Production of CXC and CC chemokines by human antigen-presenting cells in response to Lassa virus or closely related immunogenic viruses, and in cynomolgus monkeys with lassa fever.

International audienceThe pathogenesis of Lassa fever (LF), a hemorrhagic fever endemic to West Africa, remains unclear. We previously compared Lassa virus (LASV) with its genetically close, but nonpathogenic homolog Mopeia virus (MOPV) and demonstrated that the strong activation of antigen-presenting cells (APC), including type I IFN production, observed in response to MOPV probably plays a crucial role in controlling infection. We show here that human macrophages (MP) produce large amounts of CC and CXC chemokines in response to MOPV infection, whereas dendritic cells (DC) release only moderate amounts of CXC chemokines. However, in the presence of autologous T cells, DCs produced CC and CXC chemokines. Chemokines were produced in response to type I IFN synthesis, as the levels of both mediators were strongly correlated and the neutralization of type I IFN resulted in an inhibition of chemokine production. By contrast, LASV induced only low levels of CXCL-10 and CXCL-11 production. These differences in chemokine production may profoundly affect the generation of virus-specific T-cell responses and may therefore contribute to the difference of pathogenicity between these two viruses. In addition, a recombinant LASV (rLASV) harboring the NP-D389A/G392A mutations, which abolish the inhibition of type I IFN response by nucleoprotein (NP), induced the massive synthesis of CC and CXC chemokines in both DC and MP, confirming the crucial role of arenavirus NP in immunosuppression and pathogenicity. Finally, we confirmed, using PBMC samples and lymph nodes obtained from LASV-infected cynomolgus monkeys, that LF was associated with high levels of CXC chemokine mRNA synthesis, suggesting that the very early synthesis of these mediators may be correlated with a favourable outcome

Relations structure fonction et inhibitions des co-recepteurs CCR5 et CXCR4 dans l'entrée du VIH-1

PARIS7-Bibliothèque centrale (751132105) / SudocCACHAN-ENS (940162301) / SudocSudocFranceF

The exonuclease domain of Lassa virus nucleoprotein is involved in antigen-presenting-cell-mediated NK cell responses

International audienceLassa virus is an Old World Arenavirus which causes Lassa hemorrhagic fever in humans, mostly in West Africa. Lassa fever is an important public health problem, and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), low type I interferon (IFN) production, and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro, including production of IFN-γ and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands major histocompatibility complex (MHC) class I-related chains A and B and that they may produce interleukin-12 (IL-12), IL-15, and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures, suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen-presenting cell-mediated NK cell responses. IMPORTANCE: The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit the type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a recombinant Lassa virus in which the exonuclease functions have been abolished and demonstrated that NK cells are strongly activated and presented effective functions. These results show that the strategy developed by Lassa virus to evade innate immunity is also effective on NK cells, explaining the weak NK cell activation observed with the wild-type virus. By providing a better understanding of the interactions between Lassa virus and the host immune system, these results are important for the field of arenavirus biology and may be useful for a vaccine approach against Lassa fever

Exonuclease domain of the Lassa virus nucleoprotein is critical to avoid RIG-I signaling and to inhibit the innate immune response

International audienceLassa virus (LASV), which causes a viral hemorrhagic fever, inhibits the innate immune response. The exonuclease (ExoN) domain of its nucleoprotein (NP) is implicated in the suppression of retinoic acid-inducible gene I (RIG-I) signaling. We show here that a LASV in which ExoN function has been abolished strongly activates innate immunity and that this effect is dependent on RIG-I signaling. These results highlight the key role of NP ExoN function in the immune evasion that occurs during LASV infection

Role of the cytosolic tails of Rift Valley fever virus envelope glycoproteins in viral morphogenesis

International audienceThe correct folding, heterodimerization and trafficking of Gn/Gc envelope glycoproteins of Rift Valley fever virus, RVFV (Bunyaviridae and Phlebovirus genus) are essential for Golgi assembly and budding of viral particles. The Gn and Gc carboxy-terminus contain a Golgi targeting and an ER-retrieval signal, respectively. We generated RVFV-like particles with mutations in the cytosolic tails of Gn or Gc and identified regions important for release of infectious particles. The role of specific amino-acids in these regions was further investigated by creating recombinant mutant viruses by reverse-genetics. Residues outside the suspected Golgi targeting motif, i.e. the di-lysine K29-K30 motif and the N43, R44 and I46 residues of the Gn cytosolic domain, appeared important for Golgi localization and RNP packaging. Concerning the Gc tail, replacement of K2 or K3 in the di-lysine motif, had a drastic impact on Gn trafficking and induced an important organelle redistribution and cell remodeling, greatly affecting particle formation and release

Non-Pathogenic Mopeia Virus Induces More Robust Activation of Plasmacytoid Dendritic Cells than Lassa Virus

Lassa virus (LASV) causes a viral haemorrhagic fever in humans and is a major public health concern in West Africa. An efficient immune response to LASV appears to rely on type I interferon (IFN-I) production and T-cell activation. We evaluated the response of plasmacytoid dendritic cells (pDC) to LASV, as they are an important and early source of IFN-I. We compared the response of primary human pDCs to LASV and Mopeia virus (MOPV), which is very closely related to LASV, but non-pathogenic. We showed that pDCs are not productively infected by either MOPV or LASV, but produce IFN-I. However, the activation of pDCs was more robust in response to MOPV than LASV. In vivo, pDC activation may support the control of viral replication through IFN-I production, but also improve the induction of a global immune response. Therefore, pDC activation could play a role in the control of LASV infection

Release of chemokines into the supernatants of DC and MP infected with LASV and MOPV.

<p>The levels of CC and CXC chemokines were quantified by ELISA in the supernatants of MP (A) and DC (B) 24 and 72 h after mock (white bars), MOPV (gray bars), and LASV (black bars) infection. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed in pg/ml and ng/ml for CC and CXC chemokines, respectively. Significant differences are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01), and *** (<i>p</i><0.001).</p

Arenaviridae exoribonuclease presents genomic RNA edition capacity.

The Arenaviridae is a large family of viruses causing both acute and persistent infections and causing significant public health concerns in afflicted regions. A “trademark” of infection is the quick and efficient immuno-suppression mediated in part by a 3’-5’ RNA exonuclease domain (ExoN) of the Nucleoprotein (NP). Mopeia virus, the eastern African counterpart of Lassa virus, carries such ExoN domain, but does not suppress the host innate immunity. We have recently reported the crystal structure of the Mopeia virus ExoN domain, which presents a conserved fold and active site. In the present study, we show that the ExoN activity rules out a direct link between ExoN activity and alteration of the host innate immunity. We found that the Arenavirus ExoN, however, is able to excise mis-incorporated bases present at the 3’-end of double stranded RNA. ExoN(-) arenaviruses cultured in cells dampened in innate immunity still replicated in spite of a significant reduction in the viral charge over several passages. The remaining ExoN(-) virus population showed an increased base substitution rate on a narrow nucleotide spectrum, linking the ExoN activity to genome editing. Since, the Arenavirus ExoN belongs to the same nuclease family as that of the nsp14 coronavirus ExoN; which has been recently shown to promote viral RNA synthesis proofreading; we propose that Arenavirus ExoN is involved in a “limited RNA editing” mechanism mainly controlled by structural constraints and a low mutational/fitness ratio

Production of chemokines by APC infected with recombinant LASV.

<p>The production of CC and CXC chemokines by DC (A, B) and MP (C, D) was assessed after mock infection (white bars), or infection with recombinant wild-type LASV (gray bars) or rLASV NP-D389A/G392A (rNP LASV) (black bars). (A, C) The synthesis of mRNAs was analyzed by RT-qPCR 24 h after infection. (B, D) The protein levels released in the supernatants were quantified by ELISA 24 h (only for CXCL9, 10 and 11) and 48 h after infection. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± SE of 4 (A, C) and 3–7 (B, D) independent experiments. Significant differences are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (p<0.001).</p