Interférence entre les virus Chikungunya et Dengue pour l'utilisation de voies cellulaires communes chez les insectes vecteurs lors de co-infection

Emergence and geographical extension of dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses increase simultaneous outbreak in an increasing number of countries. To date, no vaccine or cure have yet been developed against these diseases those cause a tremendous impact on human health and in the economy worldwide. During recent simultaneous outbreaks, up to 12% of patients have been diagnosed to be co-infected by CHIKV and DENV. In addition, it was shown that the mosquitoes Aedes albopictus could carry and transmit simultaneously CHIKV and DENV. However, the pathology, as well as the epidemiology of a pathogen, relies on the interactions between several infectious agents present within an organism or a community in the environment. It is crucial to consider to which extent a host infected by a first microorganism is modified and whether its reaction to the infection by a second microorganism is consequently altered. However, there is no extensive report of Alphavirus-Flavivirus or Flavivirus- Flavivirus interactions. Our global objective is to characterize these co-infections in both mosquitoes and humans, at the cell and molecular level. To this aim, we started this project by performing sequential co- infection in different cell lines from Aedes albopictus and Aedes aegypti. We found that the permissiveness and production of DENV is enhanced in presence of CHIKV. On the contrary, there is no effect of DENV pre-infection on subsequent CHIKV co-infection. We generalized the synergistic phenomena and we showed that CHIKV pre-infection also increased the infection by DENV-1, DENV-3 and DENV-4, but also by two others re-emerging Flaviviruses, the Yellow Fever Virus (YFV), and the Zika Virus (ZIKV). Remarkably, we succeeded to establish a mosquito model of co-infection of Aedes aegypti mosquito after by different two feedings at 4 days interval. Using this sequential co-infection, we were able to show that a pre-infection of Aedes aegypti by CHIKV increase the level of DENV-2 RNA in salivary glands compare to mono-infected mosquitos. This phenotype is reminiscent of the phenotype we observed in vitro during successive infections. Altogether, our study paves the way to the characterization of molecular interaction between Flaviviruses and Alphaviruses in mosquito in vitro and in vivo. This study can be crucial for a better understanding of disease and epidemiology during simultaneous outbreaksAu cours des dernières années, de nombreuses épidémies ont emergé ou ré émergé, et sont causées par des arbovirus (arthropod-borne viruses), des virus transmis à des vertébrés par des insectes piqueurs vecteurs. Avec l'augmentation de la densité humaine dans certaines zones géographiques et le réchauffement climatique qui contribuent à l'expansion géographique des vecteurs, les maladies induites par ces virus (arboviroses) ont un impact de plus en plus important sur la santé humaine et l'économie mondiale. Il est donc déterminant d'augmenter nos connaissances sur les systèmes mis en jeux pour garantir la sécurité sanitaire des populations exposées. Les enjeux actuels reposent aussi bien sur la compréhension des virus que sur la compréhension de l'alternance d'hôtes, directement responsables de l'émergence et la dissémination des agents infectieux. Les moustiques sont des vecteurs majeurs des arbovirus comme la dengue (genre Flavivirus) et le Chikungunya (genre Alphavirus). Transmis par les mêmes moustiques Aedes aegypti et Aedes albopictus, le virus de la Dengue (DENV) est responsable de la plus importante arbovirose en zone tropicale, et le virus Chikungunya (CHIKV) est responsable dans le monde entier de centaines de milliers de cas d'infection, et les épidémies récentes ont touché les pays européens. Ainsi, il a été observé que le moustique Ae. albopictus pouvait porter simultanément CHIKV et DENV, et des cas de co-infections humaines ont été observés en Afrique. Toutefois, bien qu'en théorie les deux virus soient capables d'infecter les mêmes cellules chez l'insecte ou l'homme, il n'y a aucune étude détaillée sur les interactions au niveau cellulaire entre CHIKV et DENV lors de la co-infection d'une cellule. C'est pourquoi il est indispensable d'accroitre nos connaissances sur l'interférence éventuelle entre les virus Chikungunya et Dengue pour l'utilisation de voies cellulaires communes chez les insectes vecteurs et l'hôte humain lors de co-infectio

The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity

International audienceCrimean-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

Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes.

International audienceArthropod-borne virus (arbovirus) infections cause several emerging and resurgent infectious diseases in humans and animals. Chikungunya-affected areas often overlap with dengue-endemic areas. Concurrent dengue virus (DENV) and chikungunya virus (CHIKV) infections have been detected in travelers returning from regions of endemicity. CHIKV and DENV co-infected Aedes albopictus have also been collected in the vicinity of co-infected human cases, emphasizing the need to study co-infections in mosquitoes. We thus aimed to study the pathogen-pathogen interaction involved in these co-infections in DENV/CHIKV co-infected Aedes aegypti mosquitoes. In mono-infections, we detected CHIKV antigens as early as 4 days post-virus exposure in both the midgut (MG) and salivary gland (SG), whereas we detected DENV serotype 2 (DENV-2) antigens from day 5 post-virus exposure in MG and day 10 post-virus exposure in SG. Identical infection rates were observed for singly and co-infected mosquitoes, and facilitation of the replication of both viruses at various times post-viral exposure. We observed a higher replication for DENV-2 in SG of co-infected mosquitoes. We showed that mixed CHIKV and DENV infection facilitated viral replication in Ae. aegypti. The outcome of these mixed infections must be further studied to increase our understanding of pathogen-pathogen interactions in host cells

A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism.

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses

BIS uncovers a coevolving signal between E1 and the Stem region that regulate HCV fusion.

<p>(<b>A</b>) Position of the three amino acid residues that differs between H77 (blue) and A40 (red) and are hypothesized to coevolve according to BIS prediction (gt1a cluster 5). The three H77 amino acids will be replaced by A40 residues individually or altogether to challenge BIS prediction. (<b>B</b>) Impact of the E1/Stem coevolution signal on HCV entry. Infectious titers of HCVpp viral particles harboring H77 (blue), A40 (red) and H77/A40 E1E2 chimera were determined. Two E1 H77 residues (S112, I117), a single H77 E2 residue (D462) or both (S112, I117, D462) were introduced into E1E2 A40. The different envelopes were incorporated at the surface of HCVpp, then used to infect Huh7.5. Infectious titers were quantified 72h post infection by flow cytometry (mean ± SD; n = 3). *<i>p</i><0.05, ns non-significant. (<b>C</b>) H77/A40 E1 and E2 chimera expression and incorporation onto HCVpp. Expression in transfected 293T cells (Cell lysates) and incorporation onto concentrated pseudoparticles (Viral Pellets) of E1 and E2 from the different H77/A40 chimera. Detection of E1 and E2 onto pseudoparticles harboring no envelope glycoproteins was used as negative control. MLV-Capsid (CA) was detected to control equivalent HCVpp production between chimera. (<b>D</b>). Impact of the E1/Stem coevolution signal on HCV fusion. LTRhiv-luciferase vector transduced 293T cells expressing the different E1E2 H77/A40 chimeric envelope glycoproteins were co-cultured with Tat-expressing Huh7.5 cells. Co-cultured cells were exposed to an acid shock (pH5, orange) or not (pH7, red) and luciferase activities were determined 72h post-exposure. Results are presented in relative light units (RLU) for each experimental condition (mean ± SD; n = 3). *<i>p</i><0.05, ***<i>p</i><0.001.</p

BIS as a methodology to decrypt virus entry mechanisms.

<p>Schematic representation of the experimental approach employed in this study, from BIS computational analysis to the design and challenge of a mechanistic model of viral fusion. Following sequence analysis, matrix of E1E2 amino-acid coevolution were generated by BIS for different HCV genotypes. Plotting of matrix coevolution networks onto E2core structure unveiled a potential scenario of E1 and E2 rearrangements during HCV fusion, which involved the BL domain of E2. At the protein domain level, the construction of a soluble form of the BL and the conduction of several experimental assays supported such hypothesis. In parallel, at the amino acid level, the experimental validation of coevolution signals between specific residues of E1 and of the BL highlighted the critical role of E1-BL networks in regulating fusogenic rearrangements (and more generally, the critical role of coevolving networks between E1 and E2 C-terminal regions). Altogether, this approach allows us to propose a HCV fusion model where BL movements and E1 refolding are critical in the induction of E1E2 interdependent, fusogenic rearrangements. By being applicable to other viral proteins and viruses, such approach provides opportunities to uncover undescribed viral-mediated mechanisms and design innovative translational strategies for their inhibition.</p

BIS analysis of dengue E-Pr coevolving residues.

<p>(<b>A</b>) Tridimensional representation of DENV Pr (Black, PDB 3C6R) and E (multi-color, PDB 1K4R). A linear representation of the PrM-E polyprotein is depicted below the protein structures. Starting and ending residue positions of each protein (Pr, M and E) and E domain are indicated. E domains are annotated by distinct colors: DI, domain I (red); DII, domain II (yellow); DIII, domain III (blue); Tmd, transmembrane (black). (<b>B</b>) Organization and positions of the PrM-E cluster 2 (orange), 7 (blue) and 9 (pink) on tridimensional representations of the DENV E and Pr proteins. Cluster 2 and 9 are depicted on a dimeric or trimeric E-Pr structure respectively at low pH condition (PDB 3C6R). Cluster 7 is depicted on a trimeric E-Pr structure at neutral pH condition (PDB 3XIY). Linear representation of the two proteins are also depicted on the top of each structure and cluster block location are indicated (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s003" target="_blank">S1 Table</a></b>). The close proximity between DENV E and Pr cluster 2 blocks is enlarged.</p

BIS analysis of dengue E coevolving networks during pre- and post-fusion steps.

<p>(<b>A</b>) Linear representation of Dengue E protein. Starting and ending residue positions of each E domain are indicated. E domains are annotated by distinct colors: DI, domain I (red); DII, domain II (yellow); DIII, domain III (blue); Tmd, transmembrane (black). (<b>B</b>) Organization and positions of the DENV E cluster 8 blocks (dark blue) on a mature E dimeric structure (PDB 1K4R). A linear representation of E is depicted and cluster block locations are indicated (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s004" target="_blank">S2 Table</a></b>). Cluster 8 coevolving residues located in areas where the two E monomers are in close contact are enlarged (green square). Structural proximity of two-coevolving cluster 8 internal loops located into two distinct Domain II (DII) sub-domains on the linear structure is also highlighted (red square). (<b>C</b>) Positions of E cluster 5 (red) and 10 (green) blocks on a mature tridimensional E monomer at a pre-fusion state (left, PDB 1K4R) and post-fusion state (right, PDB 1OK8). Stem and transmembrane domains are represented by a grey dotted line. A linear representation of E and cluster block locations are indicated for each cluster (precise cluster positions are reported in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.s004" target="_blank">S2 Table</a></b>). At the top of the panel, a schematic represents the current experimentally-validated fusion model of DENV, and how DENV E rearranges during this process. DENV E domains are colored into distinct colors (red, domain I; yellow, domain II; blue, domain III) as in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g001" target="_blank">Fig 1A</a></b>.</p

A transmembranous form of BLd-H77 inhibits HCV infection.

<p>(<b>A</b>) BLd acts onto viral particles. H77 HCVpp or H77/JFH-1 HCVcc particles were pre-incubated with BLd-H77 (50μg/ml or 35μg/ml, respectively) or with PBS. Prior to infection of Huh7.5 cells, HCV particles and BLd-H77 mixes were diluted (1/5; +) or not (-) with cell culture media resulting in a BLd-H77 concentrations inhibiting infectivity by less than 20% (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g004" target="_blank">Fig 4F</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006908#ppat.1006908.g004" target="_blank">Fig 4H</a></b>). Viral titers of primary infection were determined 72h (HCVpp) or 4 days (HCVcc) post-infection and expressed as percentages of infection according to PBS control experiments (mean ± SD; n = 3). Statistical significances <i>(</i>*<i>p</i><0.05, ***<i>p</i><0.001) were determined for each experimental condition versus control condition (100%). (<b>B</b>) Schematic representation of the BLd-H77 transmembrane form (BLd-tm) and its possible mode of action. An IgG2 hinge region (Hinge) and the transmembrane region of the CD34 molecule (MSD) were added to the C-terminal of BLd-H77. SP, signal peptide. (<b>C</b>) A BLd-H77 transmembranous form inhibits HCVcc infection. H77/JFH-1 HCVcc were used to infect non-transduced Huh7.5, Huh7.5-C46 (C46) and Huh7.5-BLd-tm (BLd-tm). Four days post-infection, FFU titers were determined for each cell type and percentages of primary infection were normalized according to the viral titers determined following Huh7.5 infection (mean ± SD; n = 3). Statistical significances <i>(</i>***<i>p</i><0.001, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>D</b>). Propagation of HCVcc viral particles in Huh7.5-BLd-tm cell cultures. Huh7.5 and Huh7.5 BLd-tm cells were infected with HCVcc H77/JFH-1 at a m.o.i. of 0.1. At day, 1, 3 and 5 post-infection, cell culture supernatants were harvested and used to infect naïve Huh7.5 cells. Viral titers of secondary infection were determined by NS5A immunostaining four days post-infection (mean ± SD; n = 3). (<b>E</b>) BLd-tm inhibits cell entry. Non-transduced Huh7.5 and Huh7.5-BLd-tm were infected with HCVpp H77 and VSVpp. 72h post infection, amount of GFP positive cells were quantified. Percentage of infection of Huh7.5-BLd-tm is normalized for each type of particle on the percentage of infection of non-transduced Huh7.5 (mean ± SD; n = 3). Statistical significances <i>(</i>**<i>p</i><0.01, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>F</b>) Soluble E2 binding to Huh7.5-BLd-tm cells. Different doses of soluble E2 were mixed with different concentrations of Huh7.5 and Huh7.5-BLd-tm cells and E2 binding was quantified by flow cytometry. Results represent E2 ability to bind Huh7.5-BLd-tm cells for each condition relatively to the basal E2 ability to bind Huh7.5 cells (determined as 0% binding) for the same condition (mean ± SD; n = 3). *<i>p</i><0.05. (<b>G</b>) Interaction between BLd-H77 and sE2 detected by ELISA. Different amounts of mouse IgG isotype, AR3B and BLd-H77 were coated overnight into 96-well plates. Coated peptides and antibodies were then incubated with 10ng of soluble E2 (sE2) or not. After washing, soluble E2 was detected using the rat anti-E2 antibody 3/11 and an anti-rat HRP antibody. After measurement of the optical density (O.D.) at 450nm, relative E2 binding was determined by calculating the ratio of O.D. between condition with 10ng of sE2 and no sE2, for each coating condition (mean ± SD; n = 3). **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001, ns non-significant. (<b>H</b>) BLd-H77 does not affect HCVcc binding. JC1 HCVcc particles were pre-incubated with BLd-H77 (35 μg/ml), Heparine (250 μg/ml) or PBS and mixed with Huh7.5 cells for 2h at 4. After washing, amounts of cell-associated viral particles were determined by RT-qPCR (mean ± SD; n = 3). Data are shown as percentage of binding, according to binding of HCVcc particles in control condition (PBS). Statistical significances <i>(</i>*<i>p</i><0.05, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>I</b>) BLd-H77 can inhibit HCV entry following particle binding. HCVpp particles (HCVpp-H77) were incubated with Huh7.5 in presence of BLd-H77 (50μg/ml) or AR4A (25 μg/ml) during binding (1h at 4°C; 2), entry (4h at 37°C following binding; 3), or following entry (72h at 37°C following media change; 4). GFP levels were quantified 72h post infection. Huh7.5 infected in a similar manner but non-treated with BLd-H77 or AR4A were used as control (1). Percentages of infection were calculated based on viral titer obtained from control conditions. (mean ± SD; n = 3). Statistical significances <i>(</i>***<i>p</i><0.001, ns non-significant) were determined for each experimental condition versus control condition (100%). (<b>J</b>) Effect of BLd-H77 on cell-cell fusion. LTR-luciferase-transduced 293T cells expressing HCV-H77 E1E2 or VSVG glycoproteins were co-cultivated with Tat-expressing Huh7.5 cells. Following pre-incubation with PBS or with 50μg/ml of BLd-H77, co-cultured cells were exposed to an acid shock (pH5) or not (pH7) and luciferase activities were determined 72h post-exposure. Percentage of fusion of HCV and VSVG glycoproteins at pH5 between control (PBS) or BLd-H77 are indicated (mean ± SD; n = 3). Statistical significances <i>(**p</i><0.01, ***<i>p</i><0.001) were determined for each experimental condition versus control condition (100%). (<b>K</b>) Effect of BLd-H77 on virus-liposome fusion assays. H77 HCVpp particles were pre-incubated with different dose of BLd-H77 (50μg/ml, green; 100 μg/ml, blue; 150 μg/ml, red) or not (PBS, black), and mixed with R18-labelled liposomes. Dequenching of R18 was quantified following sample acidification (pH5). Data are represented as non-linear polynomial fitted curves for each experimental condition and display the evolution of the fusion rate (%) over time. Curves are representative of three independent experiments.</p