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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.
New World arenaviruses represent an important group of zoonotic pathogens that pose a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever and neurological symptoms, other closely related family members exhibit little or no pathogenicity. For instance, Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, while the closely related Tacaribe virus (TCRV) is avirulent in humans. Little is known about host cell responses to infection, or how they contribute to virulence; however, TCRV strongly induces caspase-dependent apoptosis (i.e. non-inflammatory programmed cell death) in infected cells, whereas JUNV does not.
In order to better understand the connection between apoptosis and pathogenesis, we sought to unravel the regulation of pro- and anti-apoptotic signaling in response to arenavirus infection. We demonstrated that apoptosis induced by TCRV proceeds over the mitochondrial-regulated intrinsic pathway and involves activation of p53 (accumulation and phosphorylation), activation of the pro-apoptotic BH3-only factors Puma and Noxa (accumulation), as well as inactivation of another pro-apoptotic factor called Bad (phosphorylation). The regulation of these factors in response to TCRV infection is accompanied by other classical hallmarks of intrinsic apoptosis, such as disorganization of the mitochondrial network, cytochrome c release, PS flipping, caspase cleavage and nuclear condensation. The involvement of the BH3-only factors as key players in regulating TCRV-induced apoptosis could also be validated in knockout cells, which showed either suppressed or increased apoptosis depending on the respective activation (i.e. Puma and Noxa) or inactivation (i.e. Bad) status of the respective BH3 protein. Interestingly, while JUNV does not trigger late stages of apoptosis induction (i.e. caspase activation, nuclear condensation and cell death), we could show that it activates similar upstream pro-apoptotic signaling events including activation of p53, Puma and Noxa. This supports the current hypothesis that JUNV actively evades the induction of apoptosis through the involvement of a mechanism targeting late steps in the apoptotic cascade. Specifically, this model proposes that intrinsic activation is suppressed at the level of caspase activation by JUNV NP, which serves as an alternative substrate for caspase cleavage.
Additionally, in order to identify viral factors associated with the induction of apoptosis, a full genome sequencing of TCRV was performed and contributed to the validation and correction of substantial errors reported in existing sequences for TCRV. With the help of this sequence, correct expression plasmids containing the viral genes for NP, GP and Z were constructed and tested for their ability to induce apoptosis in vitro. This revealed that both TCRV and JUNV Z are triggers for apoptosis, which further supports our finding that JUNV also induces activation of pro-apoptotic factors. Again, consistent with a model where JUNV NP blocks caspase activation directly, co-expression of JUNV Z and NP abrogated caspase activation, while simultaneous expression of TCRV NP and Z still resulted in cell death.
Finally, identification of the specific apoptotic factors involved in regulating TCRV-induced apoptosis (i.e. Bad, Puma and Noxa) and the generation of the respective knockout cell lines allowed us to investigate what influence apoptosis induction has on virus infection. Interestingly, knockout of these factors showed no direct impact on virus growth in Vero cells. However, TCRV particles produced in cells with the individual pro-apoptotic (i.e. Puma and Noxa) or anti-apoptotic (i.e. Bad) factors knocked out showed altered infectivity in primary human monocytes and macrophages, which represent important target cells for arenaviruses. Since TCRV particles that originate from the different knockout cells would be expected to contain different amounts of PS in their envelope (depending on the level of apoptosis taking place), this suggests a role of apoptosis in facilitating PS-receptor-mediated entry and/or PS-receptor signaling through downstream kinases, either of which could be contributing to successful infection in professional phagocytic cells. In particular, phosphorylation of some of the identified factors involved in regulating TCRV-induced apoptosis indicates the involvement of upstream kinases from diverse signaling pathways, some of which also play a role in regulating cytokine production – another host cell reaction that differs significantly between TCRV- and JUNV-infected monocytes and macrophages. As such, these findings represent an exciting basis for a possible connection between apoptotic responses and the regulation of pro- and anti-inflammatory cytokine responses via their associated upstream signaling processes and provide a starting point for future studies that will help us to better understand how these processes contribute to arenavirus pathogenicity.