RNA viruses: hijacking the dynamic nucleolus

Abstract

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.

Figure 1. The structure of the nucleolus.

a | Confocal microscopy image of a mammalian cell showing the relative positions of the cytoplasm, nucleus and nucleolus. The nucleus and the nucleolus are separated from the cytoplasm by the nuclear envelope. b | Bright-field microscopy image of a live mammalian cell showing the nucleolus as a highly refractive structure in the nucleus. c–d | Electron microscopy images of the mammalian nucleolus highlighting distinct structures, including the fibrillar centre (FC), dense fibrillar component (DFC), granular component (GC) and perinucleolar region (PNR). Many diagrams of the nucleolus show only a single FC and DFC when, in fact, there are many FCs and DFCs in one nucleolus. e–f | Confocal images of the nucleolus showing how fluorescent fusion proteins of nucleolar proteins can be used as markers for the different regions of the nucleolus including the DFC (EGFP-fibrillarin) (e) and the GC and DFC (DS-Red-B23) (f).

Figure 2. RNA viruses and the nucleolus.

Schematic representation of a generic replication strategy used by a positive-strand RNA virus showing localization of the capsid protein to the nucleolus. The virus enters the cell and the genome is uncoated and then translated by ribosomes to produce the viral RNA-dependent RNA polymerase (RdRp). The viral genome is therefore messenger sense and also functions as a template for the synthesis of negative-strand RNA. The RdRp uses the negative strand (or anti-genome) as a catalytic template for the production of new genomic RNA. Therefore, there is usually a molar excess (between 10:1 and 100:1) of positive-strand RNA over negative-strand RNA in infected cells. Positive-strand RNA viruses can either be enveloped or non-enveloped. However, they all produce capsid proteins for binding to viral RNA. These proteins have different names, including capsid, nucleocapsid and nucleoproteins, but all have similar functions. Negative-strand RNA viruses have a more complex life cycle in that the viral genome is not messenger sense and therefore the virus contains the enzymes needed to initiate the transcription of mRNAs and to facilitate replication.

Figure 3. HIV and the nucleolus – a potential therapeutic strategy?

Our knowledge of the interaction between HIV and the nucleolus is leading to the design of novel therapeutic strategies, and has been pioneered by John Rossi's group. Although these approaches are currently targeted at patients with both AIDS and lymphoma they might be applicable to all individuals infected with HIV-1. Haematopoietic stem cells are taken from infected individuals and treated with lentiviral vectors that contain an anti-tat/rev short hairpin RNA (shRNA), an anti-CCR5 ribozyme and a nucleolar-localizing trans-activation response element (TAR) RNA decoy. TAR is a region of secondary RNA structure that is located within the HIV long terminal repeat and is present in all HIV mRNAs. The region prevents the efficient use of the mRNA unless it is bound by the HIV transactivator (Tat) protein. The engineered stem cells then form part of a bone marrow transplant back into the (now) irradiated individual. As a result T cells are produced that are resistant to HIV-1 infection. As with anti-HIV chemotherapy, the triple combination approach is used to prevent the build up of resistant viruses.