@phdthesis{Bostedt2022,
  author    = {Linus Bostedt},
  title     = {Contributions of NP isoforms to the regulation of the Junín virus replication cycle and interactions with the host cell},
  journal    = {Einfluss der NP-Isoformen auf die Regulierung des Replikationszyklus des Jun{\´i}n-Virus und die Interaktion mit der Wirtszelle},
  url       = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-78941},
  pages     = {140},
  year      = {2022},
  abstract  = {Coding constraints imposed by the very small genome sizes of negative-strand RNA viruses (NSVs) have led to the development of numerous strategies that increase viral protein diversity, enabling the virus to both establish a productive viral replication cycle and effectively control the host antiviral response. Arenaviruses are no exception to this, and previous findings have demonstrated that the nucleoprotein (NP) of the highly pathogenic Junín virus (JUNV) exists as three additional N-terminally truncated isoforms of 53 kD (NP53kD), 47 kD (NP47kD), and 40 kD (NP40kD). The two smaller isoforms (i.e. NP47kD and NP40kD) have been characterized as products of caspase cleavage, which appears to serve a decoy function to inhibit apoptosis induction. However, whether they have additional functions in the viral replication cycle remains unknown. Further, the origin and function of NP53kD has not yet been described. In order to first identify the mechanism responsible for production of the NP53kD variant, a possible role of additional caspase cleavage sites was first excluded using a site mutagenesis approach. Subsequently, alanine mutagenesis was then used to identify a region responsible for NP53kD production. As a result, three methionine residues were identified within the characterized sequence segment of NP, linking the production of NP53kD to an alternative in-frame translation initiation. Further site-directed mutagenesis of the previously identified putative in-frame methionine codons (i.e. M78, M80 and M100) finally led to the identification of translation initiation at M80 as being predominantly responsible for the production of NP53kD. Once the identity of all three NP isoforms was known, it was then of further interest to more deeply characterize their functional roles. Consistent with the N-terminal domain containing RNA binding and homotrimerization motifs that are relevant for the viral RNA synthesis process, it could be demonstrated that all three truncated NP isoforms lost the ability to support viral RNA synthesis in a minigenome assay. However, they also did not interfere with viral RNA synthesis by full-length NP, nor did they affect the ability of the matrix protein Z to inhibit viral RNA synthesis. Moreover, it was observed that loss of the oligomerization motifs in the N-terminus also affected the subcellular localization of all three NP isoforms, which were no longer localized in discrete perinuclear inclusion bodies, but rather showed a diffuse distribution throughout the cytoplasm, with the smallest isoform NP40kD also being able to enter the nucleus. Surprisingly, the 3'-5' exonuclease function of NP, which is associated with the C-terminal domain and plays a role in inhibiting interferon induction by digestion of double-stranded RNAs, was found to be retained only by the NP40kD isoform, despite that all three isoforms retained the associated domain. Finally, previous studies using transfected NP and chemical induction of apoptosis have suggested that cleavage of NP at the caspase motifs responsible for generating NP47kD and NP40kD plays a role in controlling activation of the apoptosis pathway. Therefore, to further characterize the connection between the generation of NP isoforms and the regulation of apoptosis in a viral context, recombinant JUNVs deficient in the respective isoforms were generated. Unlike infections with wild-type JUNV, mutations of the caspase cleavage sites resulted in the induction of caspases activation. Surprisingly, however, this was also the case for mutation of the alternate start codon responsible for NP53kD generation. Taken together, the data from this study suggest a model whereby JUNV generates a pool of smaller NP isoforms with a predominantly cytoplasmic distribution. As a result of this altered localization, NP53kD appears to be able to serve as the substrate for further generation of NP47kD and NP40kD by caspase cleavage. Not only does this cleavage inhibit apoptosis induction during JUNV infection, it also results in a cytoplasmic isoform of NP that retains strong 3'-5' exonuclease activity (i.e. NP40kD) and thus may play an important role in preventing viral double-stranded RNA accumulation in the cytoplasm, where it can lead to activation of IFN signaling. Overall, such results emphasize the relevance of alternative protein isoforms in virus biology, and particularly in regulation of the host response to infection.},
  language  = {en}
}