New role of NP Exoribonuclease in Lassa virus infection

Mammarenavirus Lassa virus (LASV) causes Lassa fever (LF) which is endemic in West Africa. Vaccines andtreatments for LF are limited. LASV is a Category A Priority Pathogen in the USA and the prototype pathogen inarenavirus family in the Pandemic Preparedness Plan of NIAID. Despite the significant public health risk posedby LASV critical knowledge gaps exist in LASV basic biology. For RNA virus the RNA synthesis is error-proneas viral RNA-dependent RNA polymerase (RdRp) lacks proofreading activity. While the high mutation rate ofRNA virus provides evolutionary benefits RNA fidelity is essential for virus fitness and pathogenicity. For someRNA 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 RNAvirus is largely governed by viral RdRp. Interestingly the preliminary data indicate that LASV utilizes a proteinother than RdRp to reduce nucleotide substitution in LASV RNA which is arguably the first example in (-) RNAviruses. Specifically LASV NP has a DEDDh 3-5 exoribonuclease motif (ExoN) that has been known key toLASV immune suppression via dsRNA degradation. Abrogation of NP ExoN led to increased nucleotidesubstitution 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 serialpassaging of ExoN-deficient LASV led to improved virus titer and RNA level which was associated with putativecompensatory mutations in RdRp motif G and the Cap-Binding Domain (CBD). The virus passaging data indicatethat LASV NP ExoN and RdRp collaborate to ensure optimal viral RNA replication and fitness. The function ofLASV 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 RNAand promotes RNA synthesis efficiency and fidelity. Our working model is that LASV NP ExoN motif has novelfunction that ensures viral RNA fidelity reduces nsVG RNAs production and promotes viral RNA replicationefficiency and fitness. In this project we will: 1). leverage relevant animal models to investigate the impact ofabrogation of NP ExoN on LASV RNA mutation nsVG RNAs production and pathogenicity in vivo; 2). investigatewhether the RdRp mutations selected during serial passaging could improve ExoN- rLASV RNA synthesisefficacy and RNA fidelity; 3) perform biochemical studies to investigate the mechanism underlying the role of NPExoN in reducing RNA substitution. This project may define previously unrecognized roles of NP ExoN in LASVinfection in vitro and in vivo and provide insights into the function of LASV RdRp motif G and CBD domain. LASVNP 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.