Sixth European seminar in virology on virus\u2013host interaction at single cell and organism level

The 6th European Seminar in Virology (EuSeV) was held in Bertinoro, Italy, 22\u201324 June 2018, and brought together international scientists and young researchers working in the field of Virology. Sessions of the meeting included: virus\u2013host-interactions at organism and cell level; virus evolution and dynamics; regulation; immunity/immune response; and disease and therapy. This report summarizes lectures by the invited speakers and highlights advances in the field

Chemical and genetically-based approaches for the treatment of HIV-1 infection

Advances in HIV-1 drug therapy drastically decreased mortality and significantly improved quality of life for HIV-1-infected patients since the early days of epidemic. However, in spite of favorable outcomes provided by the newer therapies, HAART is not curative and patients are at risk of developing HIV-associated disorders. Thus, the development of new inhibitors and alternative therapeutic strategies able to interfere with the early stages of the viral replication would be mandatory for the future anti-HIV-1 treatments. In this PhD project, on one side, we are screening natural extracts and bioactive compounds in order to assess their antiviral activity in appropriate cell models. On the other side, the safety and the feasibility of third generation self-inactivating lentiviral vectors expressing anti-HIV-1 transgenes have been investigated. Specifically, these vectors exploit the RNA interference machinery to target either viral proteins (i.e. Tat, Rev and Vif) and the cellular co-receptor CCR5, along with the maC46 fusion inhibitor, capable of rendering HIV-1 target cells stably resistant to the viral infection. The ultimate aim is to explore the use of genetic medicines to modify CD4+ T lymphocytes (and their precursors: hematopoietic stem cells), in order to treat AIDS-related lymphoma patients, who offer a unique opportunity to evaluate gene therapy-based, anti-HIV strategies in an ethically acceptable clinical setting.I notevoli progressi nella terapia farmacologica per infezione da HIV-1 hanno drasticamente ridotto la mortalità legata al decorso della patologia e significativamente migliorato la qualità della vita dei pazienti affetti. Tuttavia, questo trattamento non è in grado di eradicare l'infezione, ed è spesso accompagnato dall'insorgenza di specie virali farmaco-resistenti e tossicità d’organo. Da ciò, deriva una costante ricerca ed ottimizzazione di strategie alternative e/o complementari per la cura dell’infezione da HIV-1. Questo progetto di dottorato si inserisce in questa ricerca globale di nuove terapie, proponendo due diversi approcci. Da un lato, diverse molecole derivanti da estratti botanici sono state prese come oggetto di studio al fine di valutare la loro attività antivirale. Dall'altro lato, sono stati sviluppati e testati vettori lentivirali di terza generazione, in grado di esprimere dei fattori che interferiscono con diverse fasi del ciclo replicativo di HIV-1. Nello specifico, questi vettori sfruttano il meccanismo di interferenza a RNA per colpire alcune proteine ​​virali (Tat, Rev e Vif) e il co-recettore cellulare CCR5, insieme ad un inibitore di fusione (maC46), con il fine di rendere le cellule bersaglio di HIV-1 stabilmente resistenti al virus. L’obiettivo ultimo consiste nell'accertare l’efficacia e la sicurezza di tali vettori in modelli animali, per giungere infine alla manipolazione genetica di cellule staminali ematopoietiche, derivanti da pazienti HIV-positivi ed affetti da linfoma, per i quali il trapianto di cellule staminali ematopoietiche geneticamente modificate rappresenterebbe l’unico contesto clinico eticamente accettabile

Sixth European Seminar in Virology on Virus–Host Interaction at Single Cell and Organism Level

The 6th European Seminar in Virology (EuSeV) was held in Bertinoro, Italy, 22–24 June 2018, and brought together international scientists and young researchers working in the field of Virology. Sessions of the meeting included: virus–host-interactions at organism and cell level; virus evolution and dynamics; regulation; immunity/immune response; and disease and therapy. This report summarizes lectures by the invited speakers and highlights advances in the field

Targeting the Regulatory Subunit R2Alpha of Protein Kinase A in Human Glioblastoma through shRNA-Expressing Lentiviral Vectors

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies

Gene therapy of AIDS: a novel approach combining anti HIV-1 siRNAs and a fusion inhibitor

Background: Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, combinatorial approaches, aimed at interfering with different steps of viral replication, represent powerful strategies. To this end, we previously developed a series of self-inactivating lentiviral vectors expressing multiple small interfering (si)-RNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1). The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. We identified two effective anti HIV combinatorial vectors that conferred protection against R5 and X4 tropic viruses in human primary CD4+ T lymphocytes. Methods: In the present study, these two vectors were further modified by the inclusion of a membrane-anchored peptide (maC46), which has been shown to efficiently block the entry of both CXCR-4 and CCR5 using viruses. The maC46 was inserted under the transcriptional control of the human Elongation Factor 1 promoter. Additionally, an optimized version of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE*) was also inserted in the new generation of lentiviral vectors. The antiviral activity of the optimized vectors was assessed in different cellular models. Results: The developed vectors conferred to the transduced cells a sustained and significant protection against HIV-1 challenge. Conclusion: Overall our results contribute to gain further insights in the design of combinatorial gene therapy approaches against HIV-1 for clinical application

Combination of anti HIV-1 siRNAs and a fusion inhibitor for genetic treatment of AIDS.

Despite the remarkable success of highly active antiretroviral therapy (HAART) in lowering the plasma viral load in patients with HIV -1 infection and in slowing down the disease progression, many obstacles still remain, including the generation of drug-resistant viral species, the difficulty in eradicating latent reservoirs and the high costs of the therapy. The development of complementary strategies to completely eradicate or to control HIV-1 infection without daily drug intake is therefore a priority. In this context, anti HIV-1 gene therapy based on intracellular immunization of autologous T cells or their progenitors, i.e. the CD34+ hematopoietic stem cells, resistant to infection, appears a particularly promising approach to repopulate the immune system.In order to interfere with different steps of viral replication, we previously developed a series of self-inactivating lentiviral vectors expressing multiple small interfering (si)-RNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1). The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Two effective anti- HIV combinatorial vectors that conferred protection against R5 and X4 tropic virusesin human primary CD4+ T lymphocytes were identified. Further modification of the combinatorial vectors were accomplished by the inclusion of a membrane-anchored peptide (maC46), which has been shown to efficiently block the entry of both CXCR-4 and CCR5-using viruses. The maC46 was inserted under the transcriptional control of the human Elongation Factor 1 promoter. Additionally, an optimized version of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE*) was also inserted in the new generation of lentiviral vectors. The antiviral activity of the optimized vectors was assessed in different cellular models. Overall our results contribute to gain further insights in the design of combinatorial gene therapy approaches against HIV-1 for clinical application

Anti HIV-1 gene therapy approach combining multiple siRNA with the membrane anchored fusion inhibitor C peptide maC46

Background HIV-1 infection is normally characterized by sustained viral replication and a progressive loss of CD4+ T cells, leading to AIDS. Combined antiretroviral therapy (cART) suppresses viral replication and drastically reduces mortality. However, cART alone is not able to eradicate the virus, which persists in reservoirs, and it is still associated with considerable comorbidities and the emergence of drug-resistant escape mutants. A cure for HIV-1 infection is needed to bypass the limitations of the current therapy and restore health. Advances in the fields of gene-targeting strategies, based on intracellular immunization of autologous T cells or their progenitors, i.e. the CD34+ hematopoietic stem cells (HSC), appear encouraging to repopolate the immune system. Materials and Methods Self-inactivating (SIN) lentiviral vectors were generated in order to express multiple HIV-1 inhibitors. Recombinant particles were produced in HEK 293T cells and titrated by flow cytometry and Reverse Transcriptase (RT) activity assay. To address the antiviral activity of the combinatorial vectors, CD4+ T lymphoblastoid Jurkat cells were transduced with lentiviral particles, then underwent to MTT cell viability assay and infected with the HIV-1 HXBc2 Vpr+/Vpu+/Nef+ strain (MOI=0.1 TCID50/cell). In order to test the mutagenic potential of this SIN lentiviral platform, the in vitro immortlization (IVIM) assay was employed. Results The generated vectors were characterized by different combinations of siRNA targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, along with or without an HIV membrane-anchored fusion inhibitor (maC46) , which has been shown to efficiently block the entry of both CXCR-4 and CCR5-using viruses. Specifically, the siRNA were either expressed as single transcriptional unit under the control of different human RNA polymerase III promoters (U6, 7SK, H1), or simultaneously, as an extended shRNA. The maC46, which derives from the C-terminal heptad repeat (HR2) domain of gp41, was cloned under the transcriptional control of the human elongation factor 1 alpha (EF1 alpha) promoter. Finally, an optimized version of the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE*), able to reduce the risk of oncogenicity, was introduced in all vectors. The EF1 alpha promoter led to a high level of maC46 expression, with a correct localization of the peptide at the plasma membrane. The developed lentiviral particles efficiently transduce Jurkat cells, without cytotoxic effect, and potently block HIV-1 replication up to 4 weeks post-infection. Furthermore, no impact on the viability or the proliferation of transduced murine cells with these vectors was observed in the IVIM assay. Conclusions The new generation vectors, combining a potent fusion inhibitor with 3 shRNAs, appear to be extremely promising as anti-HIV-1 vectors. Once challenge experiments in different cell lines will be completed, the best performing vectors will be assayed in primary CD4+ cells and CD34+ HSCs in order to further validate this approach for clinical application in selected HIV-1+ patients

Multi target mode of HIV-1 inhibition by the bioactive molecule Lupeol extracted from the Indian plant Hemidesmus indicus R.Br.

In the effort of identifying novel therapeutic approaches for the discovery of new anti-infective drugs, the traditional Indian medicine and the use of natural compounds active against various diseases, including viral infections, receive nowadays considerable attention. Hemidesmus indicus R.Br. is known for its ethno-botanical use and its efficacy in the treatment of bone-loss diseases, and, in the Indian traditional medicine, is widely used for the treatment of blood diseases, dyspepsia, loss of taste, dyspnea, cough, poison, menorrhagia, fever and diarrhea. In the last thirty years, many natural substances have shown antiviral activity, but very few of them showed multi target mode of action. For this reason, we assessed the Hemidesmus indicus R.Br. decoction against i) the Human Immunodeficiency Virus type 1 (HIV -1) Reverse Transcriptase (RT)-associated functions and ii) the \u3b1-glucosidase activity, with the aim to identify a molecule capable to inhibit with a multiple mode of action different promising targets. Hemidesmus indicus R.Br. decoction has been shown to be active on the inhibition of both RT-associated functions and \u3b1-glucosidase activity. Active decoction was fractionated up to obtain the bioactive components which have been isolated, tested and identified as Lupeol, Lupeol Acetate, Caffeic Acid, Chlorogenic Acid and \u3b2-amirine acetate. Among these, Lupeol inhibited the HIV-1 RT -associated RNase H function, and the \u3b1 -glucosidase activity. Our results suggest that this bioactive molecule is a potential lead for further development of drugs with multi target mechanism of action

Type-I interferon signatures in SARS-CoV-2 infected Huh7 cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has caused a global health emergency. A key feature of COVID-19 is dysregulated interferon-response. Type-I interferon (IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in the pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identified that SARS-CoV-2 infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to inhibit RIG-I mediated IFN-β production. Our results also confirm the recent findings that IFN-I pretreatment is able to reduce the susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Moreover, senescent Huh7 cells, in spite of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection, and the virus effectively inhibited IFIT1 in these cells. Finally, proteomic comparison between SARS-CoV-2, SARS-CoV, and MERS-CoV revealed a distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Investigation of different interferon-stimulated genes and their role in the inhibition of SARS-CoV-2 pathogenesis may direct novel antiviral strategies

Cell-type-resolved quantitative proteomics map of interferon response against SARS-CoV-2

The commonly used laboratory cell lines are the first line of experimental models to study the pathogenicity and performing antiviral assays for emerging viruses. Here, we assessed the tropism and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different human cell lines, compared their growth characteristics, and performed quantitative proteomics for the susceptible cell lines. Overall, Calu-3, Caco2, Huh7, and 293FT cell lines showed a high-to-moderate level of susceptibility to SARS-CoV-2. In Caco2 cells, the virus can achieve high titers in the absence of any prominent cytopathic effect. The protein abundance profile during SARS-CoV-2 infection revealed cell-type-specific regulation of cellular pathways. Type-I interferon signaling was identified as the common dysregulated cellular response in Caco2, Calu-3, and Huh7 cells. Together, our data show cell-type specific variability for cytopathogenicity, susceptibility, and cellular response to SARS-CoV-2 and provide important clues to guide future studies