Publications by authors named "Cristina Parolin"

63 Publications

Molecular Pathology Analysis of SARS-CoV-2 in Syncytiotrophoblast and Hofbauer Cells in Placenta from a Pregnant Woman and Fetus with COVID-19.

Pathogens 2021 Apr 15;10(4). Epub 2021 Apr 15.

Department of Pathology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.

A small number of neonates delivered to women with SARS-CoV-2 infection have been found to become infected through intrauterine transplacental transmission. These cases are associated with a group of unusual placental pathology abnormalities that include chronic histiocytic intervillositis, syncytiotrophoblast necrosis, and positivity of the syncytiotrophoblast for SARS-CoV-2 antigen or RNA. Hofbauer cells constitute a heterogeneous group of immunologically active macrophages that have been involved in transplacental infections that include such viral agents as Zika virus and human immunodeficiency virus. The role of Hofbauer cells in placental infection with SARS-CoV-2 and maternal-fetal transmission is unknown. This study uses molecular pathology techniques to evaluate the placenta from a neonate infected with SARS-CoV-2 via the transplacental route to determine whether Hofbauer cells have evidence of infection. We found that the placenta had chronic histiocytic intervillositis and syncytiotrophoblast necrosis, with the syncytiotrophoblast demonstrating intense positive staining for SARS-CoV-2. Immunohistochemistry using the macrophage marker CD163, SARS-CoV-2 nucleocapsid protein, and double staining for SARS-CoV-2 with RNAscope and anti-CD163 antibody, revealed that no demonstrable virus could be identified within Hofbauer cells, despite these cells closely approaching the basement membrane zone of the infected trophoblast. Unlike some other viruses, there was no evidence from this transmitting placenta for infection of Hofbauer cells with SARS-CoV-2.
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http://dx.doi.org/10.3390/pathogens10040479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071113PMC
April 2021

Animal Hosts and Experimental Models of SARS-CoV-2 Infection.

Chemotherapy 2021 26;66(1-2):8-16. Epub 2021 Mar 26.

National HIV/AIDS Research Center, Istituto Superiore di Sanità, Rome, Italy.

Viruses arise through cross-species transmission and can cause potentially fatal diseases in humans. This is the case of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which recently appeared in Wuhan, China, and rapidly spread worldwide, causing the outbreak of coronavirus disease 2019 (COVID-19) and posing a global health emergency. Sequence analysis and epidemiological investigations suggest that the most likely original source of SARS-CoV-2 is a spillover from an animal reservoir, probably bats, that infected humans either directly or through intermediate animal hosts. The role of animals as reservoirs and natural hosts in SARS-CoV-2 has to be explored, and animal models for COVID-19 are needed as well to be evaluated for countermeasures against SARS-CoV-2 infection. Experimental cells, tissues, and animal models that are currently being used and developed in COVID-19 research will be presented.
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http://dx.doi.org/10.1159/000515341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8089426PMC
March 2021

TaSCA, an Agile Survey on Chemosensory Impairments for Self-Monitoring of COVID-19 Patients: A Pilot Study.

Front Neurol 2021 24;12:633574. Epub 2021 Feb 24.

Padova Neuroscience Center, Padua, Italy.

During the COVID-19 pandemic, smell and taste disorders emerged as key non-respiratory symptoms. Due to widespread presence of the disease and to difficult objective testing of positive persons, the use of short surveys became mandatory. Most of the existing resources are focused on smell, very few on taste or trigeminal chemosensation called chemesthesis. However, it is possible that the three submodalities are affected differently by COVID-19. We prepared a short survey (TaSCA) that can be administered at the telephone or through online resources to explore chemosensation. It is composed of 11 items on olfaction, taste, and chemesthesis, in order to discriminate the three modalities. We avoided abstract terms, and the use of semiquantitative scales because older patients may be less engaged. Statistical handling included descriptive statistics, Pearson's chi-squared test and cluster analysis. The survey was completed by 83 persons (60 females and 23 males), which reported diagnosis of COVID-19 by clinical ( = 7) or molecular ( = 18) means, the others being non-COVID subjects. Cluster analysis depicted the existence of two groups, one containing mostly asymptomatic and one mostly symptomatic subjects. All swab-positive persons fell within this second group. Only one item, related to trigeminal temperature perception, did not discriminate between the two groups. These preliminary results indicate that TaSCA may be used to easily track chemosensory symptoms related to COVID-19 in an agile way, giving a picture of three different chemosensory modalities.
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http://dx.doi.org/10.3389/fneur.2021.633574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943440PMC
February 2021

Why Cells and Viruses Cannot Survive without an ESCRT.

Cells 2021 Feb 24;10(3). Epub 2021 Feb 24.

Department of Molecular Medicine, University of Padua, 35121 Padua, Italy.

Intracellular organelles enwrapped in membranes along with a complex network of vesicles trafficking in, out and inside the cellular environment are one of the main features of eukaryotic cells. Given their central role in cell life, compartmentalization and mechanisms allowing their maintenance despite continuous crosstalk among different organelles have been deeply investigated over the past years. Here, we review the multiple functions exerted by the endosomal sorting complex required for transport (ESCRT) machinery in driving membrane remodeling and fission, as well as in repairing physiological and pathological membrane damages. In this way, ESCRT machinery enables different fundamental cellular processes, such as cell cytokinesis, biogenesis of organelles and vesicles, maintenance of nuclear-cytoplasmic compartmentalization, endolysosomal activity. Furthermore, we discuss some examples of how viruses, as obligate intracellular parasites, have evolved to hijack the ESCRT machinery or part of it to execute/optimize their replication cycle/infection. A special emphasis is given to the herpes simplex virus type 1 (HSV-1) interaction with the ESCRT proteins, considering the peculiarities of this interplay and the need for HSV-1 to cross both the nuclear-cytoplasmic and the cytoplasmic-extracellular environment compartmentalization to egress from infected cells.
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http://dx.doi.org/10.3390/cells10030483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995964PMC
February 2021

Molecular Epidemiology of HIV-1 in African Countries: A Comprehensive Overview.

Pathogens 2020 Dec 21;9(12). Epub 2020 Dec 21.

National HIV/AIDS Research Center, Istituto Superiore di Sanità, 00162 Rome, Italy.

The human immunodeficiency virus type 1 (HIV-1) originated in non-human primates in West-central Africa and continues to be a major global public health issue, having claimed almost 33 million lives so far. In Africa, it is estimated that more than 20 million people are living with HIV/Acquired Immunodeficiency Syndrome (AIDS) and that more than 730,000 new HIV-1 infections still occur each year, likely due to low access to testing. The high genetic variability of HIV-1, due to a fast replication cycle and high mutation rate, may cause the generation of many viral variants in a single infected patient during a single day. Therefore, the active monitoring and characterization of the HIV-1 subtypes and recombinant forms circulating through African countries poses a significant challenge to more specific diagnoses, treatments, care, and intervention strategies. In this review, a concise characterization of all the subtypes and recombinant forms circulating in Africa is presented to highlight the magnitude of the HIV-1 threat among the African countries and to understand virus genetic diversity and dispersion dynamics better.
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http://dx.doi.org/10.3390/pathogens9121072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766877PMC
December 2020

Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line.

Viruses 2020 09 18;12(9). Epub 2020 Sep 18.

Department of Molecular Medicine, University of Padua, 35121 Padua, Italy.

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 HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.
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http://dx.doi.org/10.3390/v12091044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551853PMC
September 2020

Targeting HIV-1 RNase H: -(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants.

Viruses 2020 07 6;12(7). Epub 2020 Jul 6.

Department of Life and Environmental Sciences University of Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato, Italy.

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 -(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound ), an N-acylhydrazone derivative that inhibited viral replication (EC = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 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 revealed that compound binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.
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http://dx.doi.org/10.3390/v12070729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412550PMC
July 2020

Alix-Mediated Rescue of Feline Immunodeficiency Virus Budding Differs from That Observed with Human Immunodeficiency Virus.

J Virol 2020 05 18;94(11). Epub 2020 May 18.

Department of Molecular Medicine, University of Padua, Padua, Italy

The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains-the matrix (MA), capsid (CA), and nucleocapsid (NC) domains-drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the CS double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding. FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.
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http://dx.doi.org/10.1128/JVI.02019-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269445PMC
May 2020

Coronaviruses: a paradigm of new emerging zoonotic diseases.

Pathog Dis 2019 12;77(9)

Department of Molecular Medicine, University of Padova, Via Gabelli 63, 35121, Padova, Italy.

A novel type of coronavirus (2019-nCoV) infecting humans appeared in Wuhan, China, at the end of December 2019. Since the identification of the outbreak the infection quickly spread involving in one month more than 31,000 confirmed cases with 638 death. Molecular analysis suggest that 2019-nCoV could be originated from bats after passaging in intermediate hosts, highlighting the high zoonotic potential of coronaviruses.
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http://dx.doi.org/10.1093/femspd/ftaa006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108526PMC
December 2019

Alternate NF-κB-Independent Signaling Reactivation of Latent HIV-1 Provirus.

J Virol 2019 09 28;93(18). Epub 2019 Aug 28.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy

Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-κB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-κB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4 T cells that stably expressed the NF-κB superrepressor IκB-α 2NΔ4 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-κB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer κB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-κB activator kinase IκB kinase β (IKK-β), did not significantly diminish reactivation in a primary CD4 T central memory (T) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies. The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-κB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-κB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-κB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-κB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.
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http://dx.doi.org/10.1128/JVI.00495-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714815PMC
September 2019

Antiviral treatment and virological monitoring of oseltamivir-resistant influenza virus A(H1N1)pdm09 in a patient with chronic B lymphocytic leukemia.

J Infect Chemother 2019 Jul 20;25(7):543-546. Epub 2019 Apr 20.

Department of Molecular Medicine, University of Padova, Padova, Italy; Microbiology and Virology Unit, Azienda Ospedaliera di Padova, Padova, Italy. Electronic address:

We report the virological monitoring and the antiviral therapy adopted for the treatment of a patient affected by chronic B lymphocytic leukemia, who experienced a severe pneumonia with long-term shedding of influenza virus A(H1N1)pdm09, characterized by an early development of oseltamivir resistance.
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http://dx.doi.org/10.1016/j.jiac.2018.11.008DOI Listing
July 2019

Lentiviral Vectors as Tools for the Study and Treatment of Glioblastoma.

Cancers (Basel) 2019 Mar 24;11(3). Epub 2019 Mar 24.

Department of Molecular Medicine, University of Padova, 35121 Padova, Italy.

Glioblastoma (GBM) has the worst prognosis among brain tumors, hence basic biology, preclinical, and clinical studies are necessary to design effective strategies to defeat this disease. Gene transfer vectors derived from the most-studied lentivirus-the Human Immunodeficiency Virus type 1-have wide application in dissecting GBM specific features to identify potential therapeutic targets. Last-generation lentiviruses (LV), highly improved in safety profile and gene transfer capacity, are also largely employed as delivery systems of therapeutic molecules to be employed in gene therapy (GT) approaches. LV were initially used in GT protocols aimed at the expression of suicide factors to induce GBM cell death. Subsequently, LV were adopted to either express small noncoding RNAs to affect different aspects of GBM biology or to overcome the resistance to both chemo- and radiotherapy that easily develop in this tumor after initial therapy. Newer frontiers include adoption of LV for engineering T cells to express chimeric antigen receptors recognizing specific GBM antigens, or for transducing specific cell types that, due to their biological properties, can function as carriers of therapeutic molecules to the cancer mass. Finally, LV allow the setting up of improved animal models crucial for the validation of GBM specific therapies.
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http://dx.doi.org/10.3390/cancers11030417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468594PMC
March 2019

Ebola Virus Entry: From Molecular Characterization to Drug Discovery.

Viruses 2019 03 19;11(3). Epub 2019 Mar 19.

Department of Molecular Medicine, University of Padova, IT-35121 Padova, Italy.

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the family. The recent large outbreak of EVD in Western Africa (2013⁻2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle , allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.
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http://dx.doi.org/10.3390/v11030274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466262PMC
March 2019

Perspectives on immunotherapy via oncolytic viruses.

Infect Agent Cancer 2019 11;14. Epub 2019 Feb 11.

Department of Molecular Medicine, University of Padua, Via A. Gabelli, 63, 35121 Padua, Italy.

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.
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http://dx.doi.org/10.1186/s13027-018-0218-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371415PMC
February 2019

Meeting report: Fourth Summer School on Innovative Approaches for Identification of Antiviral Agents (IAAASS).

Antiviral Res 2019 02 23;162:110-117. Epub 2018 Dec 23.

Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria SS544, 090542 Monserrato, Italy.

The 4th Summer School on Innovative Approaches for the Identification of Antiviral Agents (IAAASS) was held at the Sardegna Ricerche Research Park in Santa Margherita di Pula, Sardinia, Italy from September 24-28, 2018. The Summer School assembled 21 internationally recognized experts and 46 graduate and postgraduate students, with the goal of discussing advances in antiviral drug discovery from the perspective of high-throughput screening, medicinal chemistry, computational chemistry, virology, molecular and structural biology. The meeting format involved three components: (a) morning sessions of plenary talks/overviews from invited speakers, (b) afternoon sessions of posters and short presentations from student participants, and (c) informal small-group discussions between students and participating faculty. Plenary talks also featured a roundtable discussion of the pros and cons of moving into an academic career versus employment in the pharmaceutical industry, featuring individuals with experience in one or both arenas. The success of the IAAASS has come from placing emphasis on informal interactions, through which speakers made themselves available to students throughout the Summer School. This report provides a summary of scientific contributions presented by the lecturers at the 4th IAAASS, with enclosed a supplementary file containing the abstracts of selected oral presentations and poster presentations.
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http://dx.doi.org/10.1016/j.antiviral.2018.12.013DOI Listing
February 2019

Amiodarone affects Ebola virus binding and entry into target cells.

New Microbiol 2018 Apr 2;41(2):162-164. Epub 2018 Mar 2.

Ebola Virus Disease is one of the most lethal transmissible infections characterized by a high fatality rate. Several research studies have aimed to identify effective antiviral agents. Amiodarone, a drug used for the treatment of arrhythmias, has been shown to inhibit filovirus infection in vitro by acting at the early step of the viral replication cycle. Here we demonstrate that amiodarone reduces virus binding to target cells and slows down the progression of the viral particles along the endocytic pathway. Overall our data support the notion that amiodarone interferes with Ebola virus infection by affecting cellular pathways/ targets involved in the viral entry process.
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April 2018

Multi-target activity of Hemidesmus indicus decoction against innovative HIV-1 drug targets and characterization of Lupeol mode of action.

Pathog Dis 2017 08;75(6)

Department of Life and Environmental Sciences, University of Cagliari, 09042 Monserrato, Italy.

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 multitarget active drug supplement, we also investigated the anti-HIV-1 activity of Hemidesmus indicus, an Ayurveda medicinal plant containing Lupeol. Results supported the potential of this plant as a valuable multitarget active drug source. In fact, by virtue of its numerous active metabolites, H. indicus was able to inhibit not only the RT-associated RNase H function, but also the HIV-1 RT-associated RNA-dependent DNA polymerase activity and the cellular α-glucosidase.
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http://dx.doi.org/10.1093/femspd/ftx065DOI Listing
August 2017

Antiviral activity of cationic amphiphilic drugs.

Expert Rev Anti Infect Ther 2017 05 20;15(5):483-492. Epub 2017 Mar 20.

a Department of Molecular Medicine , University of Padova , Padova , Italy.

Introduction: Emerging and reemerging viral infections represent a major concern for human and veterinary public health and there is an urgent need for the development of broad-spectrum antivirals. Areas covered: A recent strategy in antiviral research is based on the identification of molecules targeting host functions required for infection of multiple viruses. A number of FDA-approved drugs used to treat several human diseases are cationic amphiphilic drugs (CADs) that have the ability to accumulate inside cells affecting several structures/functions hijacked by viruses during infection. In this review we summarized the CADs' chemical properties and effects on the cells and reported the main FDA-approved CADs that have been identified so far as potential antivirals in drug repurposing studies. Expert commentary: Although there have been concerns regarding the efficacy and the possible side effects of the off-label use of CADs as antivirals, they seem to represent a promising starting point for the development of broad-spectrum antiviral strategies. Further knowledge about their mechanism of action is required to improve their antiviral activity and to reduce the risk of side effects.
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http://dx.doi.org/10.1080/14787210.2017.1305888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103695PMC
May 2017

Identification of novel X-linked gain-of-function RPGR-ORF15 mutation in Italian family with retinitis pigmentosa and pathologic myopia.

Sci Rep 2016 12 20;6:39179. Epub 2016 Dec 20.

Department of Molecular Medicine, University of Padova, Padova, 35121, Italy.

The aim of this study was to describe a new pathogenic variant in the mutational hot spot exon ORF15 of retinitis pigmentosa GTPase regulator (RPGR) gene within an Italian family with X-linked retinitis pigmentosa (RP), detailing its distinctive genotype-phenotype correlation with pathologic myopia (PM). All members of this RP-PM family underwent a complete ophthalmic examination. The entire open reading frames of RPGR and retinitis pigmentosa 2 genes were analyzed by Sanger sequencing. A novel frame-shift mutation in exon ORF15 of RPGR gene (c.2091_2092insA; p.A697fs) was identified as hemizygous variant in the male proband with RP, and as heterozygous variant in the females of this pedigree who invariably exhibited symmetrical PM in both eyes. The c.2091_2092insA mutation coherently co-segregated with the observed phenotypes. These findings expand the spectrum of X-linked RP variants. Interestingly, focusing on Caucasian ethnicity, just three RPGR mutations are hitherto reported in RP-PM families: one of these is located in exon ORF15, but none appears to be characterized by a high penetrance of PM trait as observed in the present, relatively small, pedigree. The geno-phenotypic attributes of this heterozygosity suggest that gain-of-function mechanism could give rise to PM via a degenerative cell-cell remodeling of the retinal structures.
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http://dx.doi.org/10.1038/srep39179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5171904PMC
December 2016

Meeting report: Third Summer School on Innovative Approaches for Identification of Antiviral Agents (IAAASS).

Antiviral Res 2017 03 8;139:13-17. Epub 2016 Dec 8.

Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria SS554, 09042 Monserrato, Italy.

The third Summer School on Innovative Approaches for Identification of Antiviral Agents (IAAASS) was held from September 28th to October 2nd, 2016 at the Sardegna Ricerche Research Park in Santa Margherita di Pula, Sardinia, Italy. The school brought together graduate students and postdoctoral fellows early in their careers with a faculty of internationally recognized experts, to encourage the sharing of knowledge and experience in virology research and drug development in an informal and interactive environment. The first IAAASS was held in Sardinia in 2012 and the second in 2014. The meetings provide a unique combination of plenary lectures on topics in virology, biochemistry, molecular modeling, crystallography and medicinal chemistry with small group sessions, in which students have the opportunity to ask questions and put forward their own ideas, and senior researchers offer advice, based on their own experience. This report summarizes presentations and presentations at the 3rd IAAASS.
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http://dx.doi.org/10.1016/j.antiviral.2016.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345510PMC
March 2017

Novel variants of RPGR in X-linked retinitis pigmentosa families and genotype-phenotype correlation.

Eur J Ophthalmol 2017 Mar 21;27(2):240-248. Epub 2016 Oct 21.

 Department of Molecular Medicine, University of Padua, Padua - Italy.

Purpose: To identify novel mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene and retinitis pigmentosa 2 (RP2) gene underlying X-linked retinitis pigmentosa (XLRP) and assess genotype-phenotype correlations.

Methods: The patient cohort, consisting of 13 individuals from 3 unrelated XLRP families, underwent comprehensive ophthalmologic examination. The open reading frames of RPGR and RP2 were analyzed with Sanger sequencing in each patient. The identified genetic variants were defined as mutations or polymorphisms on the basis of their pathological effect.

Results: We found 3 genetic variants: a novel mutation c.1591G>T in exon 14 and a novel polymorphism c.1105C>T in exon 10, resulting in p.Glu531* and p.Arg369Cys of RPGR gene, respectively, and one already known mutation c.413A>G in exon 2, resulting in a p.Glu138Gly of RP2 gene. Considering our XLRP probands, RPGR-related phenotypic damages were similar and less severe than those of the patient with the RP2 mutation. On the other hand, the female carriers of XLRP variants showed different RPGR-related consequences, ranging from rods hypofunctionality in c.1591G>T nonsense heterozygosity to no retinal changes in c.1105C>T polymorphic heterozygosity.

Conclusions: These findings broaden the spectrum of RPGR mutations and phenotypic variability of the disease, which will be useful for genetic consultation and diagnosis in the future.
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http://dx.doi.org/10.5301/ejo.5000879DOI Listing
March 2017

Sennoside A, derived from the traditional chinese medicine plant Rheum L., is a new dual HIV-1 inhibitor effective on HIV-1 replication.

Phytomedicine 2016 Nov 10;23(12):1383-1391. Epub 2016 Aug 10.

Department of Life and Environmental Sciences, University of Cagliari, Cittadella di Monserrato SS554, 09042, Monserrato, Cagliari, Italy; Genetics and Biomedical Research institute, National Research Council (CNR), Cittadella di Monserrato SS554, 09042, Monserrato, Cagliari, Italy. Electronic address:

Background: 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.

Methods: 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.

Results: 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.

Conclusion: Sennoside A is a new scaffold for the development of HIV-1 dual RT inhibitors.
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http://dx.doi.org/10.1016/j.phymed.2016.08.001DOI Listing
November 2016

Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

Stem Cells Transl Med 2016 Aug 5;5(8):1098-105. Epub 2016 May 5.

Fondazione Banca degli Occhi del Veneto, Venice, Italy Department of Molecular Medicine, University of Padua, Padua, Italy

Unlabelled: : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches.

Significance: This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome.
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http://dx.doi.org/10.5966/sctm.2015-0358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4954457PMC
August 2016

Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

Mol Ther Nucleic Acids 2016 Apr 19;5:e312. Epub 2016 Apr 19.

Department of Molecular Medicine, University of Padova, Padova, Italy.

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 combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.
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http://dx.doi.org/10.1038/mtna.2016.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014525PMC
April 2016

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

Stem Cells 2016 06 16;34(6):1588-600. Epub 2016 Mar 16.

Research Centre, Fondazione Banca degli Occhi del Veneto, 30174, Venice, Italy.

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 lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600.
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http://dx.doi.org/10.1002/stem.2343DOI Listing
June 2016

Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates.

Bioconjug Chem 2016 Jan 24;27(1):247-56. Epub 2015 Dec 24.

Department of Chemistry, State University of New York , Albany, New York 12222, United States.

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA.
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http://dx.doi.org/10.1021/acs.bioconjchem.5b00627DOI Listing
January 2016

Small RNAs targeting the 5' end of the viral polymerase gene segments specifically interfere with influenza type A virus replication.

J Biotechnol 2015 Sep 16;210:85-90. Epub 2015 Jun 16.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. Electronic address:

Human and avian influenza A viruses, associated with seasonal epidemics and occasionally with pandemics, have a high impact on public health. The development of new antivirals to counteract the emergence of drug resistant influenza virus variants is a main concern. The aim of this study was to develop systems for the efficient and stable expression of small therapeutic RNAs into influenza virus infected cells in order to get further insights on the efficacy of nucleic acid-based antiviral strategies. To this end, lentiviral vectors expressing either microRNAs or antisense-RNAs targeting the 5' end of the PA, PB1 and PB2 influenza virus genomic sequences were generated. Derivative recombinant lentiviral particles were employed to transduce the influenza virus highly susceptible human alveolar basal epithelial A549 cells. The expression of both RNA molecules led to a reduction up to 3 logs of the viral titer when transduced A549 cells were challenged with different human and avian subtypes of influenza type A virus. Importantly, no inhibition of influenza type B virus was observed. Overall our data support the development of nucleic acid-based antiviral strategies to control human and avian influenza A virus infection.
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http://dx.doi.org/10.1016/j.jbiotec.2015.06.391DOI Listing
September 2015

Amiodarone and metabolite MDEA inhibit Ebola virus infection by interfering with the viral entry process.

Pathog Dis 2015 Jul 30;73(5). Epub 2015 Apr 30.

Department of Microbiology, The Public Health Agency of Sweden, Solna 171 82, Sweden Department for Laboratory Medicine, Karolinska Institute, Huddinge/Stockholm 141 83, Sweden National Veterinary Institute, Uppsala 751 89, Sweden.

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, 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 that the antiviral activity is correlated with drug ability to accumulate into and interfere with the endocytic pathway. Considering that it is well tolerated, especially in the acute setting, amiodarone appears to deserve consideration for clinical use in EVD.
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http://dx.doi.org/10.1093/femspd/ftv032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108539PMC
July 2015

From the traditional Chinese medicine plant Schisandra chinensis new scaffolds effective on HIV-1 reverse transcriptase resistant to non-nucleoside inhibitors.

J Microbiol 2015 Apr 4;53(4):288-93. Epub 2015 Mar 4.

Institute of Medicinal Plant Development (IMPLAD), 151 Malianwa North Road Haidian District, 100193, Beijing, P. R. China.

HIV-1 reverse transcriptase (RT) is still an extremely attractive pharmaceutical target for the identification of new inhibitors possibly active on drug resistant strains. Medicinal plants are a rich source of chemical diversity and can be used to identify novel scaffolds to be further developed by chemical modifications. We investigated the ability of the main lignans from Schisandra chinensis (Turcz.) Baill. fruits, commonly used in Traditional Chinese Medicine, to affect HIV-1 RT functions. We purified 6 lignans from Schisandra chinensis fruits and assayed their effects on HIV-1 RT and viral replication. Among the S. chinensis fruit lignans, Schisandrin B and Deoxyschizandrin selectively inhibited the HIV-1 RT-associated DNA polymerase activity. Structure activity relationship revealed the importance of cyclooctadiene ring substituents for efficacy. In addition, Schisandrin B was also able to impair HIV-1 RT drug resistant mutants and the early phases of viral replication. We identified Schisandrin B and Deoxyschizandrin as new scaffold for the further development of novel HIV-1 RT inhibitors.
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http://dx.doi.org/10.1007/s12275-015-4652-0DOI Listing
April 2015