Nucleolin-RNA interaction modulates rotavirus replication.

Rotavirus infection is a leading cause of gastroenteritis in children worldwide; the genome of this virus is composed of 11 segments of dsRNA packed in a triple-layered protein capsid. Here, we investigated the role of nucleolin, a protein with diverse RNA-binding domains, in rotavirus infection. Knocking down the expression of nucleolin in MA104 cells by RNA interference resulted in a remarkable 6.3-fold increase in the production of infectious rhesus rotavirus (RRV) progeny, accompanied by an elevated synthesis of viral mRNA and genome copies. Further analysis unveiled an interaction between rotavirus segment 10 (S10) and nucleolin, potentially mediated by G-quadruplex domains on the viral genome. To determine whether the nucleolin-RNA interaction regulates RRV replication, MA104 cells were transfected with AGRO100, a compound that forms G4 structures and selectively inhibits nucleolin-RNA interactions by blocking the RNA-binding domains. Under these conditions, viral production increased by 1.5-fold, indicating the inhibitory role of nucleolin on the yield of infectious viral particles. Furthermore, G4 sequences were identified in all 11 RRV dsRNA segments, and transfection of oligonucleotides representing G4 sequences in RRV S10 induced a significant increase in viral production. These findings show that rotavirus replication is negatively regulated by nucleolin through the direct interaction with the viral RNAs by sequences forming G4 structures.IMPORTANCEViruses rely on cellular proteins to carry out their replicative cycle. In the case of rotavirus, the involvement of cellular RNA-binding proteins during the replicative cycle is a poorly studied field. In this work, we demonstrate for the first time the interaction between nucleolin and viral RNA of rotavirus RRV. Nucleolin is a cellular protein that has a role in the metabolism of ribosomal rRNA and ribosome biogenesis, which seems to have regulatory effects on the quantity of viral particles and viral RNA copies of rotavirus RRV. Our study adds a new component to the current model of rotavirus replication, where cellular proteins can have a negative regulation on rotavirus replication.

Ubiquitination-dependent degradation of nucleolin mediated by porcine circovirus type 3 capsid protein.

IMPORTANCE: Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes multisystem disease in pigs and poses a severe threat to the swine industry. However, the mechanisms of how PCV3 uses host proteins to regulate its own life cycle are not well understood. In this study, we found that PCV3 capsid protein interacts with nucleolin and degrades it. Degradation of nucleolin by the PCV3 capsid protein requires recruitment of the enzyme RNF34, which is transported to the nucleolus from the cytoplasm in the presence of the PCV3 capsid protein. Nucleolin also decreases PCV3 replication by promoting the release of interferon ß. These findings clarify the mechanism by which nucleolin modulates PCV3 replication in cells, thereby facilitating to provide an important strategy for preventing and controlling PCV3 infection.

Structural Integrity of Nucleolin Is Required to Suppress TDP-43-Mediated Cytotoxicity in Yeast and Human Cell Models.

The Transactivating response (TAR) element DNA-binding of 43 kDa (TDP-43) is mainly implicated in the regulation of gene expression, playing multiple roles in RNA metabolism. Pathologically, it is implicated in amyotrophic lateral sclerosis and in a class of neurodegenerative diseases broadly going under the name of frontotemporal lobar degeneration (FTLD). A common hallmark of most forms of such diseases is the presence of TDP-43 insoluble inclusions in the cell cytosol. The molecular mechanisms of TDP-43-related cell toxicity are still unclear, and the contribution to cell damage from either loss of normal TDP-43 function or acquired toxic properties of protein aggregates is yet to be established. Here, we investigate the effects on cell viability of FTLD-related TDP-43 mutations in both yeast and mammalian cell models. Moreover, we focus on nucleolin (NCL) gene, recently identified as a genetic suppressor of TDP-43 toxicity, through a thorough structure/function characterization aimed at understanding the role of NCL domains in rescuing TDP-43-induced cytotoxicity. Using functional and biochemical assays, our data demonstrate that the N-terminus of NCL is necessary, but not sufficient, to exert its antagonizing effects on TDP-43, and further support the relevance of the DNA/RNA binding central region of the protein. Concurrently, data suggest the importance of the NCL nuclear localization for TDP-43 trafficking, possibly related to both TDP-43 physiology and toxicity.