The nucleolus and viral infection

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.     

Identification of nucleolar effects in JNK-deficient cells

The c-Jun N-terminal kinase (JNK) signalling pathway has an established role in cellular stress signalling, cell survival and tumorigenesis. Here, we demonstrate that inhibition of JNK signalling results in partial delocalization of the RNA helicase DDX21 from the nucleolus to the nucleoplasm, increased nucleolar mobility of DDX21 and inhibition of rRNA processing. Furthermore, our results show that JNK signalling regulates DDX21 phosphorylation and protein expression. In conclusion, the results presented in this study reveal a previously unidentified cellular role for JNK signalling in the regulation of nucleolar functions. Based on these results, we propose that JNK-mediated effects on nucleolar homeostasis and rRNA processing should be considered when interpreting cellular phenotypes observed in JNK-deficient cell and animal models.     

The nucleolus: a model for the organization of nuclear functions

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production. The organization of the nucleolus reveals the functional compartmentation of the nucleolar machineries that depends on nucleolar activity. When this activity is blocked, disrupted or impossible, the nucleolar proteins have the capacity to interact independently of the processing activity. In addition, nucleoli are dynamic structures in which nucleolar proteins rapidly associate and dissociate with nucleolar components in continuous exchanges with the nucleoplasm. At the time of nucleolar assembly, the processing machineries are recruited in a regulated manner in time and space, controlled by different kinases and form intermediate structures, the prenucleolar bodies. The participation of stable pre-rRNAs in nucleolar assembly was demonstrated after mitosis and during development but this is an intriguing observation since the role of these pre-rRNAs is presently unknown. A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis.Robert Feulgen Lecture presented at the 48th Symposium of the Society for Histochemistry in Stresa, Lake Maggiore, Italy, 7–10 September 2006.     

RNA viruses: hijacking the dynamic nucleolus

The nucleolus is a dynamic subnuclear structure with roles in ribosome subunit biogenesis, mediation of cell-stress responses and regulation of cell growth. The proteome and structure of the nucleolus are constantly changing in response to metabolic conditions. RNA viruses interact with the nucleolus to usurp host-cell functions and recruit nucleolar proteins to facilitate virus replication. Investigating the interactions between RNA viruses and the nucleolus will facilitate the design of novel anti-viral therapies, such as recombinant vaccines and therapeutic molecular interventions, and also contribute to a more detailed understanding of the cell biology of the nucleolus.     

Nucleolar targeting: the hub of the matter

The nucleolus is a dynamic structure that has roles in various processes, from ribosome biogenesis to regulation of the cell cycle and the cellular stress response. Such functions are frequently mediated by the sequestration or release of nucleolar proteins. Our understanding of protein targeting to the nucleolus is much less complete than our knowledge of membrane‐spanning translocation systems—such as those involved in nuclear targeting—and the experimental evidence reveals that few parallels exist with these better‐characterized systems. Here, we discuss the current understanding of nucleolar targeting, explore the types of sequence that control the localization of a protein to the nucleolus, and speculate that certain subsets of nucleolar proteins might act as hub proteins that are able to bind to multiple protein targets. In parallel to other subnuclear structures, such as PML bodies, the proteins that are involved in the formation and maintenance of the nucleolus are inexorably linked to nucleolar trafficking.     

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

ABSTRACT: BACKGROUND: Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. RESULTS: Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. CONCLUSION: NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin.     

Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.     

Elucidation of the avian nucleolar proteome by quantitative proteomics using SILAC and changes in cells infected with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear compartment involved in ribosome subunit biogenesis, regulation of cell stress and modulation of cellular growth and the cell cycle, among other functions. The nucleolus is composed of complex protein/protein and protein/RNA interactions. It is a target of virus infection with many viral proteins being shown to localize to the nucleolus during infection. Perturbations to the structure of the nucleolus and its proteome have been predicted to play a role in both cellular and infectious disease. Stable isotope labeling with amino acids in cell culture coupled to LC‐MS/MS with bioinformatic analysis using Ingenuity Pathway Analysis was used to investigate whether the nucleolar proteome altered in virus‐infected cells. In this study, the avian nucleolar proteome was defined in the absence and presence of virus, in this case the positive strand RNA virus, avian coronavirus infectious bronchitis virus. Data sets, potential protein changes and the functional consequences of virus infection were validated using independent assays. These demonstrated that specific rather than generic changes occurred in the nucleolar proteome in infectious bronchitis virus‐infected cells.