45 results on '"Barrado-Gil, Lucía"'
Search Results
2. Overview of Modern Commercial Kits for Laboratory Diagnosis of African Swine Fever and Swine Influenza A Viruses
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Consejo Superior de Investigaciones Científicas (España), European Federation of Academies of Sciences and Humanities, Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), European Commission, Muzykina, Larysa, Barrado-Gil, Lucía, González De Bulnes, Antonio, Crespo-Piazuelo, Daniel, Cerón, José Joaquín, Alonso Martí, Covadonga, Montoya, María, Consejo Superior de Investigaciones Científicas (España), European Federation of Academies of Sciences and Humanities, Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), European Commission, Muzykina, Larysa, Barrado-Gil, Lucía, González De Bulnes, Antonio, Crespo-Piazuelo, Daniel, Cerón, José Joaquín, Alonso Martí, Covadonga, and Montoya, María
- Abstract
Rapid and early detection of infectious diseases in pigs is important, especially for the implementation of control measures in suspected cases of African swine fever (ASF), as an effective and safe vaccine is not yet available in most of the affected countries. Additionally, analysis for swine influenza is of significance due to its high morbidity rate (up to 100%) despite a lower mortality rate compared to ASF. The wide distribution of swine influenza A virus (SwIAV) across various countries, the emergence of constantly new recombinant strains, and the danger of human infection underscore the need for rapid and accurate diagnosis. Several diagnostic approaches and commercial methods should be applied depending on the scenario, type of sample and the objective of the studies being implemented. At the early diagnosis of an outbreak, virus genome detection using a variety of PCR assays proves to be the most sensitive and specific technique. As the disease evolves, serology gains diagnostic value, as specific antibodies appear later in the course of the disease (after 7–10 days post-infection (DPI) for ASF and between 10–21 DPI for SwIAV). The ongoing development of commercial kits with enhanced sensitivity and specificity is evident. This review aims to analyse recent advances and current commercial kits utilised for the diagnosis of ASF and SwIAV.
- Published
- 2024
3. Insights into the function of ESCRT complex and LBPA in ASFV infection
- Author
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Barrado-Gil, Lucía, primary, García-Dorival, Isabel, additional, Galindo, Inmaculada, additional, Alonso, Covadonga, additional, and Cuesta-Geijo, Miguel Ángel, additional
- Published
- 2023
- Full Text
- View/download PDF
4. Insights into the function of ESCRT complex and LBPA in ASFV infection
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European Commission, Agencia Estatal de Investigación (España), Fundación la Caixa, Ministerio de Ciencia e Innovación (España), Barrado-Gil, Lucía [0000-0002-1053-2997], García-Dorival, Isabel [0000-0002-5654-5662], Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía, García-Dorival, Isabel, Galindo Barreales, Inmaculada, Alonso Martí, Covadonga, Cuesta-Geijo, Miguel Ángel, European Commission, Agencia Estatal de Investigación (España), Fundación la Caixa, Ministerio de Ciencia e Innovación (España), Barrado-Gil, Lucía [0000-0002-1053-2997], García-Dorival, Isabel [0000-0002-5654-5662], Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía, García-Dorival, Isabel, Galindo Barreales, Inmaculada, Alonso Martí, Covadonga, and Cuesta-Geijo, Miguel Ángel
- Abstract
The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by the endosomal sorting complexes required for transport (ESCRT). The ESCRT machinery is mainly involved in the coordination of membrane dynamics; hence, several viruses exploit this complex and its accessory proteins VPS4 and ALIX for their own benefit. In this work, we found that shRNA-mediated knockdown of VPS4A decreased ASFV replication and viral titers, and this silencing resulted in an enhanced expression of ESCRT-0 component HRS. ASFV infection slightly increased HRS expression but not under VPS4A depletion conditions. Interestingly, VPS4A silencing did not have an impact on ALIX expression, which was significantly overexpressed upon ASFV infection. Further analysis revealed that ALIX silencing impaired ASFV infection at late stages of the viral cycle, including replication and viral production. In addition to ESCRT, the accessory protein ALIX is involved in endosomal membrane dynamics in a lysobisphosphatydic acid (LBPA) and Ca2+-dependent manner, which is relevant for intraluminal vesicle (ILV) biogenesis and endosomal homeostasis. Moreover, LBPA interacts with NPC2 and/or ALIX to regulate cellular cholesterol traffic, and would affect ASFV infection. Thus, we show that LBPA blocking impacted ASFV infection at both early and late infection, suggesting a function for this unconventional phospholipid in the ASFV viral cycle. Here, we found for the first time that silencing of VPS4A and ALIX affects the infection later on, and blocking LBPA function reduces ASFV infectivity at early and later stages of the viral cycle, while ALIX was overexpressed upon infection. These data suggested the relevance of ESCRT-related proteins in ASFV infection.
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- 2023
5. Pharmacological modulation of the interaction between tubulin and a SARS-CoV-2 protein
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Ministerio de Ciencia e Innovación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fuertes-Monge, Laura [0000-0002-9764-3111], Schaerlaekens, Sofie [0000-0002-4410-2311], Lucena-Agell, Daniel [0000-0001-7314-8696], García-Dorival, Isabel [0000-0002-5654-5662], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Alonso, Covandoga [0000-0002-0862-6177], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Fuertes-Monge, Laura, Schaerlaekens, Sofie, Lucena-Agell, Daniel, García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Inmaculada, Alonso, Covadonga, Díaz, José Fernando, Oliva, María A., Ministerio de Ciencia e Innovación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fuertes-Monge, Laura [0000-0002-9764-3111], Schaerlaekens, Sofie [0000-0002-4410-2311], Lucena-Agell, Daniel [0000-0001-7314-8696], García-Dorival, Isabel [0000-0002-5654-5662], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Alonso, Covandoga [0000-0002-0862-6177], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Fuertes-Monge, Laura, Schaerlaekens, Sofie, Lucena-Agell, Daniel, García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Inmaculada, Alonso, Covadonga, Díaz, José Fernando, and Oliva, María A.
- Abstract
The cytoskeleton is the main communication/transport route within cells and many viruses abuse on this cellular machine to fulfil their cycle. We initially identified the interaction of a SARS-CoV-2 protein with tubulin using a proteomic analysis. We next confirmed the interaction and identified the viral protein domain involved through in vitro co-immunoprecipitation assays and analytical ultracentrifugation experiments. Then, we focused on unveiling the molecular mechanism of the interaction to determine if the SARS-CoV-2 protein promote a stable microtubule assembly (as roads for motors) or induce microtubule dynamics (as main force generation for transport). We have combined biochemical, biophysical and structural studies to determine the ratio of protein-protein interaction and the resulting effect on tubulin assembly. We have found that this protein domain is able to promote microtubule depolymerization into rings and tubulin assembly into non-functional filaments likely because a preference for tubulin curved-conformation. Importantly, this effect is not dependent on nucleotide or nucleotide hydrolysis. Finally, tubulin is a well-known target in cancer diseases and there are four of the seven tubulin druggable sites exploited on chemotherapy. Hence, we have analyzed the ability of microtubule stabilizing (MSA) and destabilizing agents (MDA) on disrupting the interaction of SARS-CoV-2 protein with tubulin. We have found that MSAs keep microtubule structures even in the presence of the viral protein, while the effect of MDAs varies depending on their mechanism of action.
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- 2022
6. Effect of Clinically Used Microtubule Targeting Drugs on Viral Infection and Transport Function
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Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, La Caixa, Club Escola Hungaresa de Esgrima de Pontevedra, Oliva, María A. [0000-0002-2215-4639], Tosat-Bitrian, Carlota [0000-0002-0525-3512], Barrado-Gil, Lucía [0000-0002-1053-2997], Bonato, Francesca [0000-0002-0579-2874], Galindo, Inmaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], París-Ogáyar, Rebeca [0000-0002-7953-0949], Lucena-Agell, Daniel [0000-0001-7314-8696], Giménez-Abián, Juan F. [0000-0002-9220-286X], Urquiza, Jesús [0000-0002-7870-4580], Díaz, José Fernando [0000-0003-2743-3319], Palomo, Valle [0000-0002-1473-4086], Alonso, Covadonga [0000-0002-0862-6177], Oliva, María A., Tosat-Bitrian, Carlota, Barrado-Gil, Lucía, Bonato, Francesca, Galindo Barreales, Inmaculada, Garaigorta, Urtzi, Álvarez-Bernad, Beatriz, París-Ogáyar, Rebeca, Lucena-Agell, Daniel, Giménez-Abián, Juan F., García-Dorival, Isabel, Urquiza, Jesús, Gastaminza, Pablo, Díaz, José Fernando, Palomo, Valle, Alonso Martí, Covadonga, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, La Caixa, Club Escola Hungaresa de Esgrima de Pontevedra, Oliva, María A. [0000-0002-2215-4639], Tosat-Bitrian, Carlota [0000-0002-0525-3512], Barrado-Gil, Lucía [0000-0002-1053-2997], Bonato, Francesca [0000-0002-0579-2874], Galindo, Inmaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], París-Ogáyar, Rebeca [0000-0002-7953-0949], Lucena-Agell, Daniel [0000-0001-7314-8696], Giménez-Abián, Juan F. [0000-0002-9220-286X], Urquiza, Jesús [0000-0002-7870-4580], Díaz, José Fernando [0000-0003-2743-3319], Palomo, Valle [0000-0002-1473-4086], Alonso, Covadonga [0000-0002-0862-6177], Oliva, María A., Tosat-Bitrian, Carlota, Barrado-Gil, Lucía, Bonato, Francesca, Galindo Barreales, Inmaculada, Garaigorta, Urtzi, Álvarez-Bernad, Beatriz, París-Ogáyar, Rebeca, Lucena-Agell, Daniel, Giménez-Abián, Juan F., García-Dorival, Isabel, Urquiza, Jesús, Gastaminza, Pablo, Díaz, José Fernando, Palomo, Valle, and Alonso Martí, Covadonga
- Abstract
Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians.
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- 2022
7. New insights into the role of endosomal proteins for African swine fever virus infection
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La Caixa, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, European Commission, Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], García-Dorival, Isabel [0000-0002-5654-5662], Puerto, Ana del [0000-0002-5119-6395], Urquiza, Jesús [0000-0002-7870-4580], Galindo, Immaculada [0000-0001-8257-9797], Barrado-Gil, Lucía [0000-0002-1053-2997], Lasala, Fátima [0000-0001-8183-1866], Sorzano, Carlos Óscar S. [0000-0002-9473-283X], Gil, Carmen [0000-0002-3882-6081], Delgado, Rafael [0000-0002-6912-4736], Alonso, Covandoga [0000-0002-0862-6177], Cuesta-Geijo, Miguel Ángel, García-Dorival, Isabel, Puerto, Ana del, Urquiza, Jesús, Galindo Barreales, Inmaculada, Barrado-Gil, Lucía, Lasala, Fátima, Cayuela, Ana, Sorzano, Carlos Óscar S., Gil, Carmen, Delgado, Rafael, Alonso Martí, Covadonga, La Caixa, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, European Commission, Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], García-Dorival, Isabel [0000-0002-5654-5662], Puerto, Ana del [0000-0002-5119-6395], Urquiza, Jesús [0000-0002-7870-4580], Galindo, Immaculada [0000-0001-8257-9797], Barrado-Gil, Lucía [0000-0002-1053-2997], Lasala, Fátima [0000-0001-8183-1866], Sorzano, Carlos Óscar S. [0000-0002-9473-283X], Gil, Carmen [0000-0002-3882-6081], Delgado, Rafael [0000-0002-6912-4736], Alonso, Covandoga [0000-0002-0862-6177], Cuesta-Geijo, Miguel Ángel, García-Dorival, Isabel, Puerto, Ana del, Urquiza, Jesús, Galindo Barreales, Inmaculada, Barrado-Gil, Lucía, Lasala, Fátima, Cayuela, Ana, Sorzano, Carlos Óscar S., Gil, Carmen, Delgado, Rafael, and Alonso Martí, Covadonga
- Abstract
African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.
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- 2022
8. Elucidation of the Cellular Interactome of African Swine Fever Virus Fusion Proteins and Identification of Potential Therapeutic Targets
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García-Dorival, Isabel, primary, Cuesta-Geijo, Miguel Ángel, additional, Galindo, Inmaculada, additional, del Puerto, Ana, additional, Barrado-Gil, Lucía, additional, Urquiza, Jesús, additional, and Alonso, Covadonga, additional
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- 2023
- Full Text
- View/download PDF
9. Elucidation of the Cellular Interactome of African Swine Fever Virus Fusion Proteins and Identification of Potential Therapeutic Targets
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European Commission, Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Fundación la Caixa, Galindo Barreales, Inmaculada [0000-0001-8257-9797], García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Galindo Barreales, Inmaculada, Puerto, Ana del, Barrado-Gil, Lucía, Urquiza, Jesús, Alonso Martí, Covadonga, European Commission, Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Fundación la Caixa, Galindo Barreales, Inmaculada [0000-0001-8257-9797], García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Galindo Barreales, Inmaculada, Puerto, Ana del, Barrado-Gil, Lucía, Urquiza, Jesús, and Alonso Martí, Covadonga
- Abstract
African swine fever virus (ASFV) encodes more than 150 proteins, most of them of unknown function. We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins, which potentially mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for those ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Rab geranyl geranylation emerged as a significant hit, and also Rab proteins, which are crucial regulators of the endocytic pathway and interactors of both p34 and E199L. Rab proteins co-ordinate a tight regulation of the endocytic pathway that is necessary for ASFV infection. Moreover, several interactors were proteins involved in the molecular exchange at ER membrane contacts. These ASFV fusion proteins shared interacting partners, suggesting potential common functions. Membrane trafficking and lipid metabolism were important categories, as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages.
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- 2023
10. Elucidation of the cellular interactome of African Swine Fever Virus fusion proteins and identification of potential therapeutic targets
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Garcia Dorival, Isabel, Cuesta Geijo, Miguel Ángel, Galindo Barreales, Inmaculada, Del Puerto, Ana, Barrado Gil, Lucía, Urquiza López, Jesús, and Alonso Martí, Covadonga
- Subjects
Infectious Diseases ,Virology ,virus-host interaction ,interactome ,drug target ,fusion proteins ,lipid metabolism enzymes ,African swine fever virus ,ASFV ,virus–host interaction - Abstract
African swine fever virus (ASFV) encodes more than 150 proteins, most of them of unknown function. We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins located at the inner viral membrane of the virion. These proteins mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Some of these proteins shared interacting partners, suggesting potential common functions. Rab proteins geranyl geranylation emerged as a significant hit, interactor of both p34 and E199L. Our results point out that ASFV fusion proteins could act coordinately to ensure a tight regulation of the endocytic pathway, and the molecular exchange at ER membrane contacts. Membrane trafficking and lipid metabolism were important categories as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages., Supplementary figures tables Manuscript ID: viruses-2356321 Funding: PCI2021-121939/AEI/10.13039/501100011033; PID2021-122825OB; CPP2021-008618; LCF/PR/HR19/52160012
- Published
- 2023
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11. Supplementary figures
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Garcia Dorival, Isabel, Cuesta Geijo, Miguel Ángel, Galindo Barreales, Inmaculada, Del Puerto, Ana, Barrado Gil, Lucía, Urquiza López, Jesús, and Alonso Martí, Covadonga
- Subjects
virus-host interaction ,interactome ,drug target ,fusion proteins ,lipid metabolism enzymes ,African swine fever virus ,ASFV - Abstract
African swine fever virus (ASFV) encodes more than 150 proteins, most of them of unknown function. We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins located at the inner viral membrane of the virion. These proteins mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Some of these proteins shared interacting partners, suggesting potential common functions. Rab proteins geranyl geranylation emerged as a significant hit, interactor of both p34 and E199L. Our results point out that ASFV fusion proteins could act coordinately to ensure a tight regulation of the endocytic pathway, and the molecular exchange at ER membrane contacts. Membrane trafficking and lipid metabolism were important categories as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages.
- Published
- 2023
- Full Text
- View/download PDF
12. Pharmacological modulation of the interaction between tubulin and a SARS-CoV-2 protein
- Author
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Fuertes, Laura, Schaerlaekens, Sofie, Lucena-Agell, Daniel, García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Immaculada, Alonso, Covandoga, Díaz, José Fernando, Oliva, María A., Ministerio de Ciencia e Innovación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fuertes, Laura, Schaerlaekens, Sofie, Lucena-Agell, Daniel, García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Immaculada, Alonso, Covandoga, Díaz, José Fernando, and Oliva, María A.
- Abstract
1p.-6 fig., The cytoskeleton is the main communication/transport route within cells and many viruses abuse on this cellular machine to fulfil their cycle. We initially identified the interaction of a SARS-CoV-2 protein with tubulin using a proteomic analysis. We next confirmed the interaction and identified the viral protein domain involved through in vitro co-immunoprecipitation assays and analytical ultracentrifugation experiments. Then, we focused on unveiling the molecular mechanism of the interaction to determine if the SARS-CoV-2 protein promote a stable microtubule assembly (as roads for motors) or induce microtubule dynamics (as main force generation for transport). We have combined biochemical, biophysical and structural studies to determine the ratio of protein-protein interaction and the resulting effect on tubulin assembly. We have found that this protein domain is able to promote microtubule depolymerization into rings and tubulin assembly into non-functional filaments likely because a preference for tubulin curved-conformation. Importantly, this effect is not dependent on nucleotide or nucleotide hydrolysis. Finally, tubulin is a well-known target in cancer diseases and there are four of the seven tubulin druggable sites exploited on chemotherapy. Hence, we have analyzed the ability of microtubule stabilizing (MSA) and destabilizing agents (MDA) on disrupting the interaction of SARS-CoV-2 protein with tubulin. We have found that MSAs keep microtubule structures even in the presence of the viral protein, while the effect of MDAs varies depending on their mechanism of action., This research work was funded by Ministerio de Ciencia e Innovación and CSIC. It was also funded by the European Commission – NextGenerationEU (Regulation EU2020/2094), through CSIC's Global Health Platform (PTI Salud Global).
- Published
- 2022
13. COVID-19: Drug Targets and Potential Treatments
- Author
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Gil, Carmen, Ginex, Tiziana, Maestro, Inés, Nozal, Vanesa, Barrado-Gil, Lucía, Cuesta-Geijo, Miguel Ángel, Urquiza, Jesús, Ramírez, David, Alonso, Covadonga, Campillo, Nuria E., Martinez, Ana, Consejo Superior de Investigaciones Científicas (España), Fundación 'la Caixa', Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Comisión Nacional de Investigación Científica y Tecnológica (Chile), Fondo Nacional de Desarrollo Científico y Tecnológico (Chile), European Commission, Gil, Carmen [0000-0002-3882-6081], Ginex, Tiziana [0000-0002-5739-8713], Maestro, Inés [0000-0002-5026-5803], Nozal, V. [0000-0001-5260-5683], Barrado-Gil, Lucía [0000-0002-1053-2997], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Urquiza, Jesús [0000-0002-7870-4580], Ramírez, David [0000-0003-0002-1189], Alonso, Covandoga [0000-0002-0862-6177], Campillo, Nuria E. [0000-0002-9948-2665], Martínez, Ana [0000-0002-2707-8110], Gil, Carmen, Ginex, Tiziana, Maestro, Inés, Nozal, V., Barrado-Gil, Lucía, Cuesta-Geijo, Miguel Ángel, Urquiza, Jesús, Ramírez, David, Alonso, Covandoga, Campillo, Nuria E., and Martínez, Ana
- Subjects
Drug ,Drug targets ,viruses ,media_common.quotation_subject ,Druggability ,Disease ,medicine.disease_cause ,Bioinformatics ,Antiviral Agents ,01 natural sciences ,03 medical and health sciences ,Drug Discovery ,Pandemic ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Enzyme Inhibitors ,030304 developmental biology ,Coronavirus ,media_common ,0303 health sciences ,Innate immune system ,SARS-CoV-2 ,Drug discovery ,Chemistry ,Drug Repositioning ,COVID-19 ,virus diseases ,Antivirals ,Immunity, Innate ,COVID-19 Drug Treatment ,0104 chemical sciences ,3. Good health ,010404 medicinal & biomolecular chemistry ,Drug repositioning ,Perspective ,Molecular Medicine - Abstract
92 p.-22 fig.-1 tab.-1 graph. abst., Currently, we are immersed in a pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which severely threatens public health worldwide. Until now, no drug or vaccine has been approved to treat the severe disease caused by this coronavirus, COVID-19. We will focus on the main virus-based and host-based targets that can guide medicinal chemistry efforts to discover new drugs for this devastating disease. In principle, all CoVs enzymes and proteins involved in viral replication and the control of host cellular machineries are potentially druggable targets in the search for therapeutic options for SARS-CoV-2. This perspective provides an overview of the main targets from a structural point of view, together with reported therapeutic compounds with activity against SARS-CoV-2 and/or other CoVs. Also, the role of innate immune response to coronavirus infection and the related therapeutic options will be presented., Funding from CSIC (201980E024 and 202020E103) is acknowledged. This research was partially supported through "la Caixa" Banking Foundation (HR18-00469), Instituto de Salud Carlos III (ISCIII-COV20/01007), Spanish Ministry of Science and Innovation (RTI2018-097305-R-I00), CONICYT-PCI (REDES190074 to D. R. and A. M.) and FONDECYT (11180604 to D.R.). I. M. was funded by H2020-MSCA-ITN-2017 (grant no. 765912), V. N. holds a pre-doctoral FPU grant (FPU16/04466) and J. U. was financed by FPI-SGIT2018-04.
- Published
- 2020
14. Host cell targets for African swine fever virus
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Muñoz-Moreno, Raquel, Galindo, Inmaculada, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, and Alonso, Covadonga
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- 2015
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15. African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis
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Galindo, Inmaculada, Cuesta-Geijo, Miguel Angel, Hlavova, Karolina, Muñoz-Moreno, Raquel, Barrado-Gil, Lucía, Dominguez, Javier, and Alonso, Covadonga
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- 2015
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16. Identification of Niemann-Pick C1 protein as a potential novel SARS-CoV-2 intracellular target
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European Commission, La Caixa, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), García-Dorival, Isabel [0000-0002-5654-5662], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Urquiza, Jesús [0000-0002-7870-4580], Puerto, Ana del [0000-0002-5119-6395], Campillo, Nuria E. [0000-0002-9948-2665], Martínez, Ana [0000-0002-2707-8110], Gil, Carmen [0000-0002-3882-6081], Alonso, Covadonga [0000-0002-0862-6177], García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, Garaigorta, Urtzi, Urquiza, Jesús, Puerto, Ana del, Campillo, Nuria E., Martínez Gil, Ana, Gastaminza, Pablo, Gil, Carmen, Alonso Martí, Covadonga, European Commission, La Caixa, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), García-Dorival, Isabel [0000-0002-5654-5662], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Urquiza, Jesús [0000-0002-7870-4580], Puerto, Ana del [0000-0002-5119-6395], Campillo, Nuria E. [0000-0002-9948-2665], Martínez, Ana [0000-0002-2707-8110], Gil, Carmen [0000-0002-3882-6081], Alonso, Covadonga [0000-0002-0862-6177], García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, Garaigorta, Urtzi, Urquiza, Jesús, Puerto, Ana del, Campillo, Nuria E., Martínez Gil, Ana, Gastaminza, Pablo, Gil, Carmen, and Alonso Martí, Covadonga
- Abstract
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.
- Published
- 2021
17. African Swine Fever Virus Ubiquitin-Conjugating Enzyme Interacts With Host Translation Machinery to Regulate the Host Protein Synthesis
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Agencia Estatal de Investigación (España), European Commission, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), Fundación la Caixa, Comunidad de Madrid, Biotechnology and Biological Sciences Research Council (UK), Barrado-Gil, Lucía [0000-0002-1053-2997], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Galindo Barreales, Inmaculada [0000-0001-8257-9797], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Urquiza, Jesús [0000-0002-7870-4580], Nistal-Villán, Estanislao [0000-0003-2458-8833], Maluquer de Motes, Carlos [0000-0003-4712-4601], Alonso Martí, Covadonga [0000-0002-0862-6177], Barrado-Gil, Lucía, Del Puerto, Ana, Muñoz-Moreno, Raquel, Galindo Barreales, Inmaculada, Cuesta-Geijo, Miguel Ángel, Urquiza, Jesús, Nistal-Villán, Estanislao, Maluquer de Motes, Carlos, Alonso Martí, Covadonga, Agencia Estatal de Investigación (España), European Commission, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), Fundación la Caixa, Comunidad de Madrid, Biotechnology and Biological Sciences Research Council (UK), Barrado-Gil, Lucía [0000-0002-1053-2997], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Galindo Barreales, Inmaculada [0000-0001-8257-9797], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Urquiza, Jesús [0000-0002-7870-4580], Nistal-Villán, Estanislao [0000-0003-2458-8833], Maluquer de Motes, Carlos [0000-0003-4712-4601], Alonso Martí, Covadonga [0000-0002-0862-6177], Barrado-Gil, Lucía, Del Puerto, Ana, Muñoz-Moreno, Raquel, Galindo Barreales, Inmaculada, Cuesta-Geijo, Miguel Ángel, Urquiza, Jesús, Nistal-Villán, Estanislao, Maluquer de Motes, Carlos, and Alonso Martí, Covadonga
- Abstract
African Swine Fever virus (ASFV) causes one of the most relevant emerging diseases affecting swine, now extended through three continents. The virus has a large coding capacity to deploy an arsenal of molecules antagonizing the host functions. In the present work, we have studied the only known E2 viral-conjugating enzyme, UBCv1 that is encoded by the I215L gene of ASFV. UBCv1 was expressed as an early expression protein that accumulates throughout the course of infection. This versatile protein, bound several types of polyubiquitin chains and its catalytic domain was required for enzymatic activity. High throughput mass spectrometry analysis in combination with a screening of an alveolar macrophage library was used to identify and characterize novel UBCv1-host interactors. The analysis revealed interaction with the 40S ribosomal protein RPS23, the cap-dependent translation machinery initiation factor eIF4E, and the E3 ubiquitin ligase Cullin 4B. Our data show that during ASFV infection, UBCv1 was able to bind to eIF4E, independent from the cap-dependent complex. Our results provide novel insights into the function of the viral UBCv1 in hijacking cellular components that impact the mTORC signaling pathway, the regulation of the host translation machinery, and the cellular protein expression during the ASFV lifecycle.
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- 2020
18. Identification of NPC1 as a novel SARS-CoV-2 intracellular target
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Fundación la Caixa, Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Urquiza, Jesús [0000-0002-7870-4580], Puerto, Ana del [0000-0002-5119-6395], Gil, Carmen [0000-0002-3882-6081], Alonso, Covadonga [0000-0002-0862-6177], García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Inmaculada, Urquiza, Jesús, Puerto, Ana del, Gil, Carmen, Campillo, Nuria E., Martínez Gil, Ana, Alonso, Covadonga, Fundación la Caixa, Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Urquiza, Jesús [0000-0002-7870-4580], Puerto, Ana del [0000-0002-5119-6395], Gil, Carmen [0000-0002-3882-6081], Alonso, Covadonga [0000-0002-0862-6177], García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Inmaculada, Urquiza, Jesús, Puerto, Ana del, Gil, Carmen, Campillo, Nuria E., Martínez Gil, Ana, and Alonso, Covadonga
- Abstract
Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol trafficking, which is crucial in the Ebola virus (EBOV) cycle. The severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) enters the cell by binding of the viral spike (S) protein to the ACE2 receptor. This requires S-protein processing either by the surface transmembrane serine protease TMPRSS2 for plasma membrane fusion or cathepsin L for endosomal entry. Additional host factors are required for viral fusion at endosomes. Here, we report a novel interaction of the SARS-CoV-2 nucleoprotein (N) with the cholesterol transporter NPC1. Moreover, small molecules interfering with NPC1 that inhibit EBOV entry, also inhibited human coronavirus. Our findings suggest an important role for NPC1 in SARS-CoV-2 infection, a common strategy shared with EBOV, and a potential therapeutic target to fight against COVID-19.
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- 2020
19. Effect of Clinically Used Microtubule Targeting Drugs on Viral Infection and Transport Function
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Oliva, María Ángela, primary, Tosat-Bitrián, Carlota, additional, Barrado-Gil, Lucía, additional, Bonato, Francesca, additional, Galindo, Inmaculada, additional, Garaigorta, Urtzi, additional, Álvarez-Bernad, Beatriz, additional, París-Ogáyar, Rebeca, additional, Lucena-Agell, Daniel, additional, Giménez-Abián, Juan Francisco, additional, García-Dorival, Isabel, additional, Urquiza, Jesús, additional, Gastaminza, Pablo, additional, Díaz, José Fernando, additional, Palomo, Valle, additional, and Alonso, Covadonga, additional
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- 2022
- Full Text
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20. New insights into the role of endosomal proteins for African swine fever virus infection
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Cuesta-Geijo, Miguel Ángel, primary, García-Dorival, Isabel, additional, del Puerto, Ana, additional, Urquiza, Jesús, additional, Galindo, Inmaculada, additional, Barrado-Gil, Lucía, additional, Lasala, Fátima, additional, Cayuela, Ana, additional, Sorzano, Carlos Oscar S., additional, Gil, Carmen, additional, Delgado, Rafael, additional, and Alonso, Covadonga, additional
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- 2022
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21. Identification of Niemann-Pick C1 protein as a potential novel SARS-CoV-2 intracellular target
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García-Dorival, Isabel, primary, Cuesta-Geijo, Miguel Ángel, additional, Barrado-Gil, Lucía, additional, Galindo, Inmaculada, additional, Garaigorta, Urtzi, additional, Urquiza, Jesús, additional, Puerto, Ana del, additional, Campillo, Nuria E., additional, Martínez, Ana, additional, Gastaminza, Pablo, additional, Gil, Carmen, additional, and Alonso, Covadonga, additional
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- 2021
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22. Endosomal proteins NPC1 and NPC2 at African swine fever virus entry/fusion
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Cuesta-Geijo, Miguel Ángel, primary, Urquiza, Jesús, additional, del Puerto, Ana, additional, Garcia-Dorival, Isabel, additional, Lasala, Fátima, additional, Barrado-Gil, Lucía, additional, Galindo, Inmaculada, additional, Delgado, Rafael, additional, and Alonso, Covadonga, additional
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- 2021
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23. African Swine Fever Virus Ubiquitin-Conjugating Enzyme Is an Immunomodulator Targeting NF-κB Activation
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Barrado-Gil, Lucía, primary, del Puerto, Ana, additional, Galindo, Inmaculada, additional, Cuesta-Geijo, Miguel Ángel, additional, García-Dorival, Isabel, additional, de Motes, Carlos Maluquer, additional, and Alonso, Covadonga, additional
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- 2021
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- View/download PDF
24. Identification of NPC1 as a novel SARS-CoV-2 intracellular target
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Garcia-Dorival, Isabel, primary, Ángel Cuesta-Geijo, Miguel, additional, Barrado-Gil, Lucía, additional, Galindo, Inmaculada, additional, Urquiza, Jesús, additional, del Puerto, Ana, additional, Gil, Carmen, additional, Campillo, Nuria, additional, Martínez, Ana, additional, and Alonso, Covadonga, additional
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- 2020
- Full Text
- View/download PDF
25. African Swine Fever Virus Ubiquitin-Conjugating Enzyme Interacts With Host Translation Machinery to Regulate the Host Protein Synthesis
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Barrado-Gil, Lucía, primary, Del Puerto, Ana, additional, Muñoz-Moreno, Raquel, additional, Galindo, Inmaculada, additional, Cuesta-Geijo, Miguel Ángel, additional, Urquiza, Jesús, additional, Nistal-Villán, Estanislao, additional, Maluquer de Motes, Carlos, additional, and Alonso, Covadonga, additional
- Published
- 2020
- Full Text
- View/download PDF
26. Redistribution of endosomal membranes to the African swine fever virus replication site
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Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, Muñoz-Moreno, Raquel, Alonso Martí, Covadonga, Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, Muñoz-Moreno, Raquel, and Alonso Martí, Covadonga
- Abstract
African swine fever virus (ASFV) infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns) synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection. © 2017 by the authors. Licensee MDPI, Basel, Switzerland.
- Published
- 2017
27. New insights in the ubiquitylation-related mechanisms in African swine fever virus infection
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Barrado Gil, Lucía, Alonso Martí, Covadonga, Galindo Barreales, Inmaculada, UAM. Departamento de Biología Molecular, and Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)
- Subjects
Biología y Biomedicina / Biología ,Virología veterinaria - Abstract
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-06-2018, Esta tesis tiene embargado el acceso al texto completo hasta el 29-06-2021, In this work, we have investigated ubiquitylation-related mechanisms in African swine fever virus (ASFV) infection. The ubiquitin-proteasome system (UPS) is a tightly regulated organelle that control many cellular processes, including viral infections in the cell. Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. We determined that inhibition of proteasome blocked a post-internalization step, affecting ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and induced the accumulation of Lys63-polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome system that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling. Determined viral proteins are able to control the host cell ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. We verified that the viral ubiquitin-conjugating enzyme (UBCv1) is an early protein that expressed throughout ASFV infection and accumulates at late times. UBCv1 is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early stages of ASFV infection. Indeed, we corroborated the conjugating activity of this viral E2 enzyme, depending on its catalytic domain, that was able to bind several types of polyubiquitin chains. We also characterized potential UBCv1 host targets by mass spectrometry. This proteomic analysis revealed that the early viral protein interacted with the initiation translation factor eIF4E. This was consistent with previous results that pointed a relation with the 40S ribosome subunit RPS23. These interactions indicated a possible function of UBCv1 in the viral regulation of host translation. Further analysis also revealed the interaction with the G protein Arf3, related with membrane traffic and organelle structure, and the interplay with the E3 ligase Cullin4B. ASFV-mediates innate immune response inhibition through a number of genes that have been previously studied in detail. Here, we contribute a new ASFV gene involved in the regulation of the innate immune response. UBCv1 impaired NF-κB and AP-1 transcription factors activation while had no effect neither in interferon β production nor in interferon regulatory factor (IRF) activation. We detected that UBCv1 induced a decrease in IκBα phosphorylation and the inhibition of p65 translocation into the nucleus. We propose that UBCv1 blocked both signalling pathway at the level of IKK kinases. Finally, our studies and results were completed with the transcriptome analysis obtained by Next Generation sequencing of ASFV infected macrophages
- Published
- 2018
28. The ubiquitin-proteasome system is required for African swine fever replication
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Barrado-Gil, Lucía, primary, Galindo, Inmaculada, additional, Martínez-Alonso, Diego, additional, Viedma, Sergio, additional, and Alonso, Covadonga, additional
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- 2017
- Full Text
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29. The ubiquitin-proteasome system is required for African swine fever replication
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Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, Martínez-Alonso, Diego, Viedma, S., Alonso Martí, Covadonga, Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, Martínez-Alonso, Diego, Viedma, S., and Alonso Martí, Covadonga
- Abstract
Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.
- Published
- 2017
30. Redistribution of Endosomal Membranes to the African Swine Fever Virus Replication Site
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Cuesta-Geijo, Miguel, primary, Barrado-Gil, Lucía, additional, Galindo, Inmaculada, additional, Muñoz-Moreno, Raquel, additional, and Alonso, Covadonga, additional
- Published
- 2017
- Full Text
- View/download PDF
31. Antiviral role of IFITM proteins in African swine fever virus infection
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Galindo Barreales, Inmaculada [0000-0001-8257-9797], García-Sastre, Adolfo [0000-0002-6551-1827], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Muñoz-Moreno, Raquel, Cuesta-Geijo, Miguel Ángel, Martínez-Romero, C., Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, García-Sastre, Adolfo, Alonso Martí, Covadonga, Galindo Barreales, Inmaculada [0000-0001-8257-9797], García-Sastre, Adolfo [0000-0002-6551-1827], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Muñoz-Moreno, Raquel, Cuesta-Geijo, Miguel Ángel, Martínez-Romero, C., Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, García-Sastre, Adolfo, and Alonso Martí, Covadonga
- Abstract
The interferon-induced transmembrane (IFITM) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. While IFITMs are widely known to inhibit several single-stranded RNA viruses, there are limited reports available regarding their effect in double-stranded DNA viruses. In this work, we have analyzed a possible antiviral function of IFITMs against a double stranded DNA virus, the African swine fever virus (ASFV). Infection with cell-adapted ASFV isolate Ba71V is IFN sensitive and it induces IFITMs expression. Interestingly, high levels of IFITMs caused a collapse of the endosomal pathway to the perinuclear area. Given that ASFV entry is strongly dependent on endocytosis, we investigated whether IFITM expression could impair viral infection. Expression of IFITM1, 2 and 3 reduced virus infectivity in Vero cells, with IFITM2 and IFITM3 having an impact on viral entry/uncoating. The role of IFITM2 in the inhibition of ASFV in Vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that IFITM2 is acting at the late endosome, preventing the decapsidation stage of ASFV. Copyright © 2016 Muñoz-Moreno et al.
- Published
- 2016
32. Cholesterol flux is required for endosomal progression of African swine fever virions during the initial establishment of infection
- Author
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Galindo Barreales, Inmaculada [0000-0001-8257-9797], Carrascosa, José L. [0000-0002-4749-3522], Alonso Martí, Covadonga [0000-0002-0862-6177], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Cuesta-Geijo, Miguel Ángel, Chiappi, M., Galindo Barreales, Inmaculada, Barrado-Gil, Lucía, Muñoz-Moreno, Raquel, Carrascosa, José L., Alonso Martí, Covadonga, Galindo Barreales, Inmaculada [0000-0001-8257-9797], Carrascosa, José L. [0000-0002-4749-3522], Alonso Martí, Covadonga [0000-0002-0862-6177], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Cuesta-Geijo, Miguel Ángel, Chiappi, M., Galindo Barreales, Inmaculada, Barrado-Gil, Lucía, Muñoz-Moreno, Raquel, Carrascosa, José L., and Alonso Martí, Covadonga
- Abstract
African swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection. © 2016, American Society for Microbiology.
- Published
- 2016
33. Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection
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Muñoz-Moreno, Raquel, primary, Cuesta-Geijo, Miguel Ángel, additional, Martínez-Romero, Carles, additional, Barrado-Gil, Lucía, additional, Galindo, Inmaculada, additional, García-Sastre, Adolfo, additional, and Alonso, Covadonga, additional
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- 2016
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34. Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection
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Cuesta-Geijo, Miguel Ángel, primary, Chiappi, Michele, additional, Galindo, Inmaculada, additional, Barrado-Gil, Lucía, additional, Muñoz-Moreno, Raquel, additional, Carrascosa, José L., additional, and Alonso, Covadonga, additional
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- 2016
- Full Text
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35. African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis
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Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Galindo Barreales, Inmaculada, Cuesta-Geijo, Miguel Ángel, Hlavova, K., Muñoz-Moreno, Raquel, Barrado-Gil, Lucía, Domínguez, Javier, Alonso Martí, Covadonga, Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Galindo Barreales, Inmaculada, Cuesta-Geijo, Miguel Ángel, Hlavova, K., Muñoz-Moreno, Raquel, Barrado-Gil, Lucía, Domínguez, Javier, and Alonso Martí, Covadonga
- Abstract
The main cellular target for African swine fever virus (ASFV) is the porcine macrophage. However, existing data about the early phases of infection were previously characterized in non-leukocyte cells such as Vero cells. Here, we report that ASFV enters the natural host cell using dynamin-dependent and clathrin-mediated endocytosis. This pathway is strongly pH-dependent during the first steps of infection in porcine macrophages. We investigated the effect of drugs inhibiting several endocytic pathways in macrophages and compared ASFV with vaccinia virus (VV), which apparently involves different entry pathways. The presence of cholesterol in cellular membranes was found to be essential for a productive ASFV infection while actin-dependent endocytosis and the participation of phosphoinositide-3-kinase (PI3K) activity were other cellular factors required in the process of viral entry. These findings improved our understanding of the ASFV interactions with macrophages that allow for successful viral replication. © 2015 Elsevier B.V.
- Published
- 2015
36. Analysis of HDAC6 and BAG3-aggresome pathways in African swine fever viral factory formation
- Author
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Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Muñoz-Moreno, Raquel, Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, Alonso Martí, Covadonga, Galindo Barreales, Inmaculada [0000-0001-8257-9797], Alonso Martí, Covadonga [0000-0002-0862-6177], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Muñoz-Moreno, Raquel, Barrado-Gil, Lucía, Galindo Barreales, Inmaculada, and Alonso Martí, Covadonga
- Abstract
African swine fever virus (ASFV) is a double-stranded DNA virus causing a hemorrhagic fever disease with high mortality rates and severe economic losses in pigs worldwide. ASFV replicates in perinuclear sites called viral factories (VFs) that are morphologically similar to cellular aggresomes. This fact raises the possibility that both VFs and aggresomes may be the same structure. However, little is known about the process involved in the formation of these viral replication platforms. In order to expand our knowledge on the assembly of ASFV replication sites, we have analyzed the involvement of both canonical aggresome pathways in the formation of ASFV VFs HDAC6 and BAG3. HDAC6 interacts with a component of the dynein motor complex (dynactin/p150Glued) and ubiquitinated proteins, transporting them to the microtubule-organizing center (MTOC) and leading to aggresome formation, while BAG3 is mediating the recruitment of non-ubiquitinated proteins through a similar mechanism. Tubacin-mediated HDAC6 inhibition and silencing of BAG3 pathways, individually or simultaneously, did not prevent ASFV VF formation. These findings show that HDAC6 and Bag3 are not required for VFs formation suggesting that aggresomes and VFs are not the same structures. However, alternative unexplored pathways may be involved in the formation of aggresomes. © 2015 by the authors; licensee MDPI, Basel, Switzerland.
- Published
- 2015
37. Host cell targets for African swine fever virus
- Author
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Alonso Martí, Covadonga [0000-0002-0862-6177], Galindo Barreales, Inmaculada [0000-0001-8257-9797], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Muñoz-Moreno, Raquel, Galindo Barreales, Inmaculada, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Alonso Martí, Covadonga, Alonso Martí, Covadonga [0000-0002-0862-6177], Galindo Barreales, Inmaculada [0000-0001-8257-9797], Muñoz-Moreno, Raquel [0000-0002-4521-0488], Muñoz-Moreno, Raquel, Galindo Barreales, Inmaculada, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, and Alonso Martí, Covadonga
- Abstract
Viruses are strict intracellular pathogens that require the cellular environment to complete a successful infection. Among them, African swine fever virus (ASFV) is an evolutionary ancient DNA virus, endemic in Africa, which is nowadays causing an emergent disease in Europe with a potential high economic impact in the pig industry. It is well known that host-cell components are critical crossroads mapping the virus path for a productive infection, some of them at the endocytic pathway. Considering that ASFV infectious cycle strongly relies in several factors from the host cell, the study of virus-host interactions remains crucial as they will reveal the obstacles, routes and tracks, hints and the target waypoint in the virus journey to destination. © 2015 Elsevier B.V.
- Published
- 2015
38. Analysis of HDAC6 and BAG3-Aggresome Pathways in African Swine Fever Viral Factory Formation
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Muñoz-Moreno, Raquel, primary, Barrado-Gil, Lucía, additional, Galindo, Inmaculada, additional, and Alonso, Covadonga, additional
- Published
- 2015
- Full Text
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39. Effect of Clinically Used Microtubule Targeting Drugs on Viral Infection and Transport Function
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María Ángela Oliva, Carlota Tosat-Bitrián, Lucía Barrado-Gil, Francesca Bonato, Inmaculada Galindo, Urtzi Garaigorta, Beatriz Álvarez-Bernad, Rebeca París-Ogáyar, Daniel Lucena-Agell, Juan Francisco Giménez-Abián, Isabel García-Dorival, Jesús Urquiza, Pablo Gastaminza, José Fernando Díaz, Valle Palomo, Covadonga Alonso, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, La Caixa, Club Escola Hungaresa de Esgrima de Pontevedra, Oliva, María A., Tosat-Bitrian, Carlota, Barrado-Gil, Lucía, Bonato, Francesca, Galindo, Inmaculada, Garaigorta, Urtzi, Álvarez-Bernad, Beatriz, París-Ogáyar, Rebeca, Lucena-Agell, Daniel, Giménez-Abián, Juan F., Urquiza, Jesús, Díaz, José Fernando, Palomo, Valle, Alonso, Covadonga, Oliva, María A. [0000-0002-2215-4639], Tosat-Bitrian, Carlota [0000-0002-0525-3512], Barrado-Gil, Lucía [0000-0002-1053-2997], Bonato, Francesca [0000-0002-0579-2874], Galindo, Inmaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], París-Ogáyar, Rebeca [0000-0002-7953-0949], Lucena-Agell, Daniel [0000-0001-7314-8696], Giménez-Abián, Juan F. [0000-0002-9220-286X], Urquiza, Jesús [0000-0002-7870-4580], Díaz, José Fernando [0000-0003-2743-3319], Palomo, Valle [0000-0002-1473-4086], and Alonso, Covadonga [0000-0002-0862-6177]
- Subjects
Mammals ,antivirals ,microtubule targeting drugs ,SARS-CoV-2 ,mebendazole-tubulin complex crystal structure ,Organic Chemistry ,Mebendazole-tubulin complex crystal structure ,General Medicine ,Antivirals ,Antiviral Agents ,Microtubules ,Catalysis ,COVID-19 Drug Treatment ,Computer Science Applications ,Inorganic Chemistry ,Mebendazole ,Tubulin ,Animals ,Microtubule targeting drugs ,Physical and Theoretical Chemistry ,Molecular Biology ,Spectroscopy - Abstract
Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians., This work was supported by Ministerio de Ciencia e Innovación PID2019-104545RBI00/ AEI/10.13039/501100011033 to JFD and RTI2018-097305-R-I00 to CA, Consejo Superior de Investigaciones Científicas PIE 201920E111 to JFD, PIE 202020E301 and the European Commission– NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global) to JFD and CA, H2020-MSCA-ITN-2019 860070 TUBINTRAIN to JFD, “La Caixa” Banking Foundation (LCF/PR/HR19/52160012) to CA, Postdoctoral Junior Leader Fellowship Programme from ‘La Caixa’ Banking Foundation (LCF/BQ/PR18/11640007) to VP and Ministerio de Ciencia e Innovacion FPI program (PRE2020-094384) to BAB and Ministerio de Educación FPU program (FPU18/06310) to CTB. We also acknowledge a generous donation from Club Deportivo Escuela Hungaresa de Pontevedra.
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- 2022
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40. New insights into the role of endosomal proteins for African swine fever virus infection
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Miguel Ángel Cuesta-Geijo, Isabel García-Dorival, Ana del Puerto, Jesús Urquiza, Inmaculada Galindo, Lucía Barrado-Gil, Fátima Lasala, Ana Cayuela, Carlos Oscar S. Sorzano, Carmen Gil, Rafael Delgado, Covadonga Alonso, La Caixa, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, European Commission, Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], García-Dorival, Isabel [0000-0002-5654-5662], Puerto, Ana del [0000-0002-5119-6395], Urquiza, Jesús [0000-0002-7870-4580], Galindo, Immaculada [0000-0001-8257-9797], Barrado-Gil, Lucía [0000-0002-1053-2997], Lasala, Fátima [0000-0001-8183-1866], Sorzano, Carlos Óscar S. [0000-0002-9473-283X], Gil, Carmen [0000-0002-3882-6081], Delgado, Rafael [0000-0002-6912-4736], Alonso, Covandoga [0000-0002-0862-6177], Cuesta-Geijo, Miguel Ángel, García-Dorival, Isabel, Puerto, Ana del, Urquiza, Jesús, Galindo, Immaculada, Barrado-Gil, Lucía, Lasala, Fátima, Sorzano, Carlos Óscar S., Gil, Carmen, Delgado, Rafael, and Alonso, Covandoga
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RNA viruses ,Swine ,Cell Lines ,viruses ,Pathology and Laboratory Medicine ,Biochemistry ,Virions ,Chlorocebus aethiops ,Medicine and Health Sciences ,Biology (General) ,0303 health sciences ,030302 biochemistry & molecular biology ,African Swine Fever Virus ,Lipids ,3. Good health ,Precipitation Techniques ,Cholesterol ,Medical Microbiology ,Filoviruses ,Viral Pathogens ,Host-Pathogen Interactions ,Viruses ,Biological Cultures ,Cellular Structures and Organelles ,Pathogens ,Ebola Virus ,Research Article ,QH301-705.5 ,Immunology ,Endosomes ,Viral Structure ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Viral Proteins ,Virology ,Genetics ,Viral Core ,Animals ,Humans ,Immunoprecipitation ,Vesicles ,African Swine Fever ,Protein Interactions ,Molecular Biology ,Vero Cells ,Microbial Pathogens ,030304 developmental biology ,Hemorrhagic Fever Viruses ,Organisms ,Biology and Life Sciences ,Proteins ,Cell Biology ,RC581-607 ,HEK293 Cells ,Parasitology ,Immunologic diseases. Allergy - Abstract
28 p.-7 fig.-- Departamento de Biotecnología., African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection., This research was partially supported through the following awards: “La Caixa” Banking Foundation award number LCF/PR/HR19/52160012, Spain, https://fundacionlacaixa.org/en/ to CA and RD, Spanish Ministry of Science and Innovation, Spain RTI2018-097305-R- I00, https://www.ciencia.gob.es/ to CA, Instituto de Investigación Carlos III, ISCIII, Spain FIS PI 1801007, https://www.isciii.es/ to RD, European Commission, Horizon 2020 Framework Programme European Union ASFVInt ERANET-2021-862605 to CA, co-funded by Spanish Ministry of Science and Innovation, Spain PCI2021-121939 https://www.ciencia.gob.es/ to CA, VIRUSCAN FETPROACT-2016- 731868 and EPIC-CROWN-2-2021-101046084 https://ec.europa.eu/ to RD.
- Published
- 2022
41. African Swine Fever Virus Ubiquitin-Conjugating Enzyme Is an Immunomodulator Targeting NF-κB Activation
- Author
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Isabel García-Dorival, Miguel Ángel Cuesta-Geijo, Ana del Puerto, Carlos Maluquer de Motes, Lucía Barrado-Gil, Covadonga Alonso, Inmaculada Galindo, Fundación 'la Caixa', Ministerio de Ciencia e Innovación (España), Barrado-Gil, Lucía, Galindo, Inmaculada, Cuesta-Geijo,Miguel Angel, García-Dorival, Isabel, de Motes, Carlos Maluquer, Alonso, Covadonga, Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Inmaculada [0000-0001-8257-9797], Cuesta-Geijo,Miguel Angel [0000-0003-4694-1968], García-Dorival, Isabel [0000-0002-2035-2404], de Motes, Carlos Maluquer [0000-0003-4712-4601], and Alonso, Covadonga [0000-0002-0862-6177]
- Subjects
0301 basic medicine ,E2 enzyme ,Swine ,Ubiquitin-conjugating enzyme ,Biology ,IFN ,Microbiology ,African swine fever virus ,Virus ,Article ,NF-κB ,Immunomodulation ,03 medical and health sciences ,chemistry.chemical_compound ,Immune system ,Ubiquitin ,Virology ,TLR ,Animals ,Humans ,innate immunity ,Innate immune system ,030102 biochemistry & molecular biology ,NF-kappa B ,biology.organism_classification ,AP-1 ,QR1-502 ,Immunity, Innate ,Cell biology ,030104 developmental biology ,Infectious Diseases ,HEK293 Cells ,chemistry ,A549 Cells ,Interferon Type I ,Ubiquitin-Conjugating Enzymes ,biology.protein ,ASFV ,IRF3 ,Signal Transduction ,STING - Abstract
African swine fever virus (ASFV) is an acute and persistent swine virus with a high economic burden that encodes multiple genes to evade host immune response. In this work, we have revealed that early viral protein UBCv1, the only known conjugating enzyme encoded by a virus, modulates innate immune and inflammatory signaling. Transient overexpression of UBCv1 impaired activation of NF-κB and AP-1 transcription factors induced by several agonists of these pathways. In contrast, activation of IRF3 and ISRE signaling upon stimulation with TRIFΔRIP, cGAS/STING or RIG-I-CARD remained unaltered. Experiments aimed at mapping UBCv1 inhibitory activity indicated that this viral protein acts upstream or at the level step of IKKβ. In agreement with this, UBCv1 was able to block p65 nuclear translocation upon cytokine stimulation, a key event in NF-ĸB signaling. Additionally, A549 stably transduced for UBCv1 showed a significant decrease in the levels of NF-ĸB dependent genes. Interestingly, despite the well-defined capacity of UBCv1 to conjugate ubiquitin chains, a mutant disabled for ubiquitylation activity retained similar immunomodulatory activity as the wild-type enzyme, suggesting that the two functions are segregated. Altogether these data suggest that ASFV UBCv1 manipulates the innate immune response targeting the NF-κB and AP-1 pathways and opens new questions about the multifunctionality of this enzyme., This research was partially supported through “La Caixa” Banking Foundation award number LCF/PR/HR19/52160012, Spain and the Ministry of Science and Innovation grants AGL2015- 69598-R and RTI2018-097305-R-I00.
- Published
- 2021
42. Identification of Niemann-Pick C1 protein as a potential novel SARS-CoV-2 intracellular target
- Author
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Urtzi Garaigorta, Isabel García-Dorival, Carmen Gil, Ana del Puerto, Nuria E. Campillo, Pablo Gastaminza, Covadonga Alonso, Miguel Ángel Cuesta-Geijo, Ana Martínez, Inmaculada Galindo, Lucía Barrado-Gil, Jesús Urquiza, European Commission, La Caixa, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), García-Dorival, Isabel [0000-0002-5654-5662], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Urquiza, Jesús [0000-0002-7870-4580], Puerto, Ana del [0000-0002-5119-6395], Campillo, Nuria E. [0000-0002-9948-2665], Martínez, Ana [0000-0002-2707-8110], Gil, Carmen [0000-0002-3882-6081], Alonso, Covadonga [0000-0002-0862-6177], García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Immaculada, Garaigorta, Urtzi, Urquiza, Jesús, Puerto, Ana del, Campillo, Nuria E., Martínez, Ana, Gil, Carmen, and Alonso, Covadonga
- Subjects
Proteases ,Endosome ,viruses ,Carbazoles ,Endosomes ,Virus Replication ,Membrane Fusion ,Article ,03 medical and health sciences ,Niemann-Pick C1 Protein ,hemic and lymphatic diseases ,Virology ,Chlorocebus aethiops ,Plasma membrane fusion ,Animals ,Humans ,Niemann-pick type C1 (NPC1) ,Vero Cells ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Chemistry ,SARS-CoV-2 ,030302 biochemistry & molecular biology ,HEK 293 cells ,Intracellular Signaling Peptides and Proteins ,Lipid bilayer fusion ,Biological Transport ,COVID-19 Drug Treatment ,3. Good health ,Cell biology ,Potential therapeutic target ,HEK293 Cells ,Viral replication ,NPC1 ,SARS-CoV-2 nucleoprotein ,Intracellular - Abstract
10 p.-4 fig., 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., This research was partially supported through “La Caixa” Banking Foundation (LCF/PR/HR19/52160012), Instituto de Salud Carlos III (ISCIII–COV20/01007), the Spanish Ministry of Science and Innovation (RTI2018-097305-R-I00), CSIC (PIE-RD-COVID-19 refs. E201980E024 and E202020E079) and the European Union-NextGenerationEU. EVA (European Virus Archive; grant agreement No 871029).
- Published
- 2021
43. Identification of NPC1 as a novel SARS-CoV-2 intracellular target
- Author
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Isabel García-Dorival, Immaculada Galindo, Lucía Barrado-Gil, Nuria E. Campillo, Ana Martínez, Covadonga Alonso, Miguel Ángel Cuesta-Geijo, Ana del Puerto, Carmen Gil, Jesús Urquiza, Fundación 'la Caixa', Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Urquiza, Jesús [0000-0002-7870-4580], Puerto, Ana del [0000-0002-5119-6395], Gil, Carmen [0000-0002-3882-6081], Alonso, Covadonga [0000-0002-0862-6177], Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Immaculada, Urquiza, Jesús, Puerto, Ana del, Gil, Carmen, and Alonso, Covadonga
- Subjects
Target ,0303 health sciences ,2019-20 coronavirus outbreak ,Coronavirus disease 2019 (COVID-19) ,SARS-CoV-2 ,viruses ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,030302 biochemistry & molecular biology ,virus diseases ,nutritional and metabolic diseases ,Library science ,Antivirals ,N protein ,NPC1 ,3. Good health ,03 medical and health sciences ,hemic and lymphatic diseases ,Political science ,Christian ministry ,European commission ,030304 developmental biology - Abstract
Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol trafficking, which is crucial in the Ebola virus (EBOV) cycle. The severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) enters the cell by binding of the viral spike (S) protein to the ACE2 receptor. This requires S-protein processing either by the surface transmembrane serine protease TMPRSS2 for plasma membrane fusion or cathepsin L for endosomal entry. Additional host factors are required for viral fusion at endosomes. Here, we report a novel interaction of the SARS-CoV-2 nucleoprotein (N) with the cholesterol transporter NPC1. Moreover, small molecules interfering with NPC1 that inhibit EBOV entry, also inhibited human coronavirus. Our findings suggest an important role for NPC1 in SARS-CoV-2 infection, a common strategy shared with EBOV, and a potential therapeutic target to fight against COVID-19., This research was partially supported through “La Caixa” Banking Foundation (HR18-00469), Instituto de Salud Carlos III (ISCIII-COV20/01007), CSIC (201980E024 and 202020E079), Spanish Ministry of Science and Innovation (RTI2018-097305-R-I00) and the European Commission Horizon 2020 Framework Programme VACDIVA-SFS482 12-2019-1-862874.
- Published
- 2020
44. African Swine Fever Virus Ubiquitin-Conjugating Enzyme Interacts With Host Translation Machinery to Regulate the Host Protein Synthesis
- Author
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Estanislao Nistal-Villán, Lucía Barrado-Gil, Jesús Urquiza, Carlos Maluquer de Motes, Covadonga Alonso, Ana del Puerto, Miguel Ángel Cuesta-Geijo, Inmaculada Galindo, Raquel Muñoz-Moreno, Agencia Estatal de Investigación (España), European Commission, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), Fundación 'la Caixa', Comunidad de Madrid, Biotechnology and Biological Sciences Research Council (UK), Barrado-Gil, Lucía, Muñoz-Moreno, R., Galindo, Inmaculada, Cuesta-Geijo, Miguel Ángel, Urquiza, Jesús, Nistal-Villán, Estanislao, Maluquer de Motes, Carlos, and Alonso Martí, Covadonga
- Subjects
Cullin 4B Cul4B ,Microbiology (medical) ,lcsh:QR1-502 ,Biology ,Ubiquitin-conjugating enzyme ,ubiquitin-conjugating enzyme ,Microbiology ,African swine fever virus ,lcsh:Microbiology ,03 medical and health sciences ,Ribosomal protein ,ribosomal protein 23 ,translation initiation factor ,viral E2 ,Initiation factor ,Original Research ,030304 developmental biology ,0303 health sciences ,030302 biochemistry & molecular biology ,EIF4E ,Translation (biology) ,biology.organism_classification ,Ubiquitin ligase ,Cell biology ,eIF4E ,biology.protein ,ASFV ,Cullin - Abstract
15 Pág., African Swine Fever virus (ASFV) causes one of the most relevant emerging diseases affecting swine, now extended through three continents. The virus has a large coding capacity to deploy an arsenal of molecules antagonizing the host functions. In the present work, we have studied the only known E2 viral-conjugating enzyme, UBCv1 that is encoded by the I215L gene of ASFV. UBCv1 was expressed as an early expression protein that accumulates throughout the course of infection. This versatile protein, bound several types of polyubiquitin chains and its catalytic domain was required for enzymatic activity. High throughput mass spectrometry analysis in combination with a screening of an alveolar macrophage library was used to identify and characterize novel UBCv1-host interactors. The analysis revealed interaction with the 40S ribosomal protein RPS23, the cap-dependent translation machinery initiation factor eIF4E, and the E3 ubiquitin ligase Cullin 4B. Our data show that during ASFV infection, UBCv1 was able to bind to eIF4E, independent from the cap-dependent complex. Our results provide novel insights into the function of the viral UBCv1 in hijacking cellular components that impact the mTORC signaling pathway, the regulation of the host translation machinery, and the cellular protein expression during the ASFV lifecycle., This research was partially supported through Spanish Ministry of Science and Innovation under Award Number RTI2018-097305-R-I00. Funding from “la Caixa” Banking Foundation under the project code HR18-00469 and EU H2020 VACDIVA SFS-12-2019-862874 is acknowledged. Fellowship Award FPI references BES-2013-066610 and BES 2010-036950 funded B-GL and RM-M Work in EN-V. laboratory was funded by project NIETO-CM B2017/BMD 3731 and in CM laboratory was funded by the United Kingdom Biotechnology and Biological Sciences Research Council (grant ref. BB/M003647/1).
- Published
- 2020
45. Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection.
- Author
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Cuesta-Geijo MÁ, Chiappi M, Galindo I, Barrado-Gil L, Muñoz-Moreno R, Carrascosa JL, and Alonso C
- Subjects
- Animals, Chlorocebus aethiops, Hydrogen-Ion Concentration, Metabolic Flux Analysis, Vero Cells, African Swine Fever Virus physiology, Cholesterol metabolism, Endosomes metabolism, Endosomes virology, Virus Internalization
- Abstract
Unlabelled: African swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection., Importance: Since its appearance in the Caucasus in 2007, African swine fever (ASF) has been spreading westwards to neighboring European countries, threatening porcine production. Due to the lack of an effective vaccine, ASF control relies on early diagnosis and widespread culling of infected animals. We investigated early stages of ASFV infection to identify potential cellular targets for therapeutic intervention against ASF. The virus enters the cell by endocytosis, and soon thereafter, viral decapsidation occurs in the acid pH of late endosomes. We found that ASFV infection requires and reorganizes the cellular lipid cholesterol. ASFV requires cholesterol to exit the endosome to gain access to the cytoplasm to establish productive replication. Our results indicate that there is a differential requirement for cholesterol efflux for vaccinia virus or adenovirus 5 compared to ASFV., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
- Full Text
- View/download PDF
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