Dissecting the Multiple Functions of the Lymphocytic Choriomeningitis Virus Nucleoprotein
The Arenaviridae are enveloped, negative-sense RNA viruses with several family members that cause hemorrhagic fevers. These viruses encode only four open reading frames and each protein expressed performs multiple functions in the viral lifecycle. This thesis focuses on elucidating the regulation and functions of Lymphocytic Choriomeningitis Virus (LCMV) nucleoprotein (NP). Phosphorylation is a common and reversible posttranslational modification. This work identifies two residues in the Lymphocytic Choriomeningitis Virus (LCMV) nucleoprotein (NP) that are as conserved in every mammalian arenavirus and are required for recombinant LCMV recovery. One of these sites, NP Y125, was confirmed as a phosphorylation site by LC-MS/MS. Y125 resides in the N-terminal region of NP that becomes disordered when RNA is bound. The other site, NP T206, was predicted to be a phosphorylation site. Immunofluorescence demonstrated that NP T206 is required for the formation of the punctate replication-transcription complexes (RTC) typically found in LCMV infection. A minigenome reporter assay in combination with NP mutants and Northern blot analysis, demonstrated that, while NP T206A remains diffuse in the cytoplasm and does not form RTC, it can facilitate the transcription and replication of a minigenome. However, in the presence of Z and GP, translation of minigenome message with NP T206A was inhibited, suggesting that RTC formation is required to regulate viral replication. Additionally, we provide immunofluorescence evidence that, unlike Old World arenaviruses, LCMV RTC colocalize with eIF4E, and that eIF4E may participate in the translation of LCMV mRNA.
In addition to nucleating RTC formation and participating in replication and transcription, NP also inhibits stimulation of the innate immune system. This activity has been attributed to the dsRNA exonuclease activity of in the C-terminal domain of NP. NP is proposed to degrade viral dsRNA, thereby eliminating its recognition by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), and preventing the subsequent signaling cascade. However, we discovered that NP blocks transcription from interferon-stimulated response element (ISRE) promoters downstream of RIG-I and MDA5, and that the exonuclease activity of NP is not required for this inhibition. We propose the exonuclease activity of NP may be conserved across mammarenaviruses because it functions in concatemer resolution during genome replication.