Single Nucleoprotein Residue Modulates Arenavirus Replication Complex Formation

ABSTRACT The Arenaviridae are enveloped, negative-sense RNA viruses with several family members that cause hemorrhagic fevers. This work provides immunofluorescence evidence that, unlike those of NewWorld arenaviruses, the replication and tran-scription complexes (RTC) of lymphocytic choriomeningitis virus (LCMV) colocalize with eukaryotic initiation factor 4E (eIF4E) and that eIF4Emay participate in the translation of LCMVmRNA. Additionally, we identify two residues in the LCMV nucleoprotein (NP) that are conserved in every mammalian arenavirus and are required for recombinant LCMV recovery. One of these sites, Y125, was confirmed to be phosphorylated by using liquid chromatography-tandemmass spectrometry (LC-MS/ MS). NP Y125 is located in the N-terminal region of NP that is disordered when RNA is bound. The other site, NP T206, was pre-dicted to be a phosphorylation site. Immunofluorescence analysis demonstrated that NP T206 is required for the formation of the punctate RTC that are typically observed during LCMV infection. Aminigenome reporter assay using NPmutants, as well as Northern blot analysis, demonstrated that although NP T206A does not form punctate RTC, it can transcribe and replicate a minigenome. However, in the presence of matrix protein (Z) and glycoprotein (GP), translation of the minigenomemessage with NP T206A was inhibited, suggesting that punctate RTC formation is required to regulate viral replication. Together, these results highlight a significant difference between New and OldWorld arenaviruses and demonstrate the importance of RTC formation and translation priming in RTC for OldWorld arenaviruses. IMPORTANCE Several members of the Arenaviridae cause hemorrhagic fevers and are classified as category A pathogens. Arena