Localization to the Nucleolus Is a Common Feature of Coronavirus Nucleoproteins, and the Protein May Disrupt Host Cell Division

Abstract

The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus Coronaviruses are enveloped RNA viruses with nonsegmented, single-stranded, positive-sense RNA genomes of 27 to 32 kb that are 5Ј capped and 3Ј polyadenylated (26). The 5Ј two-thirds of the coronavirus genome encodes the virus contribution to the replicase-transcription complex, Rep1a and Rep1b, the latter resulting from a Ϫ1 frameshift (8). During coronavirus replication, a 3Ј-coterminal nested set of subgenomic mRNAs, which encode other viral proteins, including nucleoprotein (N protein), are synthesized. In part, based on similar genome replication strategies (17, 61), the coronavirus family, Coronaviridae, has been grouped together with the arterivirus family, Arteriviridae, into the order Nidovirales (11). While gene functions and distributions for the two families are similar, there are some differences that might lead to subtle differences in replication strategies. Recently, we have reported that the coronavirus infectious bronchitis virus (IBV) N protein localizes to the cytoplasm and a structure in the nucleus proposed to be the nucleolus in both IBV-infected cells and cells transfected with a plasmid expressing IBV N protein under the control of a PolII promoter (23). A similar result was reported with the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) N protein (54), suggesting that localization of N protein to the nucleolus was probably common to these two virus families and potentially common to all Nidovirales.Coronavirus replication is generally accepted to occur in the cytoplasm of infected cells (66), although for IBV an intact cell nucleus has been proposed to be necessary for virus replication (20). In addition, proteins normally associated with the nucleus have been implicated in the replication of the murine coronavirus mouse hepatitis virus (MHV) (30). The nucleolus is a structure found within the nucleus and is only present during interphase (1). It is the site where rRNA is synthesized and where biogenesis of ribosomal subunits and polymerase III transcripts occurs (10, 57). The nucleolus also sequesters regulatory complexes and has been implicated in the regulation of the cell cycle (10). The possible involvement of the nucleolus in coronavirus replication is not exclusive to coronaviruses. As a consequence of infection or a deliberate process, a number of viruses, including adenoviruses (37) and poliovirus (65), redistribute nucleolin, ...

Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA

“…An increasing number of virus proteins, including examples from retroviruses, DNA͞RNA viruses, and plant viruses, have been reported to exhibit nucleolar localization (9,22,(29)(30)(31)(32)(33)(34)(35). However, no specific function has been assigned to the nucleolar localization of any of these proteins.…”

Identification of a novel nucleolar localization signal and a degradation signal in Survivin-deltaEx3: a potential link between nucleolus and protein degradation

“…However, more and more nonribosomal proteins including growth factors (Antoine et al, 1997;Galcheva-Gargova et al, 1998;Pederson, 1998a, b), virus proteins (Stauber and Pavlakis, 1998;Wurm et al, 2001) and regulators of apoptosis such as p53, DEDD, MDM2 and p14ARF (Stegh et al, 1998;Zhang and Xiong, 1999;Lohrum et al, 2000) were demonstrated to localize in this sub-nuclear organelle under certain conditions. It has been well documented that a cluster of basic amino acids could lead heterologous proteins into the nucleus/nucleolus (Kaffman and O'Shea, 1999), however, no consensus motifs have been characterized thus far for nucleolus targeting and the mechanism underlying nucleolar distribution still remains largely obscure.…”

Functional Implication of Nucleolin in the Mouse First Molar Development

“…In addition, nucleolin has been known to participate in the packaging and shuttling of the ribosome between the nucleus and cytoplasm (13). There have also been numerous reports regarding the functional roles of nucleolin by forming large molecular complexes with other related factors, such as casein kinase (CK) II, c-Myb, midkine (MK), histone H1, nucleophosmin, p53, and protein phosphatase 1 (PP1) (14 -20), and then either directly or indirectly playing a role in the regulation of cell proliferation and growth, cytokinesis, replication, embryogenesis, and nucleogenesis (14,(21)(22)(23)(24). In addition to the many known functions of nucleolin, it may also function as a low affinity receptor of extracellular growth factor on the cell surface (25).…”