key: cord-0929639-by6rj1f5 authors: Swain, Jitendriya; Merida, Peggy; Rubio, Karla; Bracquemond, David; Aguilar-Ordoñez, Israel; Günther, Stefan; Barreto, Guillermo; Muriaux, Delphine title: Reorganization of F-actin nanostructures is required for the late phases of SARS-CoV-2 replication in pulmonary cells date: 2022-03-21 journal: bioRxiv DOI: 10.1101/2022.03.08.483451 sha: d6ff680d8c8d63c3ecf82cdabc96e05a6f75a4bb doc_id: 929639 cord_uid: by6rj1f5 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is worldwide the main cause of the COVID-19 pandemic. After infection of human pulmonary cells, intracellular viral replication take place in different cellular compartments resulting in the destruction of the host cells and causing severe respiratory diseases. Although cellular trafficking of SARS-CoV-2 have been explored, little is known about the role of the cytoskeleton during viral replication in pulmonary cells. Here we show that SARS-CoV-2 infection induces dramatic changes of F-actin nanostructures overtime. Ring-like actin nanostructures are surrounding viral intracellular organelles, suggesting a functional interplay between F-actin and viral M clusters during particle assembly. Filopodia-like structures loaded with viruses to neighbour cells suggest these structures as mechanism for cell-to-cell virus transmission. Strikingly, gene expression profile analysis and PKN inhibitor treatments of infected pulmonary cells reveal a major role of alpha-actinins superfamily proteins in SARS-CoV-2 replication. Overall, our results highlight cell actors required for SARS-CoV2 replication that are promises for antiviral targets. Teaser Impairing regulation of actin filaments inhibits SARS-CoV-2 particle production in human pulmonary cells. cytoskeleton during viral replication in pulmonary cells. Here we show that SARS-CoV-2 23 infection induces dramatic changes of F-actin nanostructures overtime. Ring-like actin 24 nanostructures are surrounding viral intracellular organelles, suggesting a functional 25 interplay between F-actin and viral M clusters during particle assembly. Filopodia-like 26 structures loaded with viruses to neighbour cells suggest these structures as mechanism for 27 cell-to-cell virus transmission. Strikingly, gene expression profile analysis and PKN 28 inhibitor treatments of infected pulmonary cells reveal a major role of alpha-actinins 29 superfamily proteins in SARS-CoV-2 replication. Overall, our results highlight cell actors 30 required for SARS-CoV2 replication that are promises for antiviral targets. of target cells, intracellular viral replication take place consisting of a series of complex 47 processes (e.g., viral RNA translation, particle packaging, assembly and release) that are 48 tightly orchestrated to one another and often mutually exclusive (reviewed in 6). Viral 49 translation often takes place first in order to create a stock of viral proteins that will serve 50 to assemble the newly made viral particles. The SARS-CoV-2 transcriptome consists of a 51 long unspliced genomic RNA and 9 sub-genomic RNAs that are generated by alternative 52 splicing. After viral RNA translation, once the structural nucleocapsid protein N is 53 produced in the cytosol of infected cells, SARS-CoV-2 assembly continues with the 54 interactions of the N proteins with the unspliced genomic RNA. These interactions lead to 55 a ribonucleoprotein complex that will assemble at the membrane of the Endoplasmic 56 Reticulum -Golgi intermediate Compartment (ERGIC) with the structural proteins 57 transmembrane (M), envelop protein (E) and spike protein (S) (7, 8, 9) . The viral particles, 58 ranging between 90 and 200 nm as recently described (7, 8, 10, 11) , will bud from the 59 ERGIC and egress through the secretory pathway. A number of studies over the years 60 have shown that most viruses hijack the cytoskeletal network to fulfill their own 61 replication cycle, which motivated us to perform a detail study on the role of the 62 cytoskeleton during SARS-CoV-2 infection in human host pulmonary cells (12, 13, 14) . 63 The cytoskeleton is a complex and dynamic network of protein filaments in the cytoplasm 64 of the cells, extending from the cell nucleus to the cell membrane. Its primary function is 65 to give the cell its morphology and mechanical resistance to deformation (15). In addition, 66 the cytoskeleton has been related to many different cellular processes including cell 67 migration, cell signalling, endocytosis, cell division, chromosome segregation, 68 intracellular transport, etc (12, 16, 17, 18) . It can also build specialized structures, such as The G-actin monomer combines to form a polymer which continues to form the actin 74 filament. These actin filament subunits assemble into two chains that intertwine into 75 nanostructures called F-actin chains or fibers (15). F-actin fibers generate force when the 76 growing end of the actin filaments push against a barrier, such as the cell membrane. They 77 also act as tracks for the movement of myosin molecules that affix to the microfilament 78 and "move" along them. Interestingly, the network of F-actin fibers under the plasma 79 membrane can be a carrier for virus entry or transfer from one cell to other (12, 17) . 80 Although mechanisms of trafficking implicated in SARS-CoV-2 infection have been 81 explored mostly in simian Vero cell lines (19, 20) , little is known about the participation of 82 F-actin nanostructures during SARS-CoV-2 replication in human pulmonary cells. Here, 83 we implemented confocal and super-resolution 2D and 3D STED microscopy (21) to 84 study the kinetics of M cluster formation during SARS-CoV-2 particle assembly and 85 release, as well as the effects of SARS-CoV-2 infection on the morphology of human 86 pulmonary cells as consequence of intracellular rearrangements of F-actin nanostructures. 87 The human pulmonary A549-hACE2 cells implemented here are a well-established 88 experimental model for SARS-CoV-2 research due to their high susceptibility to SARS- 89 CoV-2 infection, which can be explained by the stably overexpression of the host receptor 90 protein for the viral S protein (human angiotensin-converting enzyme 2, hACE2) and the 91 presence of the the co-receptor (human transmembrane protease serine 2, TMPRSS2) (22) . 92 Our results demonstrate that the kinetics of M cluster formation during SARS-CoV-2 93 particle assembly and release correlate with rearrangements of intracellular F-actin fibers 94 and morphological changes of SARS-CoV-2-infected human pulmonary A549-hACE2 whereas the release of SARS-CoV-2 particles peaks at 72h pi. 126 We also monitored the effect of SARS-CoV-2 infection on the cytoskeleton of A549-127 hACE2 cells by confocal microscopy at different time points pi after labelling F-actin by 128 phalloidin stain ( Figure 1E and Supplementary Fig. 2 ). We observed changes in the 129 distribution of F-actin in A549-hACE2 cells at different time points SARS-CoV-2 pi. E.g., infected cells (Fig. 5E ). 238 Following the line of ideas from our previous results (Fig. 5, 2 and 3 inhibitors, respectively (Fig. 6A, Supplementary Fig. 4) . The LD50 being determined as World Health Organization, World Health Organization The trinity of COVID-19: immunity, 475 inflammation and intervention Cytoskeleton -A crucial key in host cell for 478 coronavirus infection Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads 483 and lung injury The structural and 485 accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome 486 coronavirus (MERS-CoV) are potent interferon antagonists Betacoronavirus Assembly: Clues and Perspectives for Elucidating SARS-CoV-2 Particle Formation and Egress The Architecture of Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM and 494 Sars-cov-2 (Covid-19) by the numbers. eLife Ultrastructural analysis of SARS-CoV-2 interactions with the 499 host cell via high resolution scanning electron microscopy Molecular Architecture of the 503 SARS-CoV-2 Virus Atomic force microscopy analysis of native infectious and inactivated SARS-CoV-2 506 virions Regulation of actin assembly associated with protrusion and 508 adhesion in cell migration Pixuna virus modifies host cell cytoskeleton to secure 511 infection Rabies 513 virus inactivates cofilin to facilitate viral budding and release. Biochemical and Biophysical 514 Research Communications Actin dynamics, architecture, and 516 mechanics in cell motility Building the actin cytoskeleton: Filopodia contribute to 518 the construction of contractile bundles in the lamella Role of the actin cytoskeleton during influenza virus internalization into 521 polarized epithelial cells Self-organizing actin patterns shape membrane architecture but 525 not cell mechanics The Global Phosphorylation 537 Landscape of SARS-CoV-2 Infection Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular 543 HIV-1 Gag 545 specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain 546 platform for virus assembly Functional assessment of cell entry and receptor usage for 548 SARS-CoV-2 and other lineage B betacoronaviruses Gene set enrichment 552 analysis: A knowledge-based approach for interpreting genome-wide expression profiles Low selectivity index of ivermectin and macrocyclic lactones on SARS-558 CoV2 replication in vitro argues against their therapeutic use for COVID-19 A high-resolution temporal atlas of the SARS CoV-2 translatome and transcriptome Host cytoskeleton in respiratory syncytial virus assembly 565 and budding Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial 569 Infectivity of influenza virus filaments [25] Electron micrography of the virus of influenza [2] Friend or foe: The role of the cytoskeleton in influenza a virus assembly Mechanisms for cell-to-cell transmission of HIV-1 Interaction of PKN with α -actinin Phosphoinositide Binding 582 Regulates α -Actinin Dynamics Real-time imaging of individual virion-triggered cortical actin dynamics for 588 human immunodeficiency virus entry into resting CD4 T cells We thank CEMIPAI UAR3725 service unit for initial advices on SARS-CoV-2 virus 593 production and titration and Aymeric Neyret for the TEM image. The Microscopy STED 594 was done at the Montpellier Imaging Center for Microscopy (MRI) Montpellier University through a Montpellier University of Excellence 601 (MUSE) support and by the French Agency for Research (ANR COVID19) grant 602 "NucleoCoV-2 Centre National de la Recherche Scientifique Lorraine Université d'Excellence" (LUE) and the 606 dispositive "Future Leader" and the "Deutsche Forschungsgemeinschaft BA 4036/4-1). Karla Rubio was funded by the "Consejo de Ciencia y 608 Mexico) through the initiative 609 International Laboratory EPIGEN Author contributions: PM, DB performed cell culture, BSL3 infection, viral stock 612 amplification and titer, viral RNA extraction and qRT-PCR, immunoblots. DB 613 participated to the TEM. JS performed immunofluorescence sample preparation, confocal 614 and STED 2D and 3D Microscopy and quantitative analysis. KR performed RNA 615 extraction and sequencing. KR, IA, SG and GB performed RNA sequencing manuscript writing. JS, GB and DM 617 conceptualized the study, edited the figures and wrote the manuscript. DM supervised the 618 study. DM and GB raised funding for the study Competing interests: Authors declare that they have no competing interests Data and materials availability: 623 All data are available in the main text or the supplementary materials Imaging and quantitative analysis of time course changes in viral M clusters area, 635 morphology and mean F-actin intensity of SARS-CoV-2 infected A549-hACE2 cells A549-hACE2 cells were fixed at 0h, 24h, 48h, and 72h post infection and processed for 637 immunofluorescence Phalloidin Alexa Fluor 488 were used 639 for confocal microscopy. (A) Schematic representation and confocal images of viral 640 clusters and F-actin with different time post infection 0h to 72h. (B) Images for changes in 641 viral M clusters size at different time post infection. (C) Plot for viral M clusters size and 642 viral M clusters intensity at different time post infection RNA/µL in the supernatant of infected cells with different time post infection. (E) Images 644 for changes in F-actin intensity per cell at different time post infection. (F) Plot for mean 645 intensity with or without infection at different time of post infection. (G) Western 646 blot data for global actin content of non-infected and infected cell at different time point of 647 infection. All F-actin intensity of infected and non-infected cells are calculated from Z-648 projection images Fig 2. Morphological changes of human pulmonary cells upon SARS-CoV-2 infection Imaging and quantitative analysis of time course changes in morphology of SARS-CoV-2 655 infected A549-hACE2 cells. A549-hACE2 cells were fixed at 0h, 24h, 48h, and 72h post 656 infection and processed for immunofluorescence. SARS membrane protein anti-M rabbit 657 antibody and a secondary antibody Alexa Fluor 488 were used for confocal microscopy. (A) Confocal images of Control and 659 All cell surface area, height, volume of 661 infected and non-infected cells are calculated from actin and M protein spreading at XY 662 and Z direction at different time of post infection Fig 3. Reorganization of F-actin nanostructures and intracellular actin ring formation in 672 SARS-CoV-2 infected pulmonary cells A549-hACE2 cells were fixed for control and 48h post infection 675 processed for immunofluorescence coupled to STED microscopy. For imaging virus 676 SARS membrane protein anti-M rabbit antibody and then secondary antibody Star orange 677 (green) and for F-actin Phalloidin Star red (red) were used. (A) 2D STED images and 678 color representation of orientation angle of F-actin network with (48h post infection) or 679 without infection. (B) Plot for distribution of orientation angle, with (48h post infection) 680 or without infection Plot for distribution of F-Actin rings and viral ring diameters. (E) STED 3D images of 682 intracellular F-actin rings and viral M clusters in infected pulmonary cells < n <50 cells were analyzed from at least 3 independent experiments. Statistical 684 significant analysis were evaluated using one way ANOVA tests Transmission electron microscopy (TEM) images of intracellular structures filled with 686 budding viruses in SARS-CoV-2 infected pulmonary cells Reorganization of actin fibers into virus loaded filopodia-like protrusions at the cell 691 surface of SARS-CoV-2 infected pulmonary cells STED 2D images and Quantitative data of changes in F-actin nanostructures in SARS A549-hACE2 cells were fixed for control and 48h 694 post infection processed for immunostaining and STED microscopy. For imaging viral 695 particles and clustered SARS membrane protein anti-M rabbit antibody and then 696 secondary antibody Star orange (green) and for F-actin Phalloidin Star red (red) were 697 used. (A) Merge STED 2D Images of control and C) Plot for distribution of length of individual filopodia in control and 700 infected cells. (D) 3D projection Image and plots showing viral M cluster size at cell edge 701 (Zone 1), at filopodia-like structures (Zone 2), and at cell-to-cell connections. A number 702 of 15 < n < 20 cells were analyzed from at least 3 independent experiments Cellular gene expression analysis of SARS-CoV-2 infected human pulmonary cells 710 using RNAseq reveals an upregulation of alpha-Actinins 711 (A) Heat map showing RNA-seq-based expression analysis of differentially expressed 712 transcripts in non-infected (Ctrl) and A549hACE2 cells infected with SARS-CoV-2 for 713 48h (48h pi). n=44141 differentially expressed transcripts; 2 individual cell replicates per 714 condition. (B) Top. Reactome-based Gene Set Enrichment Analysis (GSEA) of candidates 715 with FC≥2 FDR: False Discovery Rate. (C) Reactome-719 based Gene Set Enrichment Analysis (GSEA) for Rho GTPAses pathway of candidates 720 with FC≥2. (D) Histogram plots representing the basal transcription activity Reduction of SARS-CoV-2 replication in pulmonary cells upon PKN and Rho/SFR 726 inhibitors treatment accompanied with cellular F-actin and cell shape restoration 727 (A) Dose effect of Rho/SFR and PKN inhibitors on SARS-CoV-2 replication in pulmonary 728 cells using qRT-PCR and cell viability. (B) Confocal images of changes SARS-CoV-2 729 infected A549-hACE2 cells with the treatment of Remdesivir (1µM) or PKN Inhibitor 730 (0.5 µM). (C) Plot for changes in viral clustered size with or without (infected) treatment 731 of Remdesivir (1µM) or PKN A549-hACE2 cells were 733 fixed at 48h post infection and processed for immunofluorescence and laser confocal light 734 microscopy using a SARS-CoV-2 membrane protein anti-M rabbit antibody and a 735 secondary antibody Alexa Fluor 568 (in red) and for F-actin imaging Phalloidin Alexa 736 flour 488 (Green) were used