Search

The arenavirus nucleoprotein (NP) plays an important role in the virus’ ability to block interferon (IFN) production, and its exonuclease function appears to contribute to this activity. However, efforts to analyze this contribution are complicated by the functional overlap between the exonuclease active site and a neighboring region involved in IKKε-binding and subsequent inhibition of IRF3 activation, which also plays an important role in IFN production. To circumvent this issue, we mutated a residue located away from the active site that is involved in binding of the dsRNA substrate being targeted for exonuclease digestion, i.e. H426A. We found that expression of Tacaribe virus (TCRV) NP containing this RNA-binding H426A mutation was still able to efficiently block IFN-β promoter activity in response to Sendai virus infection, despite being strongly impaired in its exonuclease activity. This was in contrast to a conventional exonuclease active site mutant (E388A), which was impaired with respect to both exonuclease activity and IFN antagonism. Importantly, growth of a recombinant virus encoding the RNA-binding mutation (rTCRV-H426A) was similar to wild-type in IFN-deficient cells, unlike the active site mutant (rTCRV-E388A), which was already markedly impaired in these cells. Further, in IFN-competent cells, the TCRV-H426A RNA-binding mutant showed more robust growth and delayed IFN-β mRNA upregulation compared to the TCRV-E388A active site mutant. Taken together, this novel mutational approach, which allows us to now dissect the different contributions of the NP exonuclease activity and IKKε-binding/IRF3 inhibition to IFN antagonism, clearly suggests that conventional exonuclease mutants targeting the active site overestimate the contribution of the exonuclease function, and that rather other IFN antagonistic functions of NP play the dominant role in IFN-antagonism.

Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species. Fil: Leske, Anne. Friedrich Loeffler Institut; Alemania Fil: Waßmann, Irke. Friedrich Loeffler Institut; Alemania Fil: Schnepel, Kevin. Friedrich Loeffler Institut; Alemania Fil: Shifflett, Kyle. National Institutes of Health; Estados Unidos Fil: Holzerland, Julia. Friedrich Loeffler Institut; Alemania Fil: Bostedt, Linus. Friedrich Loeffler Institut; Alemania Fil: Bohn, Patrick. Friedrich Loeffler Institut; Alemania Fil: Mettenleiter, Thomas C.. Friedrich Loeffler Institut; Alemania Fil: Briggiler, Ana Maria. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina Fil: Brignone, Julia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina Fil: Enria, Delia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina Fil: Cordo, Sandra Myriam. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hoenen, Thomas. Friedrich Loeffler Institut; Alemania. National Institutes of Health; Estados Unidos Fil: Groseth, Allison. Friedrich Loeffler Institut; Alemania. National Institutes of Health; Estados Unidos