Lassa virus NP DEDDh 3'-5' exoribonuclease activity is required for optimal viral RNA replication and mutation control.

Lassa virus (LASV), a mammarenavirus from , is the causative agent of Lassa fever (LF) endemic in West Africa. Currently, there are no vaccines or antivirals approved for LF. The RNA-dependent RNA polymerases (RdRp) of RNA viruses are error-prone. As a negative-sense RNA virus, how LASV copes with errors in RNA synthesis and ensures optimal RNA replication are not well elucidated. LASV nucleoprotein (NP) contains a DEDDH 3'-to-5' exoribonuclease motif (ExoN), which is known to be essential for LASV evasion of the interferon response via its ability to degrade virus-derived double-stranded RNA. Herein, we present evidence that LASV NP ExoN has an additional function important for viral RNA replication. We rescued an ExoN-deficient LASV mutant (ExoN- rLASV) by using a reverse genetics system. Our data indicated that abrogation of NP ExoN led to impaired LASV growth and RNA replication in interferon-deficient cells as compared with wild-type rLASV. By utilizing PacBio Single Molecule, Real-Time (SMRT) long-read sequencing technology, we found that rLASV lacking ExoN activity was prone to producing aberrant viral genomic RNA with structural variations. In addition, NP ExoN deficiency enhanced LASV sensitivity to mutagenic nucleoside analogues in virus titration assay. Next-generation deep sequencing analysis showed increased single nucleotide substitution in ExoN- LASV RNA following mutagenic 5-flurouracil treatment. In conclusion, our study revealed that LASV NP ExoN is required for efficient viral RNA replication and mutation control. Among negative-sense RNA viruses, LASV NP is the first example that a viral protein, other than the RdRp, contributes to reduce errors in RNA replication and maintain genomic RNA integrity. These new findings promote our understanding of the basics of LASV infection and inform antiviral and vaccine development.