Characterization of #Usutu virus NS5 protein. #Polymerase activity, protein-protein interaction and cellular localization (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Characterization of Usutu virus NS5 protein. Polymerase activity, protein-protein interaction and cellular localization.

L. Albentosa-González, P. Clemente-Casares, R. Sabariegos, A. Mas

DOI: 10.1128/AAC.01573-19

 

ABSTRACT

Usutu virus (USUV) has become increasingly relevant in recent years with large outbreaks that sporadically have affected humans, being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive single-stranded RNA genome which is replicated by the activity of the non-structural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyl-transferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we have cloned and expressed the entire NS5 and the RdRpD in a heterologous system and have used purified preparations for protein characterizations. We have determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA, and that the RdRp activity is not inhibited by Sofosbuvir. To further examine the characteristics of USUV polymerase in a more biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed its subcellular location. NS5 is predominantly found in the cytoplasm, a significant proportion is directed to the nucleus and this translocation involves nuclear location signals (NLS) located, at least, between the MTase and RdRpD domains.

Copyright © 2019 Albentosa-González et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Keywords: Arbovirus; Flavivirus; Usutu virus; Viral pathogenesis.

—–

Characterization of #Usutu virus NS5 protein. #Polymerase activity, protein-protein interaction and cellular localization (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Characterization of Usutu virus NS5 protein. Polymerase activity, protein-protein interaction and cellular localization.

L. Albentosa-González, P. Clemente-Casares, R. Sabariegos, A. Mas

DOI: 10.1128/AAC.01573-19

 

ABSTRACT

Usutu virus (USUV) has become increasingly relevant in recent years with large outbreaks that sporadically have affected humans, being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive single-stranded RNA genome which is replicated by the activity of the non-structural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyl-transferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we have cloned and expressed the entire NS5 and the RdRpD in a heterologous system and have used purified preparations for protein characterizations. We have determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA, and that the RdRp activity is not inhibited by Sofosbuvir. To further examine the characteristics of USUV polymerase in a more biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed its subcellular location. NS5 is predominantly found in the cytoplasm, a significant proportion is directed to the nucleus and this translocation involves nuclear location signals (NLS) located, at least, between the MTase and RdRpD domains.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Flavivirus; Usutu Virus; Sofosbuvir.

——

Beyond Members of the #Flaviviridae Family, #Sofosbuvir Also Inhibits #Chikungunya Virus Replication (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Beyond Members of the Flaviviridae Family, Sofosbuvir Also Inhibits Chikungunya Virus Replication

André C. Ferreira, Patrícia A. Reis, Caroline S. de Freitas, Carolina Q. Sacramento, Lucas Villas Bôas Hoelz, Mônica M. Bastos, Mayara Mattos, Natasha Rocha,Isaclaudia Gomes de Azevedo Quintanilha, Carolina da Silva Gouveia Pedrosa, Leticia Rocha Quintino Souza, Erick Correia Loiola, Pablo Trindade, Yasmine Rangel Vieira,Giselle Barbosa-Lima, Hugo C. de Castro Faria Neto, Nubia Boechat, Stevens K. Rehen, Karin Brüning, Fernando A. Bozza, Patrícia T. Bozza, Thiago Moreno L. Souza

DOI: 10.1128/AAC.01389-18

 

ABSTRACT

Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in tropical and subtropical regions of the world, where control of the CHIKV vector, Aedes mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the Flaviviridae family. Among the Flaviviridae, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell-derived astrocytes was less susceptible to sofosbuvir than were hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection-dependent manner. Sofosbuvir also exhibited antiviral activity in vivo by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg of body weight/day and prevented mortality in a neonate mouse model at 40- and 80-mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Alphavirus; Chikungunya fever; Antivirals; Sofosbuvir.

——

#Yellowfever virus is susceptible to #sofosbuvir both in vitro and in vivo (PLoS Negl Trop Dis., abstract)

[Source: PLoS Neglected Tropical Diseases, full page: (LINK). Abstract, edited.]

OPEN ACCESS /  PEER-REVIEWED / RESEARCH ARTICLE

Yellow fever virus is susceptible to sofosbuvir both in vitroand in vivo

Caroline S. de Freitas , Luiza M. Higa , Carolina Q. Sacramento, André C. Ferreira, Patrícia A. Reis, Rodrigo Delvecchio, Fabio L. Monteiro, Giselle Barbosa-Lima, Harrison James Westgarth, Yasmine Rangel Vieira, Mayara Mattos, Natasha Rocha, Lucas Villas Bôas Hoelz,  [ … ], Thiago Moreno L. Souza

Published: January 30, 2019 / DOI: https://doi.org/10.1371/journal.pntd.0007072 / This is an uncorrected proof.

 

Abstract

Yellow fever virus (YFV) is a member of the Flaviviridae family. In Brazil, yellow fever (YF) cases have increased dramatically in sylvatic areas neighboring urban zones in the last few years. Because of the high lethality rates associated with infection and absence of any antiviral treatments, it is essential to identify therapeutic options to respond to YFV outbreaks. Repurposing of clinically approved drugs represents the fastest alternative to discover antivirals for public health emergencies. Other Flaviviruses, such as Zika (ZIKV) and dengue (DENV) viruses, are susceptible to sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Our data showed that sofosbuvir docks onto YFV RNA polymerase using conserved amino acid residues for nucleotide binding. This drug inhibited the replication of both vaccine and wild-type strains of YFV on human hepatoma cells, with EC50 values around 5 μM. Sofosbuvir protected YFV-infected neonatal Swiss mice and adult type I interferon receptor knockout mice (A129-/-) from mortality and weight loss. Because of its safety profile in humans and significant antiviral effects in vitro and in mice, Sofosbuvir may represent a novel therapeutic option for the treatment of YF.

Key-words: Yellow fever virus; Yellow fever, antiviral; sofosbuvir

 

Author summary

Yellow fever virus is transmitted by mosquitoes and its infection may be asymptomatic or lead to a wide clinical spectrum ranging from a mild febrile illness to a potentially lethal viral hemorrhagic fever characterized by liver damage. Although a yellow fever vaccine is available, low coverage allows 80,000–200,000 cases and 30,000–60,000 deaths annually worldwide. There are no specific therapy and treatment relies on supportive care, reinforcing an urgent need for antiviral repourposing. Here, we showed that sofosbuvir, clinically approved against hepatitis C, inhibits yellow fever virus replication in liver cell lines and animal models. In vitro, sofosbuvir inhibits viral RNA replication, decreases the number of infected cells and the production of infectious virus particles. These data is particularly relevante since the liver is the main target of yellow fever infection. Sofosbuvir also protected infected animals from mortality, weight loss and liver injury, especially prophylatically. Our pre-clinical results supports a second use of sofosbuvir against yellow fever.

___

Citation: de Freitas CS, Higa LM, Sacramento CQ, Ferreira AC, Reis PA, Delvecchio R, et al. (2019) Yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo. PLoS Negl Trop Dis 13(1): e0007072. https://doi.org/10.1371/journal.pntd.0007072

Editor: Samuel V. Scarpino, Northeastern University, UNITED STATES

Received: March 25, 2018; Accepted: December 12, 2018; Published: January 30, 2019

Copyright: © 2019 de Freitas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The financial support was provided by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ – http://www.faperj.br/ – Grant Number E-26/201.573/2014) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – http://cnpq.br/ – Grant Numbers 306389/2014-2 and 425636/2016-0). TMLS received the funds. This work has received financial support from the National Institute of Science and Technology in Dengue (INCT dengue), a scheme funded by the Brazilian National Science Council (CNPq, Brazil) and Minas Gerais Foundation for Science (FAPEMIG, Brazil). Funding was also provided by National Council for Scientific and Technological Development (CNPq), Ministry of Science, Technology, Information and Communications (no. 465313/2014-0); Ministry of Education/CAPES (no. 465313/2014-0); Research Foundation of the State of Rio de Janeiro/FAPERJ (no. 465313/2014-0) and Oswaldo Cruz Foundation/FIOCRUZ to National Institute for Science and Technology on Innovation on Diseases of Neglected Populations (INCT/IDPN), Center for Technological Development in Health (CDTS), Fiocruz, Rio de Janeiro, RJ, Brazil. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Keywords: Yellow Fever; Flavivirus; Antivirals; Sofosbuvir; Animal models.

——

Beyond members of the #Flaviviridae family, #sofosbuvir also inhibits #chikungunya virus #replication (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Beyond members of the Flaviviridae family, sofosbuvir also inhibits chikungunya virus replication

André C. Ferreira, Patrícia A. Reis, Caroline S. de Freitas, Carolina Q. Sacramento, Lucas Villas Bôas Hoelz, Mônica M. Bastos, Mayara Mattos, Natasha Rocha,Isaclaudia Gomes de Azevedo Quintanilha, Carolina da Silva Gouveia Pedrosa, Leticia Rocha Quintino Souza, Erick Correia Loiola, Pablo Trindade, Yasmine Rangel Vieira,Giselle Barbosa-Lima, Hugo C. de Castro Faria Neto, Nubia Boechat, Stevens K. Rehen, Karin Brüning, Fernando A. Bozza, Patrícia T. Bozza, Thiago Moreno L. Souza

DOI: 10.1128/AAC.01389-18

 

ABSTRACT

Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in tropical and subtropical regions of the world, where control of the CHIKV vector, Aedes mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the Flaviviridae family. Among the Flaviviridae, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell–derived astrocytes was less susceptible to sofosbuvir compared to the hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection–dependent manner. Sofosbuvir also exhibited antiviral activity in vivo by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg/day, and prevented mortality in a neonate mouse model at 40 and 80 mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.

Copyright © 2018 American Society for Microbiology. All Rights Reserved.

Keywords: Arbovirus; Alphavirus; Flavivirus; Antivirals; Sofosbuvir.

—–

Blocking #Zika virus #vertical #transmission (Sci Rep., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Sci Rep. 2018 Jan 19;8(1):1218. doi: 10.1038/s41598-018-19526-4.

Blocking Zika virus vertical transmission.

Mesci P1, Macia A1, Moore SM1, Shiryaev SA2, Pinto A2, Huang CT2, Tejwani L1, Fernandes IR1, Suarez NA1, Kolar MJ3, Montefusco S4, Rosenberg SC5,6, Herai RH7, Cugola FR8,9,10, Russo FB8,9,10, Sheets N11, Saghatelian A3, Shresta S11, Momper JD12, Siqueira-Neto JL4, Corbett KD5, Beltrão-Braga PCB13,14,15, Terskikh AV16, Muotri AR17.

Author information: 1 University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children’s Hospital San Diego, Department of Cellular & Molecular Medicine, Stem Cell Program, La Jolla, CA, 92037-0695, USA. 2 Sanford Burnham Prebys Medical Discovery Institute, 10901N. Torrey Pines Rd., La Jolla, CA, 92037, USA. 3 Salk Institute for Biological Studies, Clayton Foundation Laboratories for Peptide Biology, Helmsley Center for Genomic Medicine, La Jolla, California, USA. 4 University of California San Diego, Skaggs School of Pharmacy and Pharmaceutical Sciences, Center for Discovery and Innovation in Parasitic Diseases, 9500 Gilman Dr., La Jolla, CA, 92093, MC 0755, USA. 5 Ludwig Institute for Cancer Research, San Diego Branch, 9500 Gilman Dr., La Jolla, CA, 92093, MC 2385, USA. 6 University of California San Diego, Department of Cellular and Molecular Medicine, 9500 Gilman Dr., La Jolla, CA, 92093, MC 2385, USA. 7 Graduate Program in Health Sciences, School of Medicine, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba, Paraná, Brazil. 8 University of São Paulo, Institute of Biomedical Science, Department of Microbiology, Laboratory of Stem Cell and Disease Modeling, São Paulo, SP, 05508-000, Brazil. 9 University of São Paulo, School of Arts Sciences and Humanities, Department of Obstetrics, São Paulo, SP, 03828-000, Brazil. 10 University of São Paulo, School of Medicine, Center for Cellular and Molecular Therapy (NETCEM), São Paulo, SP, 01246-903, Brazil. 11 Division of Inflammation Biology, La Jolla Institute for Allergy & Immunology, La Jolla, CA, 92037, USA. 12 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, 92093, USA. 13 University of São Paulo, Institute of Biomedical Science, Department of Microbiology, Laboratory of Stem Cell and Disease Modeling, São Paulo, SP, 05508-000, Brazil. patriciacbbbraga@usp.br. 14 University of São Paulo, School of Arts Sciences and Humanities, Department of Obstetrics, São Paulo, SP, 03828-000, Brazil. patriciacbbbraga@usp.br. 15 University of São Paulo, School of Medicine, Center for Cellular and Molecular Therapy (NETCEM), São Paulo, SP, 01246-903, Brazil. patriciacbbbraga@usp.br. 16 Sanford Burnham Prebys Medical Discovery Institute, 10901N. Torrey Pines Rd., La Jolla, CA, 92037, USA. terskikh@sbpdiscovery.org. 17 University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children’s Hospital San Diego, Department of Cellular & Molecular Medicine, Stem Cell Program, La Jolla, CA, 92037-0695, USA. muotri@ucsd.edu.

 

Abstract

The outbreak of the Zika virus (ZIKV) has been associated with increased incidence of congenital malformations. Although recent efforts have focused on vaccine development, treatments for infected individuals are needed urgently. Sofosbuvir (SOF), an FDA-approved nucleotide analog inhibitor of the Hepatitis C (HCV) RNA-dependent RNA polymerase (RdRp) was recently shown to be protective against ZIKV both in vitro and in vivo. Here, we show that SOF protected human neural progenitor cells (NPC) and 3D neurospheres from ZIKV infection-mediated cell death and importantly restored the antiviral immune response in NPCs. In vivo, SOF treatment post-infection (p.i.) decreased viral burden in an immunodeficient mouse model. Finally, we show for the first time that acute SOF treatment of pregnant dams p.i. was well-tolerated and prevented vertical transmission of the virus to the fetus. Taken together, our data confirmed SOF-mediated sparing of human neural cell types from ZIKV-mediated cell death in vitro and reduced viral burden in vivo in animal models of chronic infection and vertical transmission, strengthening the growing body of evidence for SOF anti-ZIKV activity.

PMID: 29352135 DOI: 10.1038/s41598-018-19526-4

Keywords: Zika Virus; Antivirals; Sofosbuvir; Zika Congenital Infection.

——-

The #Italian Compassionate use of #Sofosbuvir in #HCV #patients waitlisted for #liver #transplantation: a national real-life experience (Liver Int., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Liver Int. 2017 Sep 16. doi: 10.1111/liv.13588. [Epub ahead of print]

The Italian Compassionate use of Sofosbuvir in HCV patients waitlisted for liver transplantation: a national real-life experience.

Martini S1, Donato MF2, Mazzarelli C3, Rendina M4, Visco-Comandini U5, Filì D6, Gianstefani A7, Fagiuoli S8, Melazzini M9, Montilla S9, Pani L9, Petraglia S9, Russo P9, Trotta MP9, Carrai P10, Caraceni P7; ITACOPS study group.

Author information: 1 Gastrohepatology Unit, AOU Città della Salute e della Scienza di Torino, Italy. 2 First Division of Gastroenterology, IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy. 3 Hepatology and Gastroenterology Unit, Niguarda Ca’ Granda Hospital, Milan, Italy. 4 Gastroenterology and Digestive Endoscopy, University Hospital, Bari, Italy. 5 Infectious Diseases – Hepatology Division, National Institute for Infectious Diseases Spallanzani IRCSS, Rome, Italy. 6 Hepatology Unit, Department for the Treatment and Study of Abdominal Diseases and Abdominal Transplantation, IRCCS – ISMETT (Mediterranean Institute for Transplantation and Advanced Specialized Therapies), Palermo, Italy. 7 Department of Surgical and Medical Sciences, University of Bologna, Italy. 8 Gastroenterology and Transplant Hepatology, Ospedale Papa Giovanni XXIII di Bergamo, Italy. 9 Italian Medicines Agency (AIFA), Rome, Italy. 10 Hepatobiliary Surgery and Liver Transplantation, University of Pisa, Italy.

 

Abstract

BACKGROUND & AIMS:

This study aimed to assess the real-life clinical and virological outcomes of HCV waitlisted patients for liver transplantation (LT) who received sofosbuvir/ribavirin (SOF/R) within the Italian compassionate use program.

METHODS:

Clinical and virological data were collected in 224 patients with decompensated cirrhosis and/or hepatocellular carcinoma (HCC) receiving daily SOF/R until LT or up a maximum of 48 weeks.

RESULTS:

Of 100 transplanted patients, 51 were HCV-RNA negative for >4 weeks before LT (SVR12: 88%) and 49 negative for <4 weeks or still viremic at transplant: 34 patients continued treatment after LT (bridging therapy) (SVR12: 88%), while 15 stopped treatment (SVR12: 53%). 98 patients completed SOF/R without LT (SVR12: 73%). In patients with advanced decompensated cirrhosis (basal MELD ≥15 and/or C-P ≥B8), a marked improvement of the scores occurred in about 50% of cases and almost 20% of decompensated patients without HCC reached a condition suitable for inactivation and delisting.

CONCLUSIONS:

These real-life data indicate that in waitlisted patients: 1. bridging antiviral therapy can be an option for patients still viremic or negative <4 weeks at LT; and 2. clinical improvement to a condition suitable for delisting can occur even in patients with advanced decompensated cirrhosis.

This article is protected by copyright. All rights reserved.

KEYWORDS: Direct-acting antivirals; bridging therapy; decompensated cirrhosis; delisting; hepatitis C; liver transplantation

PMID: 28921807 DOI: 10.1111/liv.13588

Keywords: Antivirals; Hepatitis C; Sofosbuvir.

——-