Lassa Virus Nucleoprotein Mutants Generated by Reverse Genetics Induce a Robust Type I Interferon Response in Human Dendritic Cells and Macrophages

Abstract

Lassa virus (LASV; Arenaviridae) is responsible for severe hemorrhagic fevers in Africa. LASV nucleoprotein (NP) plays important roles in regulating viral transcription and replication and in inhibiting type I interferon (IFN) production. The NP C-terminal domain contains a 3-to-5 exonuclease activity involved in suppressing IFN induction. We have established a murine polymerase (Pol) I reverse genetics system for LASV, showing that residues D389 and G392 of NP were critical for LASV viability, while the D389A/G392A and D389T/392A double mutants were severely altered in the ability to suppress IFN in macrophages and dendritic cells. Assessing their attenuation in vivo may open new perspectives in vaccinology.Lassa fever is a viral hemorrhagic fever caused by an Old World arenavirus, Lassa virus (LASV; family Arenaviridae) (45) transmitted by infected Mastomys natalensis, a peridomestic rodent (35). It is a major public health concern in regions of endemicity in West Africa and a threat for importation and misuse as a bioterrorism agent in industrial countries (9). The severity of the disease varies from asymptomatic infection to fatal hemorrhagic fever (19,33,34). Whether infection leads to death seems to depend on host immune responses, although the mechanisms involved remain to be clarified. LASV tropism for antigen-presenting cells (APC), such as dendritic cells (DC) and macrophages (MP), in the early stages of infection probably plays a key role in the defective cellular responses observed for severe cases (5,28,29,51). DC and MP massively release LASV but are not activated and do not produce cytokines, except for a modest type I interferon (IFN) production (5, 7).LASV is enveloped and has two single-stranded RNA genome segments (L and S) of ambisense polarity (9). The L segment codes for the small zinc-finger protein Z and for the RNA-dependent RNA polymerase (Pol) L. The S segment codes for the nucleoprotein (NP) and the precursor to the glycoproteins maturated by subtilase SKI-1/S1P (24) into GP1 and GP2. In each segment, the open reading frames (ORFs) are separated by an intergenic region forming a hairpin structure acting as a transcription termination signal for the mRNA synthesis (25,40,41,47). The two RNA segments contribute to virulence. A reassortant exchanging the L segment of LASV with that of the attenuated Mopeia arenavirus (MOPV) is attenuated and protects guinea pigs against a LASV challenge (26, 27). In the S segment, NP plays a role not only in regulating transcription and replication (41) but also in inhibiting type I IFN production by interfering with IFN regulatory factor 3 (IRF-3) activation (32). Reverse genetics systems have been developed for several arenaviruses: lymphocytic choriomeningitis virus (LCMV) (10,13,14,46), Junin virus (1, 13), Pichinde virus (22), and very recently, Lassa virus (2). Singlecycle LCMV (44)-or Junin-based chimeras (3) have been developed as well. Here, we established a reverse genetics system for LASV, enabling us to determine the role of specific amino ac...

The Exonuclease Domain of Lassa Virus Nucleoprotein Is Involved in Antigen-Presenting-Cell-Mediated NK Cell Responses

“…Infection of APC with rNP-LASV leads to the production of large amounts of type I IFN, whereas this is not the case following infection with rWT-LASV (19). Similarly, the levels of type I IFN produced in NK/APC cocultures were comparable to the level found in APC single cultures (data not shown).…”

“…D389 was previously shown to be involved in the exonuclease activity of NP as it is within the active site, and G392 was found to be crucial for IFN suppression (4,7,8). rNP-LASV, but not the recombinant wild-type virus (rWT-LASV), induces substantial production of type I IFN by DC and M (19). We show here that DC and M infected by rNP-LASV induce strong NK cell activation leading to IFN-␥ secretion.…”

“…Recombinant wild-type LASV (rWT-LASV) and NP-D389A/G392A (rNP-LASV) were generated by reverse genetics as previously described (19) and passaged twice in Vero E6 cells. Viral supernatants were harvested, titrated, and used as the infectious virus stock.…”

Pathogenic Old World Arenaviruses Inhibit TLR2/Mal-Dependent Proinflammatory Cytokines In Vitro

“…Recent in vitro studies implicated the arenavirus NP protein in the blocking of IFN regulatory factor 3 (IRF3), a major downstream regulator of RIG-I-inducible type I IFN responses (41,42). Particularly, it was shown previously that LASV NP D389A/G392A and D389T/G392A mutations were critically involved in the suppression of IFN in murine macrophages and DCs (14).…”

Inhibition of Arenavirus by A3, a Pyrimidine Biosynthesis Inhibitor

“…NP, the most abundant viral protein in both infected cells and virions, together with the L segment and the viral genome RNA forms the viral ribonucleoprotein (vRNP) core, which is active in RNA replication and gene transcription (1,4). The arenavirus NP has been also shown to counteract the type I interferon and inflammatory responses of the host against viral challenge (5)(6)(7)(8).…”