New role of NP Exoribonuclease in Lassa virus infection

Mammarenavirus Lassa virus (LASV) causes Lassa fever (LF), which is endemic in West Africa. Vaccines and treatments for LF are limited. LASV is a Category A Priority Pathogen in the USA and the prototype pathogen in arenavirus family in the Pandemic Preparedness Plan of NIAID. Despite the significant public health risk posed by LASV, critical knowledge gaps exist in LASV basic biology. For RNA virus, the RNA synthesis is error-prone as viral RNA-dependent RNA polymerase (RdRp) lacks proofreading activity. While the high mutation rate of RNA virus provides evolutionary benefits, RNA fidelity is essential for virus fitness and pathogenicity. For some RNA viruses, even a moderate increase in mutation rate may substantially affect virus fitness. As a negative-sense (-) RNA virus, how LASV controls RNA replication error to ensure fitness is unknown. The fidelity of RNA virus is largely governed by viral RdRp. Interestingly, the preliminary data indicate that LASV utilizes a protein other than RdRp to reduce nucleotide substitution in LASV RNA, which is arguably the first example in (-) RNA viruses. Specifically, LASV NP has a DEDDh 3’-5’ exoribonuclease motif (ExoN) that has been known key to LASV immune suppression via dsRNA degradation. Abrogation of NP ExoN led to increased nucleotide substitution in LASV genomic RNA, higher sensitivity to mutagenic nucleoside analogues, increased non-standard viral genomic RNAs (nsVG RNAs) production and impaired LASV RNA replication. Furthermore, serial passaging of ExoN-deficient LASV led to improved virus titer and RNA level, which was associated with putative compensatory mutations in RdRp motif G and the Cap-Binding Domain (CBD). The virus passaging data indicate that LASV NP ExoN and RdRp collaborate to ensure optimal viral RNA replication and fitness. The function of LASV RdRp motif G and CBD, and more broadly, the function of motif G of (-) RNA virus are largely unknown. In (+) RNA virus picornavirus RdRp, the ortholog motif G regulates RdRp translocation on viral template RNA and promotes RNA synthesis efficiency and fidelity. Our working model is that LASV NP ExoN motif has novel function that ensures viral RNA fidelity, reduces nsVG RNAs production and promotes viral RNA replication efficiency and fitness. In this project, we will: 1). leverage relevant animal models to investigate the impact of abrogation of NP ExoN on LASV RNA mutation, nsVG RNAs production and pathogenicity in vivo; 2). investigate whether the RdRp mutations selected during serial passaging could improve ExoN- rLASV RNA synthesis efficacy and RNA fidelity; 3) perform biochemical studies to investigate the mechanism underlying the role of NP ExoN in reducing RNA substitution. This project may define previously unrecognized roles of NP ExoN in LASV infection in vitro and in vivo and provide insights into the function of LASV RdRp motif G and CBD domain. LASV NP may be the first example in (-) RNA viruses that a viral protein other than RdRp contributes to RNA fidelity. Thus, this project may advance our understanding of arenavirus basic biology and move the field forward. Targeting LASV NP ExoN may be a novel approach for development of LF treatments and vaccines.