The interaction of viral proteins with nucleolar antigens may account for why viral proteins have been observed in the nucleolus and may also explain the viral exploitation of nucleolar function, leading to alterations in host cell transcription, translation, and disruption of the host cell cycle to facilitate viral replication. In this study we wanted to investigate whether coronavirus N proteins interacted with two nucleolar antigens, fibrillarin and nucleolin. Interaction with either or both of these antigens might explain our previous observations that coronavirus N proteins localized to the nucleolus (26, 64). Because proteins that localize to the nucleolus have been implicated in cell growth and the cell cycle (11, 40, 41), we also wished to investigate whether coronavirus N proteins affect cell division. Our data indicated that rather than adopting its normal (globular) Christmas tree-like appearance (5), in infected cells fibrillarin was distributed throughout the nucleolus and was possibly more concentrated in the nucleolar periphery. Transfection of either HeLa or Vero cells with plasmids that expressed either the IBV (type III coronavirus) or MHV (type II coronavirus) N proteins also resulted in a change in distribution of fibrillarin, in a similar manner to that observed in virus-infected cells. 	However, unlike HIV Rev protein, which localizes to the nucleolus with a pattern similar to fibrillarin (16) (determined by confocal microscopy), the coronavirus N protein localized uniformly throughout the nucleolus (see, e.g., Fig. 3F) (26, 64). The redistribution of fibrillarin, as a consequence of virus infection, is not unique to coronaviruses. Infection of cells with adenovirus also resulted in the redistribution of fibrillarin (46). To our knowledge this is the first time this has been demonstrated for an RNA virus. The reason for reorganization of fibrillarin to the perinucleolar region is unknown; however, the perinucleolar compartment has been implicated to play a role in transcription and in RNA metabolism in the host cell (28). To investigate whether the coronavirus N protein also associates with fibrillarin in the cytoplasm, experiments with a GFP-fibrillarin fusion protein indicated that both fibrillarin and IBV or MHV N protein colocalized in the perinuclear region and nucleolus (e.g., Fig. 6C and 7C, respectively). The nucleolar functions of fibrillarin are well established and include a role in ribosome assembly (60) and, as a factor of the nucleolus, in cell cycle regulation (11). Experiments that blocked fibrillarin with antibody prevented the translocation of fibrillarin to the nucleoli and resulted in the reduction or inhibition of PolI transcription (19); by redistributing fibrillarin N protein might have a similar effect. 	 Alternatively, by interacting with fibrillarin, N protein could potentially affect ribosomal biogenesis and, therefore, have a concomitant effect on host cell translation. During MHV infection, host cell translation is decreased, although translation of viral mRNAs is either unaffected or upregulated (24, 55). N protein may therefore interact with nucleolar components to improve translation of virus mRNAs, perhaps by sequestering ribosomes (or parts thereof) to viral mRNAs (26). Unfortunately, from the point of view of this study, the monoclonal antibody against nucleolin, used in immunofluorescence, only recognized human nucleolin (in accordance with the manufacturer’s guidelines [Leinco Technologies]), and this limited the study of possible redistribution of nucleolin by N protein to HeLa cells which expressed MHV N protein. While our data suggested that nucleolin was not reorganized in these cells, it is not possible to conclude that this does not occur in virus-infected cells or with other coronavirus N proteins expressed in species-specific cell types. For example, nucleolin is retained in the cytoplasm in poliovirus-infected cells (62), probably because cytoplasmic-nuclear trafficking is prevented (23), and adenovirus infection results in the redistribution of nucleolin to the cytoplasm (38). 	Binding studies with purified immobilized phosphorylated or nonphosphorylated IBV N protein and nuclear extracts prepared from Vero cells indicated that there was a direct interaction between N protein and nucleolin (Fig. 8E, lanes 1 and 3, respectively). Two recombinant His-tagged proteins were used to investigate the specificity of nucleolin binding to N protein, HIV core (p24) protein, and DcuR, a DNA-binding protein isolated from E. coli (22). Nucleolin did not bind to either of these immobilized proteins or to the NTA beads in the absence of protein, indicating that nucleolin specifically interacted with N protein. Interestingly, the data indicated that immobilized Nphos protein bound more nucleolin than Nnonphos protein, a finding that is contrary to what would be predicted if the protein associated by electrostatic charge alone. Reference & Source information: https://www.ncbi.nlm.nih.gov/ Read more on :
