Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of 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 loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion. Orthonairoviruses, like the lethal Crimean-Congo hemorrhagic fever virus (CCHFV), encode secreted glycoproteins, such as GP38, in addition to virion envelope glycoproteins (Gn and Gc) that are processed by internal cleavage of the viral M segment encoded polyprotein. CCHFV MLD-GP38 proteins (GP160/GP85) also include an N-terminal domain encompassing a mucin-like protein that is released from GP38 by Furin. The protective effect of non-neutralizing monoclonal antibodies targeting GP38 against lethal CCHFV challenge previously highlighted the importance of GP38 in CCHFV replication. CCHFV also encodes a double-membrane-spanning protein (NSm) of unknown function, located 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 transcription-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-glycosylation profiles that are advantageous for efficient virus release. These data advanced our understanding of GP38 and NSm roles and CCHFV-host interactions.

The Bunyavirales order comprises more than 500 viruses (generally defined as bunyaviruses) classified into 12 families. Some of these are highly pathogenic viruses infecting different hosts, including humans, mammals, reptiles, arthropods, birds, and/or plants. Host cell sensing of infection activates the innate immune system that aims at inhibiting viral replication and propagation. Upon recognition of pathogen-associated molecular patterns (PAMPs) by cellular pattern recognition receptors (PRRs), numerous signaling cascades are activated, leading to the production of interferons (IFNs). IFNs act in an autocrine and paracrine manner to establish an antiviral state by inducing the expression of hundreds of IFN-stimulated genes (ISGs). Some of these ISGs are known to restrict bunyavirus infection. Along with other constitutively expressed host cellular factors with antiviral activity, these proteins (hereafter referred to as “restriction factors”) target different steps of the viral cycle, including viral entry, genome transcription and replication, and virion egress. In reaction to this, bunyaviruses have developed strategies to circumvent this antiviral response, by avoiding cellular recognition of PAMPs, inhibiting IFN production or interfering with the IFN-mediated response. Herein, we review the current knowledge on host cellular factors that were shown to restrict infections by bunyaviruses. Moreover, we focus on the strategies developed by bunyaviruses in order to escape the antiviral state developed by the infected cells.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus that causes high mortality in humans. This enveloped virus harbors two surface glycoproteins (GP), Gn and Gc, that are released by processing of a glycoprotein precursor complex whose maturation takes place in the ER and is completed through the secretion pathway. Here, we characterized the trafficking network exploited by CCHFV GPs during viral assembly, envelopment, and/or egress. We identified membrane trafficking motifs in the cytoplasmic domains (CD) of CCHFV GPs and addressed how they impact these late stages of the viral life cycle using infection and biochemical assays, and confocal microscopy in virus-producing cells. We found that several of the identified CD motifs modulate GP transport through the retrograde trafficking network, impacting envelopment and secretion of infectious particles. Finally, we identified PACS-2 as a crucial host factor contributing to CCHFV GPs trafficking required for assembly and release of viral particles.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral disease with a mortality rate reaching up to 40% in humans. Currently, CCHF affects three continents: Asia, Europe, and Africa. An increase in confirmed cases in Africa has been observed since 2000. In Central Africa, several countries have reported the circulation of CCHV virus (CCHFV). However, in Gabon, there is a lack of recent data on the circulation of the virus in the Gabonese population. To provide an overview of the epidemiological situation in Gabon, we tested 3,081 human serum samples collected between 2005 and 2008 in villages throughout the country for anti-CCHFV antibodies. Using a double-antigen ELISA kit, our study found 15/3,081 samples positive for CCHFV. These positive samples were also tested using the Blackbox CCHFV IgG kit and the Luminex technique. These analyses confirmed seven and four positives for the Blackbox CCHFV IgG kit and the Luminex technique, respectively. This study suggests low circulation of CCHFV in the rural human population of Gabon. Competent authorities must survey CCHFV to identify and prevent clinical cases in the human population.

Identification and characterization of interferon-stimulated genes modulating Crimean-Congo hemorrhagic fever virus infection: Le virus de la fièvre hémorragique de Crimée-Congo (CCHFV) est un virus émergent zoonotique transmis par les tiques responsable chez l'homme d'une maladie hémorragique ayant un taux de mortalité de 10 à 40 %, sans traitement ni vaccin spécifique. À ce jour, les interactions entre le CCHFV et l'immunité innée sont mal connues. Notamment, l'interféron de type I (IFN) joue un rôle crucial dans l'établissement d'un état antiviral contre le CCHFV, probablement par la transcription de gènes stimulés par l'IFN (ISGs) présentant des propriétés antivirales. Cependant, il reste à déterminer quels ISGs modulent l'infection par le CCHFV et leurs mécanismes d'action. Nous avons réalisé un crible en gain de fonction en utilisant des pseudoparticules virales compétentes pour l'entrée et la transcription d'un minigénome rapporteur (tc-VLPs), un modèle d’étude du CCHFV, et des vecteurs lentiviraux codant individuellement >500 ISGs humains, >350 ISGs macaques et >250 ISGs bovins. Nous avons identifié plusieurs ISGs qui modulent négativement ou positivement l'infection par les tc-VLPs. Pour définir leurs mécanismes d'action, nous avons disséqué leurs impacts sur les différentes étapes du cycle viral à l'aide de diverses méthodes. Nous avons identifié des ISGs inhibant l’entrée virale et une protéine de liaison au GTP qui promeut la réplication et/ou la transcription des tc-VLPs. De plus, les effets de la surexpression de ces ISGs ont été étudiés dans le contexte de l'infection par le virus sauvage. Dans l'ensemble, ces résultats fournissent de nouvelles données sur l'interaction du CCHFV avec la réponse immunitaire innée de l'hôte et ouvrent la voie à des stratégies thérapeutiques.