The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity Natalia Freitas, Margot Enguehard, Solène Denolly, Camille Levy, Gregory Neveu, Solène Lerolle, Stephanie Devignot, Friedemann Weber, Eric Bergeron, Vincent Legros, et al. To cite this version: Natalia Freitas, Margot Enguehard, Solène Denolly, Camille Levy, Gregory Neveu, et al.. The in- terplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity. PLoS Pathogens, Public Library of Science, 2020, 16 (9), pp.e1008850. 10.1371/journal.ppat.1008850. inserm-02966583 HAL Id: inserm-02966583 https://www.hal.inserm.fr/inserm-02966583 Submitted on 14 Oct 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. PLOS PATHOGENS RESEARCH ARTICLE The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment- encoded accessory proteins and structural proteins promote virus assembly and infectivity 1 1 1 1 Natalia FreitasID *, Margot Enguehard , Solène DenollyID , Camille Levy , 1 1 2 2 a1111111111 Gregory NeveuID , Solène LerolleID , Stephanie DevignotID , Friedemann WeberID , 3 1,4 1 a1111111111 Eric BergeronID , Vincent LegrosID , FrancËois-Loïc CossetID * a1111111111 a1111111111 1 CIRI±Centre International de Recherche en Infectiologie, Univ Lyon, Universite Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, 46 alleÂe d'Italie, Lyon, France, 2 Institute for Virology, FB10- a1111111111 Veterinary Medicine, Justus-Liebig University, Gieûen, Germany, 3 Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 4 Universite de Lyon, VetAgro Sup, Marcy-l'E toile, France * [email protected] (NF); [email protected] (FLC) OPEN ACCESS Citation: Freitas N, Enguehard M, Denolly S, Levy C, Neveu G, Lerolle S, et al. (2020) The interplays Abstract between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has and structural proteins promote virus assembly become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA and infectivity. PLoS Pathog 16(9): e1008850. genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor gen- https://doi.org/10.1371/journal.ppat.1008850 erates the two envelope glycoproteins (GPs) as well as three secreted nonstructural pro- Editor: Jens H. Kuhn, Division of Clinical Research, teins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like UNITED STATES protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined Received: March 13, 2020 the relevance of each M-segment non-structural proteins in virus assembly, egress and Accepted: August 1, 2020 infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of Published: September 21, 2020 MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The Copyright: This is an open access article, free of all copyright, and may be freely reproduced, loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, distributed, transmitted, modified, built upon, or showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent otherwise used by anyone for any lawful purpose. with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans The work is made available under the Creative Commons CC0 public domain dedication. with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in parti- cle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the Data Availability Statement: All relevant data are within the manuscript and its Supporting ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm dele- Information files. tion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle for- Funding: This work performed in the laboratory of mation and secretion. NSm expression affected N-glycosylation of different viral proteins FLC was supported by the French National most likely due to increased speed of trafficking through the secretory pathway. This high- Research Agency (ANR), the IDEXLYON (ANR-16- lights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. IDEX-0005) and the LabEx Ecofect (ANR-11-LABX- Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle pro- 0048) of the ªUniversite de Lyonº (http://ecofect. universite-lyon.fr/), within the program duction and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and "Investissements d'Avenir" (ANR-11-IDEX-0007) PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008850 September 21, 2020 1 / 29 PLOS PATHOGENS Role of non-structural proteins in CCHFV assembly, release and infectivity operated by the ANR. VL received financial support Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and from the "IDEXLYON IMPULSION 2019" project virion secretion. (ANR-16-IDEX-0005) of the ``Universite de Lyonº, within the program ``Investissements d'Avenirº (ANR-16-IDEX-0005) and from the "Global Health Impulsion 2020" project of VetAgro Sup. FW is supported by the CCHVaccine consortium that has Author summary received funding from the European Union's Horizon 2020 research and innovation program Orthonairoviruses, like the lethal Crimean-Congo hemorrhagic fever virus (CCHFV), under grant agreement no. 732732, and by the encode secreted glycoproteins, such as GP38, in addition to virion envelope glycoproteins Swedish Research Council (Vetenskapsrådet, VR) (Gn and Gc) that are processed by internal cleavage of the viral M segment encoded poly- section Research Environment, Infection Biology (contract number 2018-05766). The funders had protein. CCHFV MLD-GP38 proteins (GP160/GP85) also include an N-terminal domain no role in study design, data collection and encompassing a mucin-like protein that is released from GP38 by Furin. The protective analysis, decision to publish, or preparation of the effect of non-neutralizing monoclonal antibodies targeting GP38 against lethal CCHFV manuscript. challenge previously highlighted the importance of GP38 in CCHFV replication. CCHFV Competing interests: The authors have declared also encodes a double-membrane-spanning protein (NSm) of unknown function, located that no competing interests exist. between the Gn and Gc on the polyprotein. To investigate the roles of these so-called accessory proteins encoded by the CCHFV M-segment in virus formation and infectivity, we generated several M-segment deletion mutants and tested them in a CCHFV transcrip- tion-entry-competent virus-like particle (tc-VLP) system. Here, we demonstrate that GP38 is crucial for Gc biogenesis, interaction with Gn and trafficking to the Golgi, and that its deletion abrogates formation of infectious particles. We also show that NSm increases the rate of protein trafficking through the secretory pathway with altered N-gly- cosylation profiles that are advantageous for efficient virus release. These data advanced our understanding of GP38 and NSm roles and CCHFV-host interactions. Introduction The genus Orthonairovirus belongs to the family Nairoviridae of the order Bunyavirales and consists of at least 43 viruses assigned to 15 groups/species [1]. They are tick-borne viruses that circulate in nature in enzootic cycles involving ticks and vertebrates that can act as amplifying hosts [2±4]. Ticks are considered as both the vector and the main reservoir, and transmit the virus when feeding on animals [5]. The genus is named after the Nairobi sheep disease virus (NSDV), responsible for often-fatal acute hemorrhagic gastroenteritis in sheep and goats. Its most significant human pathogen, is the Crimean-Congo hemorrhagic fever virus (CCHFV), producing a severe hemorrhagic disease with a mortality rate of up to 30%. Because of its epi- demic potential, the high rate of mortality, the possibility of nosocomial infections, and lack of specific treatment options and preventive strategies, CCHFV poses a serious threat to the world health [6]. CCHFV activity closely mirrors the geographic distribution of its vectors, and since 2000, the incidence and range of confirmed CCHF cases have markedly increased, probably due to the geographical expansion of Hyalomma spp. ticks, an important tick vector of CCHFV. CCHFV, like all orthonairoviruses, carries a tri-segmented negative single stranded RNA genome in association with virus-encoded nucleoprotein (NP) and RNA-dependent