Cristina Parolin - Academia.edu (original) (raw)

Papers by Cristina Parolin

Viruses

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C v... more Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for ...

Identification and development of effective drugs active against SARS-CoV-2 are urgently needed. ... more Identification and development of effective drugs active against SARS-CoV-2 are urgently needed. Here, we report on the anti-SARS-CoV-2 activity of MEDS433, a novel inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of thede novopyrimidines biosynthesis. MEDS433 inhibitsin vitrovirus replication in the low nanomolar range, and through a mechanism that stems from its ability to block hDHODH activity. MEDS433 thus represents an attractive candidate to develop novel anti-SARS-CoV-2 agents.

Viruses

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the ... more HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N’-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations reve...

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a h... more Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate, and caused by members of the Filoviridae family. The recent large outbreak of EVD in West Africa (2013-2016), highlighted the worldwide danger of this disease and its impact on global public health and economy. The development of highly needed anti-Filoviridae antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, and therefore screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to in vitro study of Filoviridae entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus like particles, tremendously boosted both our knowledge on viral life cycle and the identification of promising anti-Filoviridae compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds an...

Infectious Agents and Cancer

Background: With few exceptions, current chemotherapy and radiotherapy protocols only obtain a sl... more Background: With few exceptions, current chemotherapy and radiotherapy protocols only obtain a slightly prolonged survival with severe adverse effects in patients with advanced solid tumors. In particular, most solid malignancies not amenable to radical surgery still carry a dismal prognosis, which unfortunately is also the case for relapsing disease after surgery. Even though targeted therapies obtained good results, clinical experience showed that tumors eventually develop resistance. On the other hand, earlier attempts of cancer immunotherapy failed to show consistent efficacy. More recently, a deeper knowledge of immunosuppression in the tumor microenvironment (TME) allowed the development of effective drugs: in particular, monoclonal antibodies targeting the so-called immune checkpoint molecules yielded striking and lasting effects in some tumors. Unfortunately, these monoclonal antibodies are not effective in a majority of patients and are ineffective in several solid malignancies. Furthermore, due to their mechanism of action, checkpoint inhibitors often elicit autoimmune-like disease. Main body: The use of viruses as oncolytic agents (OVs) was considered in the past, while only recently OVs revealed a connection with immunotherapy. However, their antitumoral potential has remained largely unexplored, due to safety concerns and some limitations in the techniques to manipulate viruses. OV research was recently revived by a better knowledge of viral/cancer biology and advances in the methodologies to delete virulence/immune-escape related genes from even complex viral genomes or "to arm" OVs with appropriate transgenes. Recently, the first oncolytic virus, the HSV-1 based Talimogene Laherparepvec (T-VEC), was approved for the treatment of non-resectable melanoma in USA and Europe. Conclusion: OVs have the potential to become powerful agents of cancer immune and gene therapy. Indeed, in addition to their selective killing activity, they can act as versatile gene expression platforms for the delivery of therapeutic genes. This is particularly true for viruses with a large DNA genome, that can be manipulated to address the multiple immunosuppressive features of the TME. This review will focus on the open issues, on the most promising lines of research in the OV field and, more in general, on how OVs could be improved to achieve real clinical breakthroughs in cancers that are usually difficult to treat by immunotherapy.

Pathogens and disease, Jun 20, 2017

Despite the availability of several anti-retrovirals there is still an urgent need for developing... more Despite the availability of several anti-retrovirals there is still an urgent need for developing novel therapeutic strategies and finding new drugs against underexplored HIV-1 targets. Among them, there are the HIV-1 Reverse Transcriptase (RT)-associated Ribonuclease H (RNase H) function and the cellular α-glucosidase, involved in the control mechanisms of N-linked glycoproteins formation in the endoplasmic reticulum. It is known that many natural compounds, such as pentacyclic triterpenes, are a promising class of HIV-1 inhibitors. Hence, here we tested the pentacyclic triterpene Lupeol, showing that it inhibits the HIV-1 RT-associated RNase H function. We then performed combination studies of Lupeol and the active site RNase H inhibitor RDS1759, and blind docking calculations, demonstrating that Lupeol binds to an HIV-1 RT allosteric pocket. On the bases of these results and searching for potential multi-target active drug supplement, we also investigated the anti-HIV-1 activity ...

Phytomedicine, 2016

Despite the availability of effective antiretroviral therapies, drugs for HIV-1 treatment with ne... more Despite the availability of effective antiretroviral therapies, drugs for HIV-1 treatment with new mode of action are still needed. An innovative approach is aimed to identify dual HIV-1 inhibitors, small molecules that can inhibit two viral functions at the same time. Rhubarb, originated from Rheum palmatum L. and Rheum officinale Baill., is one of the earliest and most commonly used medicinal plants in Traditional Chinese Medicine (TCM) practice. We wanted to explore TCM for the identification of new chemical scaffolds with dual action abilities against HIV-1. R. palmatum L. and R. officinale Baill. extracts along with their main single isolated constituents anthraquinone derivatives were tested on both HIV-1 Reverse Transcriptase (RT)-associated DNA Polymerase (RDDP) and Ribonuclease H (RNase H) activities in biochemical assays. Active compounds were then assayed for their effects on HIV-1 mutated RTs, integrase (IN) and viral replication. Both R. palmatum L. and R. officinale Baill. extracts inhibited the HIV-1 RT-associated RNase H activity. Among the isolated constituents, Sennoside A and B were effective on both RDDP and RNase H RT-associated functions in biochemical assays. Sennoside A was less potent when tested on K103N, Y181C, Y188L, N474A and Q475A mutated RTs, suggesting the involvement of two RT binding sites for its antiviral activity. Sennoside A affected also HIV-1 IN activity in vitro and HIV-1 replication in cell-based assays. Viral DNA production and time of addition studies showed that Sennoside A targets the HIV-1 reverse transcription process. Sennoside A is a new scaffold for the development of HIV-1 dual RT inhibitors.

Chemmedchem, May 21, 2014

Molecular therapy. Nucleic acids, Jan 19, 2016

Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this c... more Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs 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) were developed. 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. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combi...

STEM CELLS, 2016

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease ca... more Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell l...

Pathogens and disease, Jan 30, 2015

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a h... more Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate and a treatment has not been developed yet. Recently, it has been shown that cationic amphiphiles, among them the antiarrhythmic drug amiodarone, inhibit filovirus infection. In the present work we investigated how amiodarone interferes with Ebola virus infection. Wild type Sudan ebolavirus and recombinant Vesicular Stomatitis virus (VSV), pseudotyped with the Zaire ebolavirus glycoprotein, were used to gain further insight into the ability of amiodarone to affect Ebola virus infection. We show that amiodarone decreases Ebola virus infection at concentrations close to those found in the sera of patients treated for arrhythmias. The drug acts by interfering with the fusion of the viral envelope with the endosomal membrane. We also show that MDEA, the main amiodarone metabolite, contributes to the antiviral activity. Finally, studies with amiodarone analogues indicate tha...

Cells, 2014

Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiqui... more Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. Indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. Due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. Thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to bypass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. In this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells.

A soluble form of the CD4 receptor (sCD4) can either enhance or inhibit the infection of cells by... more A soluble form of the CD4 receptor (sCD4) can either enhance or inhibit the infection of cells by simian immunodeficiency virus (SIV) and human immunodeficiency virus. We investigated the basis for these varying effects by studying the entry of three SIV isolates into CD4-positive and CD4-negative cells expressing different chemokine receptors. Infection of CD4-negative cells depended upon the viral envelope glycoproteins and upon the chemokine receptor, with CCR5 and gpr15 being more efficient than STRL33. Likewise, enhancement of infection by sCD4 was observed when CCR5- and gpr15-expressing target cells were used but not when those expressing STRL33 were used. The sCD4-mediated enhancement of virus infection of CD4-negative, CCR5- positive cells was related to the sCD4-induced increase in binding of the viral gp120 envelope glycoprotein to CCR5. Inhibitory effects of sCD4 could largely be explained by competition for virus attachment to cellular CD4 rather than other detrimental ...

Gene Therapy, 2001

Even in the era of highly active antiretroviral therapy (HAART), gene therapy (GT) can remain a p... more Even in the era of highly active antiretroviral therapy (HAART), gene therapy (GT) can remain a promising approach for suppressing HIV infection, especially if complemented with other forms of pharmacological and immunological intervention. A large number of vectors and targets have been studied. Here we discuss the potential of genetically treated, antigen-specific immunocompetent cells for adoptive autologous immunotherapy of HIV infection.

Gene Therapy, 2005

Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficienc... more Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficiency virus type 1 (HIV-1) could be of primary importance for the control of this viral infection. Here, we designed a chimeric cell surface receptor (105TCR) to provide primary human T-lymphocytes with antibody-type specificity for the HIV-1 envelope glycoprotein. This receptor includes the single chain Fv domain of the neutralizing anti-gp120 human monoclonal antibody F105, CD8a hinge and the transmembrane and the cytoplasmic domains of TCRz. Our results show that 105TCR is expressed at the cellular surface and is capable of recognizing the HIV-1 envelope glycoprotein inducing highly efficient effector T-cell responses, including extracellular signal-regulated kinase phosphorylation and cytokine secretion. Moreover, human primary CD8+ T-lymphocytes transduced by oncoretroviral and lentiviral vectors containing the 105TCR gene are able to mediate in vitro-specific cytolysis of envelope-expressing cells and HIV-1-infected CD4+ T-lymphocytes. These findings suggest that 105TCR is particularly suited for in vivo efficacy studies.

Journal of …, 1998

The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-... more The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.

Journal of …, 2007

The biogenesis of multivesicular bodies (MVBs) is topologically equivalent to virion budding. Hen... more The biogenesis of multivesicular bodies (MVBs) is topologically equivalent to virion budding. Hence, a number of viruses exploit the MVB pathway to build their envelope and exit from the cell. By expression of dominant negative forms of Vps4 and Vps24, two components of the MVB pathway, we observed an impairment in infectious herpes simplex virus (HSV) assembly/egress, in agreement with a recent report showing the involvement in HSV envelopment of Vps4, the MVB-specific ATPase (C. M. Crump, C. Yates, and T. Minson, J. Virol. 81:7380-7387). Furthermore, HSV infection resulted in morphological changes to MVBs. Glycoprotein B (gB), one of the most highly conserved glycoproteins across the Herpesviridae family, was sorted to MVB membranes. In cells expressing the dominant negative form of Vps4, the site of intracellular gB accumulation was altered; part of gB accumulated as an endoglycosidase H-sensitive immature form at a calreticulin-positive compartment, indicating that gB traffic was dependent on a functional MVB pathway. gB was ubiquitinated in both infected and transfected cells. Ubiquitination was in part dependent on ubiquitin lysine 63, a signal for cargo sorting to MVBs. Partial deletion of the gB cytoplasmic tail resulted in a dramatic reduction of ubiquitination, as well as of progeny virus assembly and release to the extracellular compartment. Thus, HSV envelopment/egress and gB intracellular trafficking are dependent on functional MVB biogenesis. Our data support the view that the sorting of gB to MVB membranes may represent a critical step in HSV envelopment and egress and that modified MVB membranes constitute a platform for HSV cytoplasmic envelopment or that MVB components are recruited to the site(s) of envelopment.

Journal of …, 1997

Division of Human Retrovirology, Dana-Farber Cancer Institute, and Department of Pathology, Harva... more Division of Human Retrovirology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School,1 and Department of Cancer Biology, Harvard School of Public Health,2 Boston, Massachusetts 02115; Institute of Microbiology, University of ...

To compare three methods for using HIV-1 genotype to predict antiretroviral drug susceptibility. ... more To compare three methods for using HIV-1 genotype to predict antiretroviral drug susceptibility. Methods: We applied three genotypic interpretation algorithms to 478 reverse transcriptase (RT) and 410 protease sequences for which phenotypic data were available. Sequences were obtained from clinical practice and from published sequences in the Stanford HIV-1 RT and Protease Sequence Database. The genotypic interpretation algorithms included: Stanford HIVdb program (HIVdb), the Visible Genetics/Bayer Diagnostics Guidelines 6.0 (VGI) and a genotypic interpretation program (AntiRetroScan, ARS) developed at the University of Siena, Italy. Genotypic interpretations were normalized to a three-level output: susceptible, intermediate and resistant. Discordances were defined as differences between genotype and phenotype for the same virus isolate. Discordances for which an isolate was considered susceptible by one test but resistant by another test were considered major discordances. Results: The frequency of major discordances between genotype and phenotype was 10.6, 13.7 and 15.7% for ARS, VGI and HIVdb, respectively (P < 0.0001 for ARS versus HIVdb and for ARS versus VGI; P = 0.002 for VGI versus HIVdb). The correlation between genotype and phenotype was highest for non-nucleoside RT inhibitors and lowest for nucleoside RT inhibitors. Half of the major discordances involved stavudine, didanosine and zalcitabine. The concordance among the three genotypic algorithms was high, with weighted Kappa values ranging between 0.76 and 0.84 for the pairwise comparisons between each of the algorithms. Conclusions: Genotype interpretation algorithms correctly predict phenotype in 85-90% of cases, but the rate of concordance is not uniformly distributed among different drugs. These data provide insight into the potential additional benefit derived from phenotyping.

Viruses

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C v... more Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for ...

Identification and development of effective drugs active against SARS-CoV-2 are urgently needed. ... more Identification and development of effective drugs active against SARS-CoV-2 are urgently needed. Here, we report on the anti-SARS-CoV-2 activity of MEDS433, a novel inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of thede novopyrimidines biosynthesis. MEDS433 inhibitsin vitrovirus replication in the low nanomolar range, and through a mechanism that stems from its ability to block hDHODH activity. MEDS433 thus represents an attractive candidate to develop novel anti-SARS-CoV-2 agents.

Viruses

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the ... more HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N’-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations reve...

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a h... more Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate, and caused by members of the Filoviridae family. The recent large outbreak of EVD in West Africa (2013-2016), highlighted the worldwide danger of this disease and its impact on global public health and economy. The development of highly needed anti-Filoviridae antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, and therefore screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to in vitro study of Filoviridae entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus like particles, tremendously boosted both our knowledge on viral life cycle and the identification of promising anti-Filoviridae compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds an...

Infectious Agents and Cancer

Background: With few exceptions, current chemotherapy and radiotherapy protocols only obtain a sl... more Background: With few exceptions, current chemotherapy and radiotherapy protocols only obtain a slightly prolonged survival with severe adverse effects in patients with advanced solid tumors. In particular, most solid malignancies not amenable to radical surgery still carry a dismal prognosis, which unfortunately is also the case for relapsing disease after surgery. Even though targeted therapies obtained good results, clinical experience showed that tumors eventually develop resistance. On the other hand, earlier attempts of cancer immunotherapy failed to show consistent efficacy. More recently, a deeper knowledge of immunosuppression in the tumor microenvironment (TME) allowed the development of effective drugs: in particular, monoclonal antibodies targeting the so-called immune checkpoint molecules yielded striking and lasting effects in some tumors. Unfortunately, these monoclonal antibodies are not effective in a majority of patients and are ineffective in several solid malignancies. Furthermore, due to their mechanism of action, checkpoint inhibitors often elicit autoimmune-like disease. Main body: The use of viruses as oncolytic agents (OVs) was considered in the past, while only recently OVs revealed a connection with immunotherapy. However, their antitumoral potential has remained largely unexplored, due to safety concerns and some limitations in the techniques to manipulate viruses. OV research was recently revived by a better knowledge of viral/cancer biology and advances in the methodologies to delete virulence/immune-escape related genes from even complex viral genomes or "to arm" OVs with appropriate transgenes. Recently, the first oncolytic virus, the HSV-1 based Talimogene Laherparepvec (T-VEC), was approved for the treatment of non-resectable melanoma in USA and Europe. Conclusion: OVs have the potential to become powerful agents of cancer immune and gene therapy. Indeed, in addition to their selective killing activity, they can act as versatile gene expression platforms for the delivery of therapeutic genes. This is particularly true for viruses with a large DNA genome, that can be manipulated to address the multiple immunosuppressive features of the TME. This review will focus on the open issues, on the most promising lines of research in the OV field and, more in general, on how OVs could be improved to achieve real clinical breakthroughs in cancers that are usually difficult to treat by immunotherapy.

Pathogens and disease, Jun 20, 2017

Despite the availability of several anti-retrovirals there is still an urgent need for developing... more Despite the availability of several anti-retrovirals there is still an urgent need for developing novel therapeutic strategies and finding new drugs against underexplored HIV-1 targets. Among them, there are the HIV-1 Reverse Transcriptase (RT)-associated Ribonuclease H (RNase H) function and the cellular α-glucosidase, involved in the control mechanisms of N-linked glycoproteins formation in the endoplasmic reticulum. It is known that many natural compounds, such as pentacyclic triterpenes, are a promising class of HIV-1 inhibitors. Hence, here we tested the pentacyclic triterpene Lupeol, showing that it inhibits the HIV-1 RT-associated RNase H function. We then performed combination studies of Lupeol and the active site RNase H inhibitor RDS1759, and blind docking calculations, demonstrating that Lupeol binds to an HIV-1 RT allosteric pocket. On the bases of these results and searching for potential multi-target active drug supplement, we also investigated the anti-HIV-1 activity ...

Phytomedicine, 2016

Despite the availability of effective antiretroviral therapies, drugs for HIV-1 treatment with ne... more Despite the availability of effective antiretroviral therapies, drugs for HIV-1 treatment with new mode of action are still needed. An innovative approach is aimed to identify dual HIV-1 inhibitors, small molecules that can inhibit two viral functions at the same time. Rhubarb, originated from Rheum palmatum L. and Rheum officinale Baill., is one of the earliest and most commonly used medicinal plants in Traditional Chinese Medicine (TCM) practice. We wanted to explore TCM for the identification of new chemical scaffolds with dual action abilities against HIV-1. R. palmatum L. and R. officinale Baill. extracts along with their main single isolated constituents anthraquinone derivatives were tested on both HIV-1 Reverse Transcriptase (RT)-associated DNA Polymerase (RDDP) and Ribonuclease H (RNase H) activities in biochemical assays. Active compounds were then assayed for their effects on HIV-1 mutated RTs, integrase (IN) and viral replication. Both R. palmatum L. and R. officinale Baill. extracts inhibited the HIV-1 RT-associated RNase H activity. Among the isolated constituents, Sennoside A and B were effective on both RDDP and RNase H RT-associated functions in biochemical assays. Sennoside A was less potent when tested on K103N, Y181C, Y188L, N474A and Q475A mutated RTs, suggesting the involvement of two RT binding sites for its antiviral activity. Sennoside A affected also HIV-1 IN activity in vitro and HIV-1 replication in cell-based assays. Viral DNA production and time of addition studies showed that Sennoside A targets the HIV-1 reverse transcription process. Sennoside A is a new scaffold for the development of HIV-1 dual RT inhibitors.

Chemmedchem, May 21, 2014

Molecular therapy. Nucleic acids, Jan 19, 2016

Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this c... more Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs 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) were developed. 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. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combi...

STEM CELLS, 2016

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease ca... more Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell l...

Pathogens and disease, Jan 30, 2015

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a h... more Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate and a treatment has not been developed yet. Recently, it has been shown that cationic amphiphiles, among them the antiarrhythmic drug amiodarone, inhibit filovirus infection. In the present work we investigated how amiodarone interferes with Ebola virus infection. Wild type Sudan ebolavirus and recombinant Vesicular Stomatitis virus (VSV), pseudotyped with the Zaire ebolavirus glycoprotein, were used to gain further insight into the ability of amiodarone to affect Ebola virus infection. We show that amiodarone decreases Ebola virus infection at concentrations close to those found in the sera of patients treated for arrhythmias. The drug acts by interfering with the fusion of the viral envelope with the endosomal membrane. We also show that MDEA, the main amiodarone metabolite, contributes to the antiviral activity. Finally, studies with amiodarone analogues indicate tha...

Cells, 2014

Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiqui... more Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. Indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. Due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. Thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to bypass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. In this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells.

A soluble form of the CD4 receptor (sCD4) can either enhance or inhibit the infection of cells by... more A soluble form of the CD4 receptor (sCD4) can either enhance or inhibit the infection of cells by simian immunodeficiency virus (SIV) and human immunodeficiency virus. We investigated the basis for these varying effects by studying the entry of three SIV isolates into CD4-positive and CD4-negative cells expressing different chemokine receptors. Infection of CD4-negative cells depended upon the viral envelope glycoproteins and upon the chemokine receptor, with CCR5 and gpr15 being more efficient than STRL33. Likewise, enhancement of infection by sCD4 was observed when CCR5- and gpr15-expressing target cells were used but not when those expressing STRL33 were used. The sCD4-mediated enhancement of virus infection of CD4-negative, CCR5- positive cells was related to the sCD4-induced increase in binding of the viral gp120 envelope glycoprotein to CCR5. Inhibitory effects of sCD4 could largely be explained by competition for virus attachment to cellular CD4 rather than other detrimental ...

Gene Therapy, 2001

Even in the era of highly active antiretroviral therapy (HAART), gene therapy (GT) can remain a p... more Even in the era of highly active antiretroviral therapy (HAART), gene therapy (GT) can remain a promising approach for suppressing HIV infection, especially if complemented with other forms of pharmacological and immunological intervention. A large number of vectors and targets have been studied. Here we discuss the potential of genetically treated, antigen-specific immunocompetent cells for adoptive autologous immunotherapy of HIV infection.

Gene Therapy, 2005

Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficienc... more Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficiency virus type 1 (HIV-1) could be of primary importance for the control of this viral infection. Here, we designed a chimeric cell surface receptor (105TCR) to provide primary human T-lymphocytes with antibody-type specificity for the HIV-1 envelope glycoprotein. This receptor includes the single chain Fv domain of the neutralizing anti-gp120 human monoclonal antibody F105, CD8a hinge and the transmembrane and the cytoplasmic domains of TCRz. Our results show that 105TCR is expressed at the cellular surface and is capable of recognizing the HIV-1 envelope glycoprotein inducing highly efficient effector T-cell responses, including extracellular signal-regulated kinase phosphorylation and cytokine secretion. Moreover, human primary CD8+ T-lymphocytes transduced by oncoretroviral and lentiviral vectors containing the 105TCR gene are able to mediate in vitro-specific cytolysis of envelope-expressing cells and HIV-1-infected CD4+ T-lymphocytes. These findings suggest that 105TCR is particularly suited for in vivo efficacy studies.

Journal of …, 1998

The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-... more The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.

Journal of …, 2007

The biogenesis of multivesicular bodies (MVBs) is topologically equivalent to virion budding. Hen... more The biogenesis of multivesicular bodies (MVBs) is topologically equivalent to virion budding. Hence, a number of viruses exploit the MVB pathway to build their envelope and exit from the cell. By expression of dominant negative forms of Vps4 and Vps24, two components of the MVB pathway, we observed an impairment in infectious herpes simplex virus (HSV) assembly/egress, in agreement with a recent report showing the involvement in HSV envelopment of Vps4, the MVB-specific ATPase (C. M. Crump, C. Yates, and T. Minson, J. Virol. 81:7380-7387). Furthermore, HSV infection resulted in morphological changes to MVBs. Glycoprotein B (gB), one of the most highly conserved glycoproteins across the Herpesviridae family, was sorted to MVB membranes. In cells expressing the dominant negative form of Vps4, the site of intracellular gB accumulation was altered; part of gB accumulated as an endoglycosidase H-sensitive immature form at a calreticulin-positive compartment, indicating that gB traffic was dependent on a functional MVB pathway. gB was ubiquitinated in both infected and transfected cells. Ubiquitination was in part dependent on ubiquitin lysine 63, a signal for cargo sorting to MVBs. Partial deletion of the gB cytoplasmic tail resulted in a dramatic reduction of ubiquitination, as well as of progeny virus assembly and release to the extracellular compartment. Thus, HSV envelopment/egress and gB intracellular trafficking are dependent on functional MVB biogenesis. Our data support the view that the sorting of gB to MVB membranes may represent a critical step in HSV envelopment and egress and that modified MVB membranes constitute a platform for HSV cytoplasmic envelopment or that MVB components are recruited to the site(s) of envelopment.

Journal of …, 1997

Division of Human Retrovirology, Dana-Farber Cancer Institute, and Department of Pathology, Harva... more Division of Human Retrovirology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School,1 and Department of Cancer Biology, Harvard School of Public Health,2 Boston, Massachusetts 02115; Institute of Microbiology, University of ...

To compare three methods for using HIV-1 genotype to predict antiretroviral drug susceptibility. ... more To compare three methods for using HIV-1 genotype to predict antiretroviral drug susceptibility. Methods: We applied three genotypic interpretation algorithms to 478 reverse transcriptase (RT) and 410 protease sequences for which phenotypic data were available. Sequences were obtained from clinical practice and from published sequences in the Stanford HIV-1 RT and Protease Sequence Database. The genotypic interpretation algorithms included: Stanford HIVdb program (HIVdb), the Visible Genetics/Bayer Diagnostics Guidelines 6.0 (VGI) and a genotypic interpretation program (AntiRetroScan, ARS) developed at the University of Siena, Italy. Genotypic interpretations were normalized to a three-level output: susceptible, intermediate and resistant. Discordances were defined as differences between genotype and phenotype for the same virus isolate. Discordances for which an isolate was considered susceptible by one test but resistant by another test were considered major discordances. Results: The frequency of major discordances between genotype and phenotype was 10.6, 13.7 and 15.7% for ARS, VGI and HIVdb, respectively (P < 0.0001 for ARS versus HIVdb and for ARS versus VGI; P = 0.002 for VGI versus HIVdb). The correlation between genotype and phenotype was highest for non-nucleoside RT inhibitors and lowest for nucleoside RT inhibitors. Half of the major discordances involved stavudine, didanosine and zalcitabine. The concordance among the three genotypic algorithms was high, with weighted Kappa values ranging between 0.76 and 0.84 for the pairwise comparisons between each of the algorithms. Conclusions: Genotype interpretation algorithms correctly predict phenotype in 85-90% of cases, but the rate of concordance is not uniformly distributed among different drugs. These data provide insight into the potential additional benefit derived from phenotyping.