Viral hijacking of the nucleolar DNA-damage response machinery: a novel mechanism to regulate host cell biology

Recent landmark studies indicate that nucleoli play critical roles in the DNA-damage response (DDR) via interaction of DDR machinery including NBS1 with nucleolar Treacle protein, a key mediator of ribosomal RNA (rRNA) transcription and processing, implicated in Treacher-Collins syndrome. Here, using proteomics, confocal/super-resolution imaging, and infection under BSL-4 containment, we present the first report that this nucleolar DDR pathway is targeted by infectious pathogens. We find that Treacle has antiviral activity, but that matrix protein of Henipaviruses and P3 protein of rabies virus, highly pathogenic viruses of the order Mononegavirales, interact with Treacle and inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1-Treacle interaction during a DDR. These data identify a novel mechanism for viral modulation of host cells by appropriating the nucleolar DDR; this appears to have developed independently in different viruses, and represents, to our knowledge, the first direct intra-nucleolar function for proteins of any mononegavirus.


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Fibrillarin (FBL) is a nucleolar protein that was identified by a genome-wide functional genomics screen to be 108 required for HeV production, and reported to interact with M protein, although no function has yet been 109 identified for this interaction 15 . We thus examined the subnucleolar localization of GFP-HeV M and red 110 fluorescent protein-fused FBL (RFP-FBL) in living cells using super-resolution imaging ( Fig. 2A). This 111 confirmed localization of GFP-HeV M to subnucleolar puncta, but FBL was clearly excluded from these

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Henipavirus M protein contains two nuclear localization sequences (NLSs) and two nuclear export sequences 119 (NESs) 17,27 , but the requirements for nucleolar targeting are unknown. Since NLSs and nucleolar localization 120 sequences (NoLSs) are often proximal or overlap 28 we examined the impact on nucleolar localization of GFP-fused M proteins of both HeV and NiV formed clear subnucleolar puncta in ≥ c. 90% and 80%, 125 respectively, of nucleoli examined (Fig. 3A, B). Mutation of K258 to A did not prevent accumulation of HeV or 126 NiV M protein within nucleoli, but strongly impaired accumulation within subnucleolar puncta, such that no 127 punctate localization was observed in nucleoli of cells expressing mutated HeV or NiV M protein (Fig. 3A, B).

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This suggested that mutation of K258 inhibits localization of M protein to puncta and, consistent with this, we 129 were able to detect clear exclusion of K258A-mutated M protein from subnucleolar punctate structures (Fig. 130 S1). Interestingly, although K258A mutation of NiV M protein resulted in a more cytoplasmic localization, as 131 previously reported 17 , HeV M protein mutated at K258A remained largely nuclear (Fig. 3A). Thus, it appears 132 that the requirements for subnucleolar localization are conserved between HeV and NiV M proteins, while those 133 for nucleo-cytoplasmic localization differ, in spite of high sequence conservation between the proteins (c. 90% 134 amino acid identity). Importantly, the finding that K258A mutation did not prevent nucleolar localization of 135 HeV M protein, but resulted in a failure to accumulate within subnucleolar puncta, provided the opportunity to 136 define the nature of the puncta and to examine the specific roles of punctate localization.

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The most pronounced nucleolar interactor was Treacle, which is known to localize to subnucleolar puncta 29 , 151 reminiscent of HeV M nucleolar localization (Figs. 1, 2), and so was selected for further analysis. To assess the 7 resulted in a significant (p < 0.003) increase in virus production (Fig. 4A), indicating that Treacle has antiviral 155 properties.

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Using IP and immunoblot (IB) analysis, we further confirmed that GFP-HeV M interacts with endogenous

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Treacle and with co-transfected HA-Treacle, and that mutation of K258 prevents these interactions (Fig. 4B).

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Co-localization of HeV M with endogenous Treacle within the subnucleolar puncta was also confirmed by

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Since HeV M protein appears to appropriate the DDR-Treacle pathway, we reasoned that M protein might bind 211 at the same or overlapping site(s) in Treacle as NBS1, thereby mimicking the DDR-activated response but in the 212 absence of DNA-damage. Such competitive binding would be consistent with the lack of additional effects of previous reports 4,32 , NBS1-Treacle complexes were readily detected by co-IP from control cells (Fig. 8B).

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However, the association was not detectable in IPs from HeV M protein-expressing cells, indicating that HeV M 217 protein efficiently displaces NBS1 from the complex (Fig. 8B).

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The above data provide, to our knowledge, the first evidence of a specific intra-nucleolar function for a protein  8C(i)). Following the finding that viral proteins can bind to Treacle (Fig. 4), we 234 hypothesized that such a mechanism could be exploited by viruses through the formation of a complex with 235 Treacle analogous to that formed by NBS1, thereby inducing the DDR pathway without the requirement for a 236 DNA damage signal (Fig. 8C(ii)). Consistent with this, our data indicate that M protein interaction with Treacle 237 displaces NBS1 from the complex, and appropriates the downstream pathway that would be activated during a 238 DDR. These data indicate that binding of specific proteins to Treacle can directly inhibit rRNA biogenesis, 239 consistent with the idea that specific regulation of Treacle function (Fig. 8C(i)), rather than some alternative 240 function of nucleolar NBS1 or of the NBS1-Treacle complex, is responsible for rRNA suppression in the DDR. score = 2.77) 15 .Thus, our data support both the model for DDR mediated rRNA silencing via direct inhibition of 244 Treacle-dependent rRNA biogenesis, and the proposed novel mechanism for viral subversion of this process.

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The precise mechanisms underlying Treacle function in rRNA production are not fully resolved, but likely    (Fig. 2), and FBL interaction is unaffected by 271 K258A (Fig. S3). Thus, the interaction of M protein with FBL appears distinct from the role of HeV M protein 272 in Treacle-dependent modulation of rRNA production. Taken together with the findings that other nucleolar interactors including NPM1 and NCL were unaffected by K258A mutation (Fig. 3C)

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For EU analysis, HeLa cells were seeded into 8-well chambers and mock or HeV-infected (MOI 5