Coronaviruses (CoV) nucleocapsid N proteins are key in incorporating the genomic RNA into the progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used CoV model virus, interacts with non-structural protein 3 (nsp3), a component of the RTCs. These two aspects of CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 but not with other RTCs components using a systematic yeast two-hybrid approach, and identified two distinct regions in the N protein that redundantly mediates this interaction. A selective N protein variant carrying point mutations in these two regions fail to bind nsp3 in vitro, resulting in an inhibition of its recruitment to the RTCs in vivo. Furthermore, opposite to the wild type N protein, this N protein variant impairs the stimulation of gRNA and viral mRNA transcription in vivo and in vitro, which in turn leads to an impairment in MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in CoV life cycle as it is critical for optimal viral RNA synthesis. IMPORTANCE Coronaviruses (CoV) have been regarded for a long time as a relatively harmless pathogen for humans. Two severe respiratory tract infection outbreaks caused by SARS-CoV and MERS-CoV, however, have caused high pathogenicity and mortality rates in humans. This highlighted the relevance of being able to control CoV infections. We used a CoV model virus, mouse hepatitis virus (MHV), to investigate the importance of the recruitment of nucleocapsid (N) protein, a central component of CoV virions, to intracellular platforms where CoV replicate, transcribe and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial in promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain both an important aspect of CoV life cycle and reveal an interaction between viral proteins that could be targeted in antiviral therapies.

The regulatory pathways necessary for the maintenance of adult hematopoietic stem cells (HSCs) remain poorly defined. By using loss-of-function approaches, we report a selective and cell-autonomous requirement for the p300/CBP-binding transcriptional coactivator Cited2 in adult HSC maintenance. Conditional deletion of Cited2 in the adult mouse results in loss of HSCs causing multilineage bone marrow failure and increased lethality. In contrast, conditional ablation of Cited2 after lineage specification in lymphoid and myeloid lineages has no impact on the maintenance of these lineages. Additional deletion of Ink4a/Arf (encoding p16(Ink4a) and p19(Arf)) or Trp53 (encoding p53, a downstream target of p19(Arf)) in a Cited2-deficient background restores HSC functionality and rescues mice from bone marrow failure. Furthermore, we show that the critical role of Cited2 in primitive hematopoietic cells is conserved in humans. Taken together, our studies provide genetic evidence that Cited2 selectively maintains adult HSC functions, at least in part, via Ink4a/Arf and Trp53.