Insights into the function of ESCRT complex and LBPA in ASFV infection

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

COVID-19: Drug targets and potential treatments

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.Peer reviewe

New insights in the ubiquitylation-related mechanisms in African swine fever virus infection

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-06-2018Esta tesis tiene embargado el acceso al texto completo hasta el 29-06-2021In 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 macrophage

Analysis of HDAC6 and BAG3-Aggresome Pathways in African Swine Fever Viral Factory Formation

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

Redistribution of Endosomal Membranes to the African Swine Fever Virus Replication Site

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

Identification of NPC1 as a novel SARS-CoV-2 intracellular target

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.N

The ubiquitin-proteasome system is required for African swine fever replication

<div><p>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.</p></div

Proteasome inhibition decreased core breakdown of ASF viral particles.

<p>(A) Schematics show the disposition of virion layers. The outer capsid composed by ASFV p72 major capsid protein is represented in red colour and the inner core with the viral core protein p150 in green colour. Encapsidated virions would double label to both proteins in yellow while uncoated virions lose capsid staining and would single label in green. Empty virions positive for capsid protein p72 yielded a red signal. Representative confocal microscopy images of ASFV infected cells labelled for viral major capsid protein p72 (red) and inner core protein p150 (green). Cells were pretreated with 1 μM MG132, 200 nM Bafilomycin and infected for 3 hpi at a moi of 10 pfu/cell. Bar = 10μm (B) Number of intact cores (green) and encapsidated virions (yellow) per cells in each condition 3hpi. (C) Graphical representation showing the percentages of uncoated viral cores in cells treated with DMSO, MG132 and Baf normalized to the total number of virions counted in 50 cells per condition.</p

Effect of proteasome inhibitors on ASFV infection.

<p>Percentages of ASFV Ba71V-infected cells analysed by flow cytometry at 16 hpi using monoclonal antibodies against early p30 (A) and late p72 (B) viral proteins. Vero cells treated with increasing doses of MG132 (0.1, 0.5 and 1 μM), Lactacystin (5, 10 and 20 μM) and Bortezomib (0.01, 0.1 and 0.5 μM) 1 h prior to infection or left untreated. Data normalised to controls were expressed as mean±SD of three independent experiments and compared to DMSO. Significant differences were marked with asterisks as indicated (**p<0.01; ***p<0.001). (C) Representative flow cytometry profiles of the % of cells expressing the late protein p72. (D) Cytotoxicity assay of inhibitors MG132 (1 μM), Lactacystin (20 μM) and Bortezomib (0.5 μM) used to select the non-toxic working concentrations. (E) Cell viability analysis by counting the number of cells with Trypan Blue stain. Cells were treated with the inhibitor MG132 (1 μM), Lactacystin (20 μM) and Bortezomib (0.5 μM) for 16h.</p