In TGEV-infected cells, nsp1 is distributed in both the nucleus and the cytoplasm, which is not surprising as it can freely diffuse into the nucleus because of its small molecular weight (∼9 kDa). In contrast to TGEV nsp1, both MHV nsp1 and SARS-CoV nsp1 are localized exclusively in the cytoplasm of virus-infected cells. Due to its binding to the 40S ribosomal subunit, nsp1 inhibits cellular mRNA translation in some cases (HCoV-229E and HCoV-NL63) but not in others (TGEV). In addition, nsp1 inhibits IFN induction and signaling.
SARS-CoV proteins 6 and 9b affect nucleocytoplasmic transport. Protein 6 impedes nuclear import of factors such as STAT1 and antagonizes IFN signaling pathways. Protein 9b shuttles from the nucleus by its interaction with cellular exportin 1 (Crm1), which is essential for proper protein 9b degradation, and blocking nuclear export of protein 9b induces cell apoptosis.
As noted above, N protein-recruited DDX1 functions in the RTC in facilitating TRS read-through and synthesis of long sgmRNAs. The 3b proteins of IBV and SARS-CoV, though different in nature, have also been located in part in the nuclei of transfected or infected cells. Following nuclear localization, SARS-CoV 3b protein trafficts to the outer membrane of mitochondria, where it inhibits the induction of type 1 interferon (IFN) elicited by RIG-I and the mitochondrial antiviral signaling protein. Similarly, the 4b proteins of MERS-CoV, bat coronavirus (BtCoV)-HKU4, and BtCoV-HKU5 also localize to the nucleus and inhibit type 1 IFN induction and, less efficiently, NF-kB signaling pathways.
The coronavirus protein most frequently associated with the host cell nucleus is the N protein, and its transport to the nucleus is regulated by phosphorylation. N protein nuclear localization is associated with induction of cell cycle arrest and inhibition of cytokinesis and is involved in recruitment to the cytoplasm of cell nuclear proteins, such as heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) and the helicase DDX1.
RELEVANCE OF THE CELL NUCLEUS IN CORONAVIRUS RNA SYNTHESIS
All positive strand RNA viruses that infect animals replicate in the cytoplasm of the infected host cell. However, there is ample evidence that implicated the nucleus and nuclear proteins in the replication and pathogenesis of positive-strand RNA viruses, including coronaviruses. The replication of these RNA viruses in enucleated cells is variable, ranging from 10% to 100% of that in nucleated controls. The relocation of nuclear proteins to the cytoplasm and of viral proteins to the nucleus during virus replication highlights the relevance of this organelle during the coronavirus infectious cycle and raises important question: What is the role of nuclear factors in the replication of these viruses, and do viral proteins traveling to the nucleus participate in RTC activity?
A new hypothesis postulates that stress granules are involved in an integrated stress-innate immunity activation response. In this pathway, viral RNA and proteins, along with host pathogen-sensing factors, such as the dsRNA-binding protein kinase R (PKR) and the RNA helicases retinoic acid-induced gene 1 (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), can be sequestered in stress granules. Additional insight into the relevance of stress granules and P bodies for the regulation coronavirus RNA synthesis is still required.
These granules contained the stress granule markers T cell intracellular antigen 1 (TIA-1), TIA-1-related protein (TIAR), and polypirimidine tract-binding protein (PTB) in association with with viral gRNA and sgmRNAs. TGEV-induced stress granules might contribute to the spatiotemporal regulation of viral RNA synthesis. Several stress granule proteins (including caprin and G3BP) have been associated with IBV N protein, pointing to nthe relevance of these RNA-protein complexes in the regulation of coronavirus gene expression.
For coronaviruses, MHV replication was found to be enhanced in cell deficient in stress granule formation, implying that stress granules contribute to viral inhibition. TGEV induced stress granules that persisted from 7 to 16 hpi, which was correlated with a decrease in viral replication and transcription.
During infection, RNA viruses dynamically interact with stress granules and P bodies, leading to varying stress granule phenotypes. Many virus have evolved mechanisms to antagonize the formation of stress granules, suggesting that stress granules are involved in restricting virus replication through RNA silencing. In contrast, other RNA viruses, such as respiratory suncytial virus, induce stress granule formation and take advantage of stress granule responses as part of the infectious cycle.