Mutation analysis of a recombinant NS replicon shows that influenza virus NS1 protein blocks the splicing and nucleo-cytoplasmic transport of its own viral mRNA

The genome of influenza A virus consists of eight single-stranded RNA molecules of negative polarity. Their replication and transcription take place in the nucleus of infected cells using ribonucleoprotein complexes (RNPs) as templates. Two of the viral transcripts, those generated by RNPs 7 and 8, can be spliced and lead to two alternative protein products (M1 and M2, NS1 and NEP/NS2, respectively). Previous studies have shown that when expressed from cDNA, NS1 protein alters the splicing and transport of RNA polymerase II-driven transcripts. Here we used a transient replication/transcription system, in which RNP 8 is replicated and transcribed by recombinant RNA and proteins, to study the splicing and nucleo-cytoplasmic transport of true viral transcripts. Our results show that the encoded NS1 protein inhibits the splicing of the collinear transcript. This regulation is mediated by the N-terminal region of the protein but does not involve its RNA-binding activity. We also show that NS1 protein preferentially blocks the nucleo-cytoplasmic transport of the collinear RNP 8 transcript in an RNA-binding dependent manner. These results rule out previous models to explain the regulation of mRNA processing and transport by NS1 and underlines the relevance of NS1 protein in the control of virus gene expression

Hepatitis C Virus Blocks Interferon Effector Function by Inducing Protein Kinase R Phosphorylation

SummaryHepatitis C virus (HCV) is a single-stranded RNA virus encoding a single polyprotein whose translation is driven by an internal ribosome entry site (IRES). HCV infection strongly induces antiviral interferon-stimulated gene (ISG) expression in the liver, yet it persists, suggesting that HCV can block ISG effector function. We now show that HCV infection triggers phosphorylation and activation of the RNA-dependent protein kinase PKR, which inhibits eukaryotic translation initiation factor eIF2α and attenuates ISG protein expression despite normal ISG mRNA induction. ISG protein induction is restored and the antiviral effects of interferon are enhanced when PKR expression is suppressed in interferon-treated infected cells. Whereas host protein translation, including antiviral ISGs, is suppressed by activated PKR, HCV IRES-dependent translation is not. These results suggest that the ability of HCV to activate PKR may, paradoxically, be advantageous for the virus during an IFN response by preferentially suppressing the translation of ISGs

Implicación de la proteína NS1 del virus de la gripe en la regulación de la expresión génica y en la morfogénesis viral

Tesis Doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-05-200

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

Hepatitis B Virus and DNA Damage Response: Interactions and Consequences for the Infection

Hepatitis B virus (HBV) is a major etiologic agent of acute and chronic hepatitis, and end-stage liver disease. Establishment of HBV infection, progression to persistency and pathogenesis are determined by viral and cellular factors, some of which remain still undefined. Key steps of HBV life cycle e.g., transformation of genomic viral DNA into transcriptionally active episomal DNA (cccDNA) or transcription of viral mRNAs from cccDNA, take place in the nucleus of infected cells and strongly depend on enzymatic activities provided by cellular proteins. In this regard, DNA damage response (DDR) pathways and some DDR proteins are being recognized as important factors regulating the infection. On one hand, HBV highjacks specific DDR proteins to successfully complete some of the steps of its life cycle. On the other hand, HBV subverts DDR pathways to presumably create a cellular environment that favours its replication. Direct consequences of these interactions are: HBV DNA integration into host chromosomal DNA, and accumulation of mutations in host chromosomal DNA that could eventually trigger carcinogenic processes, which would explain in part the incidence of hepatocellular carcinoma in chronically infected patients. Unravelling the interactions that HBV establishes with DDR pathways might help identify new molecular targets for therapeutic intervention

Narrazioni della catastrofe dopo Fukushima

Current interferon alpha-based treatment of hepatitis C virus (HCV) infection fails to cure a sizeable fraction of patients treated. The cause of this treatment failure remains unknown. Here using mathematical modelling, we predict treatment failure to be a consequence of the emergent properties of the interferon-signalling network. HCV induces bistability in the network, creating a new steady state where it can persist. Cells that admit the new steady state alone are refractory to interferon. Using a model of viral kinetics, we show that when the fraction of cells refractory to interferon in a patient exceeds a critical value, treatment fails. Direct-acting antivirals that suppress HCV replication can eliminate the new steady state, restoring interferon sensitivity and improving treatment response. Our study thus presents a new conceptual basis of HCV persistence and treatment response, elucidates the origin of the synergy between interferon and direct-acting antivirals, and facilitates rational treatment optimization

Identification of Aquarius and Senataxin as Restriction Host Factors for Hepatitis B Virus Infection

© 2020 by the authorsHepatitis B virus (HBV) represents an important human pathogen causing acute and chronic hepatitis. Over 240 million people are chronically infected, many of whom will die due to complications such as liver cirrhosis and hepatocellular carcinoma. Currently approved therapies are very effective in suppressing virus replication and viremia, but they are not curative, because they do not completely eliminate the nuclear episomal DNA form of HBV (cccDNA) that re-establishes infection upon interruption of therapy. Despite our understanding of many aspects of the HBV lifecycle, details of the HBV cccDNA biology remain poorly understood. Our group is pursuing a loss-of-function genetic screening approach, to identify cellular factors regulating HBV infection. A lentivirus-delivered short hairpin RNA (shRNA) library, composed of 384 shRNAs, was used to interrogate the function of 80 DNA damage repair pathway proteins in the establishment of HBV infection. The primary screening identified 10 cellular factors that regulate the HBV infection both positively or negatively. Two of those proteins, aquarius (AQR) and senataxin (SETX), were subsequently validated as factors restricting the HBV infection in independent experiments. Silencing of AQR and SETX led to an increased infection efficiency that was characterized by higher intracellular levels of HBV cccDNA, HBV mRNA, and core protein, and increased HBV e antigen (HBeAg) accumulation in the supernatants of infected cells. The expression level, glycosylation pattern, and localization of the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP), in AQR- and SETX-downregulated cells was equivalent to that of the control cells. Collectively, our results are compatible with AQR and SETX restricting early steps in the HBV lifecycle and downstream HBV entry, that affect the establishment of the HBV cccDNA pool. Experiments to unravel the function of these proteins in the context of HBV infection are currently underway.This work was supported by grants SAF2016-75169-R (AEI/FEDER, UE) from the Spanish Ministry of Economy, Industry and Competitiveness, and a CTSA Pilot Award (NIH/NCATS/STSI 5UL1 TR001114) to U.G. A.G.M is supported by the Spanish Minitry of Education (FPU17/03424)