Aspetti funzionali della compartimentalizzazione nucleare di proteine di citomegalovirus umano in un modello di infezione litica in vitro

SUMMARY The nucleolus is a nuclear domain involved in the biogenesis of ribosomes, as well as in many other important cellular regulatory activities, such as cell cycle control and mRNA transport and processing. Several literature data describe the involvement of the nucleolar compartment during the replication cycle of many viruses, including herpesviruses. In a previous study, we demonstrated the preferential targeting and accumulation of one of the major human cytomegalovirus (HCMV) tegument phosphoprotein (pp65) to the nucleolar compartment and, in particular, to the nucleolar matrix of lytically infected fibroblasts; such targeting was already evident at very early times after infection. The aim of the present study was to investigate the functional significance of HCMV pp65 targeting to the nucleolar compartment (particularly related to the HCMV gene expression over the course of the lytic program). First of all, we assessed the possible effects of rRNA synthesis inhibition on the development of HCMV lytic infection, by using either actinomycin D or cisplatin at low concentrations, that are known to selectively inhibit RNA polymerase I activity, and hence ribosome biogenesis, whilst leaving RNA polymerase II and III functions unaffected. Upon inhibition of rRNA synthesis by either agent, we observed a significant redistribution of nucleolar proteins within the nucleoplasm and a simultaneous depletion of viral pp65 from the nucleolus; this effect was highly evident in both unextracted cells and in nuclear matrices in situ. Of particular interest, even a brief suppression of rRNA synthesis resulted in a very strong inhibition of the progression of HCMV infection, as was concluded from the absence of accumulation of HCMV major immediate-early proteins within the nucleus of infected cells. These data suggest that a functional relationship might exist between rRNA synthesis, pp65 localization to the nucleolus and the normal development of HCMV lytic infection. Among nucleolar functions of potential interest for HCMV, and related to rRNA synthesis, we focused on cell cycle control, that occurs also in the nucleolus and which is modulated by HCMV. The original data arisen from the present study support the hypothesis of a potentially relevant role of pp65, and its nucleolar localization, in the control of the cell cycle by HCMV (that is known to induce a blockade of cell proliferation in G1–G1/S in many experimental models), and for the development of viral infection. Specifically, in this study we demonstrate that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phases, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S supported an efficient development of the HCMV lytic cycle, in contrast with S and G2/M phases, where infection was highly reduced. These data reinforce the hypothesis of HCMV pp65 involvement in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. In order to detect possible molecular partners of pp65 in the nucleolar compartment, we focused our attention on the relationship between this viral antigen and nucleolar proteins of interest in cell cycle and rRNA synthesis control, such as nucleolin (C23), nucleophosmin, fibrillarin and UBF (Upstream Binding factor). Confocal microscopy analysis and reciprocal co-immunoprecipitation point to C23 as a possible pp65 partner in the nucleolus. Accordingly, by preliminary experiments of C23 knockdown, pp65 was no longer found in the nucleolus of infected cells; moreover, in these experimental conditions we observed not only a significantly reduced expression of HCMV major immediate-early proteins, but also a lack of their nuclear accumulation and a poor presence in the cytoplasmic compartment.