Persistent hepatitis C virus infection in vitro: coevolution of virus and host.

The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host

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

Identification of Niemann-Pick C1 protein as a potential novel SARS-CoV-2 intracellular target

Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol traffic. This protein has been shown to play an important role for several viruses. It has been reported that SARS-CoV-2 enters the cell through plasma membrane fusion and/or endosomal entry upon availability of proteases. However, the whole process is not fully understood yet and additional viral/host factors might be required for viral fusion and subsequent viral replication. Here, we report a novel interaction between the SARS-CoV-2 nucleoprotein (N) and the cholesterol transporter NPC1. Furthermore, we have found that some compounds reported to interact with NPC1, carbazole SC816 and sulfides SC198 and SC073, were able to reduce SARS-CoV-2 viral infection with a good selectivity index in human cell infection models. These findings suggest the importance of NPC1 for SARS-CoV-2 viral infection and a new possible potential therapeutic target to fight against COVID-19

Nanobodies Protecting From Lethal SARS-CoV-2 Infection Target Receptor Binding Epitopes Preserved in Virus Variants Other Than Omicron

The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.This work was partially funded by Ministerio de Ciencia e Innovación (MICIN; https://www.ciencia.gob.es/) and the Spanish Research Council (CSIC; https://www.csic.es/) under grants PIE-RD-COVID 19 (No 202020E079) and PTI+ Salud Global REC_EU (No SGL 2103051, NextGenerationEU) to LF, JMC, PG, and UG, and (No SGL 2103053, NextGenerationEU) to MM-A. This study was partially conducted within the CSIC Antiviral Screening Network, an infrastructure supported by NextGeneration EU funds (https://ec.europa.eu/info/strategy/recovery-plan-europe_es) from the European Union and the European Virus Archive Global (EVag) of the European Union’s Horizon 2020 (https://ec.europa.eu/programmes/horizon2020/en/home) research and innovation programme (No 871029) to PG and UG. EM facilities of CNB-CSIC were supported by Ministerio de Ciencia e Innovación (MICIN; https://www.ciencia.gob.es/), EU-FEDER (https://ec.europa.eu/regional_policy/es/funding/erdf/) CRIOMECORR project (ESFRI-2019-01-CSIC-16). JMC access to the European Synchrotron Radiation Facility (ESRF) CM01 line through the Iberian-BAG, and to the Instruct Image Processing Center (I2PC, http://i2pc.es/) by projects PID16168 and PID14989. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

The Bacterial Mucosal Immunotherapy MV130 Protects Against SARS-CoV-2 Infection and Improves COVID-19 Vaccines Immunogenicity

COVID-19-specific vaccines are efficient prophylactic weapons against SARS-CoV-2 virus. However, boosting innate responses may represent an innovative way to immediately fight future emerging viral infections or boost vaccines. MV130 is a mucosal immunotherapy, based on a mixture of whole heat-inactivated bacteria, that has shown clinical efficacy against recurrent viral respiratory infections. Herein, we show that the prophylactic intranasal administration of this immunotherapy confers heterologous protection against SARS-CoV-2 infection in susceptible K18-hACE2 mice. Furthermore, in C57BL/6 mice, prophylactic administration of MV130 improves the immunogenicity of two different COVID-19 vaccine formulations targeting the SARS-CoV-2 spike (S) protein, inoculated either intramuscularly or intranasally. Independently of the vaccine candidate and vaccination route used, intranasal prophylaxis with MV130 boosted S-specific responses, including CD8+-T cell activation and the production of S-specific mucosal IgA antibodies. Therefore, the bacterial mucosal immunotherapy MV130 protects against SARS-CoV-2 infection and improves COVID-19 vaccines immunogenicity.CF was supported by AECC Foundation (INVES192DELF) and is currently funded by the Miguel Servet program (ID: CP20/00106) (ISCIII). IH-M receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 713673. AJ-C is a postgraduate fellow of the City Council of Madrid at the Residencia de Estudiantes (2020–2021). GD is supported by an European Molecular Biology Organization (EMBO) Long-term fellowship (ALTF 379-2019). This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. Project number 892965. OL and JA-C acknowledge Comunidad de Madrid (Tec4Bio-CM, S2018/NMT-4443, FEDER). Work in OL laboratory was funded by CNIO with the support of the projects Y2018/BIO4747 and P2018/NMT4443 from Comunidad de Madrid and co-funded by the European Social Fund and the European Regional Development Fund. The CNIO is supported by the Instituto de Salud Carlos III (ISCIII). Work at CNB and CISA is funded by the Spanish Health Ministry, Instituto de Salud Carlos III (ISCIII), Fondo COVID-19 grant COV20/00151, and Fondo Supera COVID-19 (Crue Universidades-Banco Santander) (to JG-A). Work in the DS laboratory is funded by the CNIC; by the European Research Council (ERC-2016-Consolidator Grant 725091); by Agencia Estatal de Investigación (PID2019-108157RB); by Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); by Fondo Solidario Juntos (Banco Santander); by a research agreement with Inmunotek S.L.; and by Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MICINN, and the Pro CNIC Foundation.Peer reviewe

The Cardinal Mazarine and The Dance Gittars Chacony in Dido and Aeneas by H. Purcell. The imposible connection?

Con frecuencia se estudia la Historia de la Música del periodo Barroco aislándola de la política, de los matrimonios de estado, de las guerras dinásticas, de la religión... En un mundo globalizado, como el de hoy, los devenires culturales-musicales de composición, interpretación y difusión son totalmente diferentes a la problemática de estos mismos factores en el siglo XVII. En este artículo planteamos una hipótesis que relaciona causa-efecto las decisiones del Cardenal Mazarino con una de las grandes obras inglesas, Dido y Eneas de Henry Purcell, y especialmente en un aspecto muy concreto; su Dance Gittars Chacony. A lo largo de este viaje hay varios protagonistas en mayor o menor medida además de los ya mencionados; Luis XIV, Carlos II de Inglaterra, Francisco Corbetta y Oliver Cromwell. Veremos al final si la conexión es tan imposible.The History of Music of the Baroque period is frequently studied, isolating it from politics, state marriages, dynastic wars, religión, etc. In a globalized world like today’s, the cultural-musical developments, both of composition, interpretation and dissemination, are totally different from the problem of these same factors in the seventeenth century. In this article, we propose a hypothesis that relates cause and effect to the decisions of Cardinal Mazarin with one of the great English works, Dido and Aeneas, by Henry Purcell, especially in a very specific aspect, his Dance Gittars Chacony. Throughout this trip there are several protagonists, to a greater or lesser extent, apart from those already mentioned: Louis XIV, Charles II of England, Francesco Corbetta and Oliver Cromwell. We will see at the end if the connection is, in fact, impossible