Ribosome biogenesis is critical for normal cell proliferation and highly responsive to metabolic, physiological and environmental challenges. However, the impact of ribosome biogenesis on virus infection biology and whether it might be linked to innate immune responses is poorly understood. Given the universal dependence of virus protein synthesis upon cellular ribosomes, it is assumed that ribosome biogenesis would support ongoing protein synthesis and virus reproduction. Here, we show that while interfering with ribosome biogenesis suppressed ongoing protein synthesis in uninfected primary fibroblasts, it did not detectably impact protein synthesis in HCMV-infected cells. By contrast, reducing ribosome biogenesis unexpectedly stimulated HCMV reproduction. Genome-wide transcriptome analysis revealed that interfering with rRNA accumulation decreased expression of genes involved in cell division, many of which were regulated by NF-Y or the DREAM complex. Among these was High Mobility Group Box 2 (HMGB2), a p53-repressed gene whose product is found both bound to chromatin and in the cytoplasm where it is likely critical for innate immune responses to dsDNA. Significantly, depleting the RNAPI specific transcription factor TIF-IA impaired IFNB1 mRNA accumulation in response to HCMV or dsDNA. Thus, preventing rRNA accumulation dampens cellular responses to dsDNA in part by regulating levels of the DNA sensor HMGB2 (Figure 7F). Moreover, it suggests that rRNA biogenesis triggers an HMGB2-dsDNA sensing pathway and reveals a surprising role for rRNA accumulation and/or nucleolar activity in cell intrinsic innate immunity.

Our results show that rRNA accumulation, ribosome biogenesis and DNA sensing can be integrated to coordinate IFNB1 production. Inhibiting ribosome biogenesis triggers nucleolar stress, which is known to stabilize p53 (Boulon et al., 2010; Chakraborty et al., 2011; Golomb et al., 2014; Deisenroth et al., 2016) and likely accounts for our observed p53-dependent repression of HMGB2 expression. This allows HMGB2 levels to be directly tuned to nucleolar stress and dampened accordingly to limit IFNB1 transcription in response to dsDNA sensing (Figure 7F). Further investigation might reveal that additional genes whose expression is regulated upon TIF-IA-depletion might also contribute to proper control of cell intrinsic innate immune responses. While RNAP III, which transcribes nuclear genes encoding 5S rRNA and tRNAs, reportedly transcribes cytoplasmic dsDNA into a RIG-I RNA substrate (Chiu et al., 2009), nucleolar or ribosome biogenesis functions of the enzymes were not implicated previously. Additional indirect evidence consistent with a role for ribosomes in controlling innate immunity, however, is mounting. Formation of a multipartite complex including STING and S6K is needed to activate IRF3 in response to DNA sensing (Wang et al., 2016) A ribosomal protein deficiency resulting in Diamond-Blackfan Anemia (DBA) impairs rRNA processing and ribosome biogenesis, resulting in activation of innate immune signaling (Danilova et al., 2018). The enigmatic cleavage of UBF by poliovirus protease 3C(pro), which inhibits pol I transcription (Banerjee et al., 2005), can now be considered a strategy to limit IFNB1 production. In addition, antagonizing RNAPI ameliorates experimental autoimmune encephalomyelitis in animal models and is associated with a benign course of multiple sclerosis (Achiron et al., 2013).

Deregulated ribosome biogenesis is associated with human diseases that may be differentially impacted by IFNB1 production. For example, proliferative diseases like cancer are associated with hyperactivated rRNA synthesis and ribosome biogenesis. In normal cells, IFNB1 produced in response to ribosome biogenesis may limit proliferation and serve to restrict tumor development. This could in part account for why many tumors have impaired nucleic acid sensing and/or IFN pathways (Critchley-Thorne et al., 2009; Vanpouille-Box et al., 2018). Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease characterized by expression of a truncated form of Lamin A called progerin, nucleolar expansion, enhanced ribosome biogenesis, and elevated interferon production (Buchwalter and Hetzer, 2017). The correlation between ribosome biogenesis and ISG product accumulation raises the possibility that HGPS may be considered an interferonopathy precipitated by pathogenic enhancement of ribosome production. In contrast, ribosomopathies result from decreased ribosome biogenesis. Patients with Shwachman-Bodian-Diamond Syndrome (SBDS), an inherited ribosomopathy characterized by pancreatic, bone marrow, and immune dysfunction (Giri et al., 2015), experience elevated rates of infectious complications, such as those involving Pneumococcus, Staphylococcus, and Parvovirus B19 (Grinspan and Pikora, 2005; Miniero et al., 1996). Poor innate immune responses, including reduced IFN production, resulting from decreased ribosome biogenesis may be a contributing factor.

While an HMGB2-dependent DNA-sensing pathway links ribosome biogenesis and IFN production, the identity of the non-microbial DNA detected remains enigmatic. Increases in rDNA instability caused by nucleolar dysfunction could trigger a DDR, which reportedly can activate cGAS to stimulate increased interferon production (Kobayashi, 2008). HMGB2 facilitates sensing of long dsDNA by cGAS (Andreeva et al., 2017). Accordingly, decreased RNAPI-induced DNA damage could in part explain why inhibiting ribosome biogenesis limits innate immune responses. However, impairing rRNA accumulation by dampening rRNA synthesis also reportedly can activate DDR pathways (Calo et al., 2018). Further investigation is needed to resolve this paradox. Nevertheless, failsafe pathways likely detect cytoplasmic dsDNA resulting from rDNA transcription activation, much like cGAS detects cytoplasmic dsDNA during senescence.

In addition to IFNB1 production, both cGAS and HMGB2 regulate senescence and HMGB2 plays a role controlling the senescence-associated secretory pathway (Aird et al., 2016; Guerrero and Gil, 2016; Yang et al., 2017; Zirkel et al., 2018). Activation of dsDNA sensing pathways by ribosome biogenesis might contribute to the reported associations between ribosome biogenesis, lifespan and aging (Sharifi and Bierhoff, 2018; Tiku and Antebi, 2018).For example, by activating cytoplasmic dsDNA-sensing, rDNA instability resulting from elevated ribosome biogenesis observed in HGPS could accelerate senescence and thereby drive the progression and severity of premature aging.

Our findings challenge notions that ribosome biogenesis is universally required to support sustained mRNA translation. While corroborating evidence was found in uninfected primary human fibroblasts, it was not the case in HCMV-infected cells. Although polysome levels and protein synthesis were not detectably altered by TIF-IA-depletion in HCMV-infected fibroblasts, precisely why infected compared to uninfected cell protein synthesis was insensitive to interfering with ribosome biogenesis remains unknown. The underlying mechanism(s) are potentially multifaceted and complex perhaps involving virus-induced increases in cellular translation factors that allow more efficient use of existing ribosomes (McKinney et al., 2012; McKinney et al., 2014), differences among ribosome populations, cis-acting elements (Mizrahi et al., 2018) in host and viral mRNAs being translated, specific virus-encoded polypeptides, and/or the differential complexity of mRNA populations in uninfected compared to HCMV-infected cells. Changes in rRNA transcription also reportedly influence proliferation and stem cell fate (Zhang et al., 2014). Notably, sustained protein synthesis despite reduced nucleolar volume and rRNA synthesis has been observed in germline stem cell differentiation in Drosophila (Neumüller et al., 2008; Sanchez et al., 2016). Likewise, rRNA synthesis is not required for growth factor-mediated hypertrophy of human primary myotubes (Crossland et al., 2017). Thus, rRNA and ribosome biogenesis can be uncoupled from protein synthesis in invertebrates and in primary human cells. Further study is required to understand how rRNA and ribosome biogenesis impinges on these different downstream effectors, including HMGB2 protein and mRNA levels. Given the large energy costs associated with ribosome building, it is perhaps no wonder that it is integrated into vital cellular responses that govern survival, including proliferation, lifespan and now cell intrinsic immunity.