Akt phosphorylation of HCV NS5B regulates polymerase activity and HCV infection

Hepatitis C virus (HCV) is a single-stranded RNA virus of positive polarity [ssRNA(+)] that replicates its genome through the activity of one of its proteins, called NS5B. This viral protein is responsible for copying the positive-polarity RNA genome into a negative-polarity RNA strand, which will be the template for new positive-polarity RNA genomes. The NS5B protein is phosphorylated by cellular kinases, including Akt. In this work, we have identified several amino acids of NS5B that are phosphorylated by Akt, with positions S27, T53, T267, and S282 giving the most robust results. Site-directed mutagenesis of these residues to mimic (Glu mutants) or prevent (Ala mutants) their phosphorylation resulted in a reduced NS5B in vitro RNA polymerase activity, except for the T267E mutant, the only non-conserved position of all those that are phosphorylated. In addition, in vitro transcribed RNAs derived from HCV complete infectious clones carrying mutations T53E/A and S282E/A were transfected in Huh-7.5 permissive cells, and supernatant viral titers were measured at 6 and 15 days post-transfection. No virus was rescued from the mutants except for T53A at 15 days post-transfection whose viral titer was statistically lower as compared to the wild type. Therefore, phosphorylation of NS5B by cellular kinases is a mechanism of viral polymerase inactivation. Whether this inactivation is a consequence of interaction with cellular kinases or a way to generate inactive NS5B that may have other functions are questions that need further experimental workMinisterio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). CP was supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CG-C was supported by the predoctoral contract PRE2018-083422 from MCIU. CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) was funded by the Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMS

DNA polymerase λ, a novel DNA repair enzyme in human cells

DNA polymerase lambda (pol λ) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5′-deoxyribose-5-phosphate lyase activity in pol λ supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol λ to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol λ inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5′-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol λ is a novel β-like DNA polymerase. However, the high affinity of pol λ for dNTPs (37-fold over pol β) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol β and pol λ have nonredundant in vivo functions.This work was supported by Ministerio de Ciencia y Tecnologı´a Grant BMC2000-1138, Comunidad Auto´noma de Madrid Grant 08.5/0063/2000 (to L. B.) and by an institutional grant from Fundacio´n Ramo´n Areces

DNA polymerase λ, a novel DNA repair enzyme in human cells

DNA polymerase lambda (pol λ) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5′-deoxyribose-5-phosphate lyase activity in pol A supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol A to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol λ inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5′-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol A is a novel β-like DNA polymerase. However, the high affinity of pol λ for dNTPs (37-fold over pol β) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol β and pol λ have nonredundant in vivo function

Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.CP is supported by the Miguel Servet program (grants CP14/00121 and CP19/00001) of the Instituto de Salud Carlos III cofinanced by FEDER. CP has received funding from Ministerio de Ciencia, Innovacio´n y Universidades (grant BFU2017-91384-EXP), from Instituto de Salud Carlos III (grants PI18/00210 and PI21/00139), from Fundación La Marato´ (grant 525/C/2021), and from CSIC (grant CSIC-COV19-014). ED has received funding from CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III), from Ministerio de Economía y Competitividad (grants SAF2014-52400-R, SAF2017-87846-R, and PID2020-113888RB-I00), and from Comunidad de Madrid/FEDER (grants S2013/ABI-2906 PLATESA, and S2018/BAA-4370 PLATESA2). AM has received funding from Ministerio de Economía y Competitividad (grants SAF2016-80451-P, PID2019-106068GB-I00, EQC2018-004420-P, and EQC2018-004631-P), and Plan Propio of Universidad de Castilla-La Mancha. A.G-P and L.DM have received funding from Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía, cofinanced by FEDER and FSE (grants BIO-264, and P10-CVI-6561) and Plan Propio of Universidad de Málaga.Peer reviewe

Purificacion y caracterizacion del mecanismo de accion de la proteina RecN de Bacillus subtilis

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai

Purificación y Caracterización del Mecanismo de Acción de la Proteína RecN de Bacillus subtilis

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-01-199

Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization

Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/ PKB. In the presence of transiently expressed NS5B or in replicon- or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options.Fundación Ramón Areces. CIBERehd is funded by Instituto de Salud Carlos III. Celia Perales is supported by the Miguel Servet program (Instituto de Salud Carlos III).Peer Reviewe

Sequence Homology Required by Human Immunodeficiency Virus Type 1 To Escape from Short Interfering RNAs

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5′ end or the 3′ end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5′ and 3′ ends are partially tolerated

Akt Interacts with Usutu Virus Polymerase, and Its Activity Modulates Viral Replication

Usutu virus (USUV) is a flavivirus that mainly infects wild birds through the bite of Culex mosquitoes. Recent outbreaks have been associated with an increased number of cases in humans. Despite being a growing source of public health concerns, there is yet insufficient data on the virus or host cell targets for infection control. In this work we have investigated whether the cellular kinase Akt and USUV polymerase NS5 interact and co-localize in a cell. To this aim, we performed co-immunoprecipitation (Co-IP) assays, followed by confocal microscopy analyses. We further tested whether NS5 is a phosphorylation substrate of Akt in vitro. Finally, to examine its role in viral replication, we chemically silenced Akt with three inhibitors (MK-2206, honokiol and ipatasertib). We found that both proteins are localized (confocal) and pulled down (Co-IP) together when expressed in different cell lines, supporting the fact that they are interacting partners. This possibility was further sustained by data showing that NS5 is phosphorylated by Akt. Treatment of USUV-infected cells with Akt-specific inhibitors led to decreases in virus titers (>10-fold). Our results suggest an important role for Akt in virus replication and stimulate further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral target

Uso de la RNasa P como agente antiviral

Filing Date: (2009-11-11).-- Priority Data: ES P200803224 (2008-11-11).-- Titulares: Consejo Superior de Investigaciones Científicas (CSIC), Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto Nacional de Investigaciones Agrarias (INIA), Universidad de Castilla La Mancha (UCLM).[EN] Use of the RNase P of Synechocysitis sp. to inhibit replication of RNA virus and for the production of medicaments for the treatment of diseases caused by RNA virus.[ES] Uso de la RNasa P de Synechocysitis sp. para inhibir la replicación de virus de RNA, y para la elaboración de medicamentos para el tratamiento de enfermedades provocadas por virus de RNA.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas, Instituto Nacional de Investigaciones Agrarias (INIA), Universidad de Castilla la ManchaA2 Solicitud de patentes sin informe sobre el estado de la técnic