located in the profusion domain.
- Published
- 2009
40. The Ectodomain of Herpes Simplex Virus Glycoprotein H Contains a Membrane α-Helix with Attributes of an Internal Fusion Peptide, Positionally Conserved in the Herpesviridae Family
- Author
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Tatiana Gianni, Rita Casadio, Pier Luigi Martelli, Gabriella Campadelli-Fiume, GIANNI T., MARTELLI P.L., CASADIO R., and CAMPADELLI-FIUME G.
- Subjects
Author's Correction ,GLYCOPROTEIN H ,Recombinant Fusion Proteins ,VIRAL FUSION ,viruses ,Molecular Sequence Data ,Immunology ,Herpesvirus 1, Human ,Biology ,Transfection ,medicine.disease_cause ,Gp41 ,Microbiology ,Protein Structure, Secondary ,Virus ,Cell Line ,Conserved sequence ,Viral Proteins ,Viral Envelope Proteins ,Cricetinae ,Virology ,medicine ,Animals ,Amino Acid Sequence ,Conserved Sequence ,Herpesviridae ,Sequence Deletion ,ALPHA HELIX ,Membrane Glycoproteins ,Base Sequence ,biology.organism_classification ,Herpesvirus glycoprotein B ,HIV Envelope Protein gp41 ,Virus-Cell Interactions ,Protein Structure, Tertiary ,Cell biology ,Membrane glycoproteins ,Herpes simplex virus ,Ectodomain ,Mutagenesis ,Vesicular stomatitis virus ,Insect Science ,COS Cells ,DNA, Viral ,INTERNAL FUSION PEPTIDE ,biology.protein ,Viral Fusion Proteins ,HERPES SIMPLEX VIRUS - Abstract
Human herpesviruses enter cells by fusion with target membranes, a process that requires three conserved glycoproteins: gB, gH, and gL. How these glycoproteins execute fusion is unknown. Neural network bioinformatics predicted a membrane α-helix contained within the ectodomain of herpes simplex virus (HSV) gH, positionally conserved in the gH of all examined herpesviruses. Evidence that it has attributes of an internal fusion peptide rests on the following lines of evidence. (i) The predicted membrane α-helix has the attribute of a membrane segment, since it transformed a soluble form of gD into a membrane-bound gD. (ii) It represents a critical domain of gH. Its partial or entire deletion, or substitution of critical residues inhibited HSV infectivity and fusion in the cell-cell fusion assay. (iii) Its replacement with the fusion peptide from human immunodeficiency virus gp41 or from vesicular stomatitis virus G partially rescued HSV infectivity and cell-cell fusion. The corresponding antisense sequences did not. (iv) The predicted α-helix located in the varicella-zoster virus gH ectodomain can functionally substitute the native HSV gH membrane α-helix, suggesting a conserved function in the human herpesviruses. We conclude that HSV gH exhibits features typical of viral fusion glycoproteins and that this property is likely conserved in the Herpesviridae family.
- Published
- 2007
41. The multipartite system that mediates entry of herpes simplex virus into the cell
- Author
-
Cristina Forghieri, Gabriella Campadelli-Fiume, Elisa Avitabile, Tatiana Gianni, Michele Amasio, Laura Menotti, Arianna Cerretani, Campadelli-Fiume G., Amasio M., Avitabile E., Cerretani A., Forghieri C., Gianni T, and Menotti L.
- Subjects
chemistry.chemical_classification ,Models, Molecular ,Viral Structural Proteins ,Herpesvirus entry mediator ,Biology ,Virus Internalization ,medicine.disease_cause ,Herpesvirus glycoprotein B ,Virology ,Cell biology ,Multipartite ,Infectious Diseases ,Herpes simplex virus ,Ectodomain ,chemistry ,medicine ,Humans ,Receptors, Virus ,Simplexvirus ,Glycoprotein ,Receptor ,Tropism ,Glycoproteins - Abstract
The multipartite entry-fusion system of herpes simplex virus is made of a quartet of glycoproteins-gD, gB, gH.gL-and three alternative gD receptors, herpesvirus entry mediator (HVEM), nectin1 and modified sites on heparan sulphate. This multipartite system recapitulates the basic steps of virus-cell fusion, i.e. receptor recognition, triggering of fusion and fusion execution. Specifically, in addition to serving as the receptor-binding glycoprotein, gD triggers fusion through a specialised domain, named pro-fusion domain (PFD), located C-terminally in the ectodomain. In the unliganded gD the C-terminal region folds around the N-terminal region, such that gD adopts a closed autoinhibited conformation. In HVEM- and nectin1-bound gD the C-terminal region is displaced (opened conformation). gD is the tool for modification of HSV tropism, through insertion of ligands to heterologous tumour-specific receptors. It is discussed whether gD responds to the interaction with the natural and the heterologous receptors by adopting similar conformations, and whether the closed-to-open switch in conformation is a generalised mechanism of activation. A peculiar recombinant highlighted that the central Ig-folded core of gD may not encode executable functions for entry and that the 219-314 aa segment may be sufficient to trigger fusion. With respect to fusion execution, gB appears to be a prospective fusogen based on its coiled-coil trimeric structure, similar to that of another fusion glycoprotein. On the other hand, gH exhibits molecular elements typical of class 1 fusion glycoproteins, in particular heptad repeats and strong tendency to interact with lipids. Whether fusion execution is carried out by gB or gH.gL, or both glycoproteins in complex or sequentially remains to be determined.
- Published
- 2007
42. The soluble ectodomain of herpes simplex virus gD contains a membrane-proximal pro-fusion domain and suffices to mediate virus entry
- Author
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Gabriella Campadelli-Fiume, Tatiana Gianni, Laura Menotti, Francesca Cocchi, Roselyn J. Eisenberg, Gary H. Cohen, Daniela Fusco, COCCHI F., FUSCO D., MENOTTI L., GIANNI T., EISENBERG R.J., COHEN G.H., and CAMPADELLI-FIUME G.
- Subjects
Herpesvirus entry mediator ,VIRAL FUSION ,viruses ,Molecular Sequence Data ,GLYCOPROTEIN D ,Biology ,medicine.disease_cause ,Membrane Fusion ,Viral Envelope Proteins ,Viral entry ,medicine ,Simplexvirus ,Amino Acid Sequence ,DNA Primers ,Infectivity ,Multidisciplinary ,Base Sequence ,Sequence Homology, Amino Acid ,Lipid bilayer fusion ,Biological Sciences ,Virology ,Herpesvirus glycoprotein B ,Cell biology ,VIRAL GLYCOPROTEINS ,PROFUSION DOMAIN ,Transmembrane domain ,Herpes simplex virus ,Ectodomain ,Mutagenesis ,HERPES SIMPLEX VIRUS - Abstract
Entry of herpes simplex virus (HSV) 1 into cells requires the interaction of HSV gD with herpesvirus entry mediator or nectin1 receptors, and fusion with cell membrane mediated by the fusion glycoproteins gB, gH, and gL. We report that the gD ectodomain in soluble form (amino acids 1-305) was sufficient to rescue the infectivity of a gD-null HSV mutant, indicating that gD does not need to be anchored to the virion envelope to mediate entry. Entry mediated by soluble gD required, in addition to the receptor-binding sites contained within residues 1-250, a discrete downstream portion (amino acids 261-305), located proximal to the transmembrane segment in full-length gD. We named it as profusion domain. The pro-fusion domain was required for entry mediated by virion-bound gD, because its substitution with the corresponding region of CD8 failed to complement the infectivity of gD -/+ HSV. Furthermore, a receptor-negative gD (gD Δ6-259 ) inhibited virus infectivity when coexpressed with wild-type gD; i.e., it acted as a dominant-negative gD mutant. The pro-fusion domain is proline-rich, which is characteristic of regions involved in protein-protein interactions. P291L-P292A substitutions diminished the gD capacity to complement gD -/+ HSV infectivity. We propose that gD forms a tripartite complex with its receptor and, by way of the proline-rich pro-fusion domain, with the fusion glycoproteins, or with one of them. The tripartite complex would serve to recruit/activate the fusion glycoproteins and bring them from a fusion-inactive to a fusion-active state, such that they execute fusion of the virion envelope with cell membrane.
- Published
- 2004
43. Hydrophobic α-Helices 1 and 2 of Herpes Simplex Virus gH Interact with Lipids, and Their Mimetic Peptides Enhance Virus Infection and Fusion
- Author
-
Giorgio Lenaz, Christian Bergamini, Gabriella Campadelli-Fiume, Romana Fato, and Tatiana Gianni
- Subjects
Author's Correction ,Recombinant Fusion Proteins ,DNA Mutational Analysis ,Molecular Sequence Data ,Immunology ,Biology ,medicine.disease_cause ,Membrane Fusion ,Microbiology ,Protein Structure, Secondary ,Virus ,Viral Envelope Proteins ,Biomimetics ,Viral entry ,Virology ,medicine ,Animals ,Simplexvirus ,Amino Acid Sequence ,Peptide sequence ,Cells, Cultured ,Sequence Deletion ,Membranes ,Cell fusion ,Lipid bilayer fusion ,Herpes Simplex ,Lipid Metabolism ,biology.organism_classification ,Lipids ,Herpesvirus glycoprotein B ,Virus-Cell Interactions ,Cell biology ,Herpes simplex virus ,Vesicular stomatitis virus ,Insect Science ,Liposomes ,Peptides ,Hydrophobic and Hydrophilic Interactions - Abstract
Entry of herpes simplex virus into cells occurs by fusion and requires four glycoproteins. gD serves as the receptor binding glycoprotein. Of the remaining glycoproteins, gH carries structural and functional elements typical of class 1 fusion glycoproteins, in particular α-helix 1 (α-H1), with properties of a candidate fusion peptide, and two heptad repeats. Here, we characterized α-H2 and compared it to α-H1. α-H2 (amino acids 513 to 531) is of lower hydrophobicity than α-H1. Its deletion or mutation decreased virus infection and cell fusion. Its replacement with heterologous fusion peptides did not rescue infection and cell fusion beyond the levels exhibited by the α-H2-deleted gH. This contrasts with α-H1, which cannot be deleted and can be functionally replaced with heterologous fusion peptides (T. Gianni et al., J. Virol. 79: 2931-2940, 2005). Synthetic peptides mimicking α-H1 and α-H2 induced fusion of nude lipid vesicles. Importantly, they increased infection of herpes simplex virus, pseudorabies virus, bovine herpesvirus 1, and vesicular stomatitis virus. The α-H1 mimetic peptide was more effective than the α-H2 peptide. Consistent with the findings that gH carries membrane-interacting segments, a soluble form of gH, but not of gD or gB, partitioned with lipid vesicles. Current findings highlight that α-H2 is an important albeit nonessential region for virus entry and fusion. α-H1 and α-H2 share the ability to target the membrane lipids; they contribute to virus entry and fusion, possibly by destabilizing the membranes. However, α-H2 differs from α-H1 in that it is of lower hydrophobicity and cannot be replaced with heterologous fusion peptides.
- Published
- 2007
44. Heptad Repeat 2 in Herpes Simplex Virus 1 gH Interacts with Heptad Repeat 1 and Is Critical for Virus Entry and Fusion
- Author
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Gabriella Campadelli-Fiume, Carlo Bertucci, Angela Piccoli, Tatiana Gianni, Gianni T., Piccoli A., Bertucci C., and Campadelli-Fiume G.
- Subjects
Author's Correction ,Amino Acid Motifs ,DNA Mutational Analysis ,Immunology ,Molecular Conformation ,Peptide ,Herpesvirus 1, Human ,Biology ,Antiviral Agents ,Membrane Fusion ,Microbiology ,Cell Line ,Protein structure ,FUSION ,Viral Envelope Proteins ,Genes, Reporter ,Viral entry ,Cricetinae ,Virology ,Animals ,chemistry.chemical_classification ,Coiled coil ,Circular Dichroism ,Molecular Mimicry ,GLYCOPROTEIN ,Lipid bilayer fusion ,beta-Galactosidase ,Herpesvirus glycoprotein B ,Virus-Cell Interactions ,Protein Structure, Tertiary ,Cell biology ,GH ,Heptad repeat ,Biochemistry ,chemistry ,Insect Science ,Antigens, Surface ,ENTRY ,Peptides ,Glycoprotein ,Protein Binding ,HERPES SIMPLEX VIRUS - Abstract
Herpes simplex virus 1 (HSV-1) entry into cells and cell-cell fusion mediated by HSV-1 glycoproteins require four glycoproteins, gD, gB, gH, gL. Of these, gH is the only one that so far exhibits structural-functional features typical of viral fusion glycoproteins, i.e., a candidate fusion peptide and, downstream of it, a heptad repeat (HR) segment able to form a coiled coil, named HR-1. Here, we show that gH carries a functional HR-2 capable of physical interaction with HR-1. Specifically, mutational analysis of gH aimed at increasing or decreasing the ability of HR-2 to form a coiled coil resulted in an increase or decrease of fusion activity, respectively. HSV infection was modified accordingly. A mimetic peptide with the HR-2 sequence inhibited HSV-1 infection in a specific and dose-dependent manner. Circular dichroism spectroscopy showed that both HR-2 and HR-1 mimetic peptides adopt mainly random conformation in aqueous solution, while a decrease in peptide environmental polarity determines a conformational change, with a significant increase of the α-helical conformation content, in particular, for the HR-1 peptide. Furthermore, HR-1 and HR-2 mimetic peptides formed a stable complex, as revealed in nondenaturing electrophoresis and by circular dichroism. The mixture of HR-1 and HR-2 peptides reversed the inhibition of HSV infection exerted by the single peptides. Complex formation between HR-1 and HR-2 was independent of the presence of adjacent gH sequences and of additional glycoproteins involved in entry and fusion. Altogether, HR-2 adds to the features typical of class 1 fusion glycoproteins exhibited by HSV-1 gH.
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