Modulatory effect of rRNA synthesis and ppUL83 nucleolar compartmentalization on human cytomegalovirus gene expression in vitro

Abstract

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 processing. Many viruses, including herpesviruses, are known to exploit the nucleolar compartment during their replication cycle. In a previous study, we demonstrated the preferential targeting and accumulation of the human cytomegalovirus (HCMV) UL83 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. Here we have investigated the possible effects of rRNA synthesis inhibition upon 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, whilst leaving RNA polymerase II function unaffected. Following the inhibition of rRNA synthesis by either of the agents used, 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 nucleolar matrix and the normal development of HCMV lytic infection.

Human Cytomegalovirus pUL83 Stimulates Activity of the Viral Immediate-Early Promoter through Its Interaction with the Cellular IFI16 Protein

“…Consistent with previous reports of their separate localizations (3,4,27), immunofluorescent analysis revealed that IFI16 and pUL83 were colocalized within the nucleus and nucleolus in infected cells at 48 hpi following infection at a low multiplicity of infection (Fig. 5A).…”

Cell‐cycle‐dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro

“…In this experiment, MRC5 fibroblasts enriched in G1, G1/S, S, and G2/M were infected with HCMV for 3 h and then double‐labeled (nucleolar proteins: green; pp65: red) by indirect IF. The expected accumulation of pp65 within the nucleolar compartment [Arcangeletti et al, 2003, 2009] was only evident in G1‐ and G1/S‐synchronized cells, while this pattern was either faintly observable in some cells (most likely cells still residing in G1) or absent in S and G2/M, where the viral protein was almost evenly distributed over the whole nucleus. It should be recalled that pp65 belongs to the “early‐late” group of HCMV‐encoded proteins; the expression of this protein does not start at 3 h p.i.…”

“…Concerning HCMV, the association of specific viral components with different nuclear domains has been demonstrated [Sanchez et al, 1998; Ahn and Hawyard, 2000; Lee et al, 2004; Lymberopoulos and Pearson, 2007; Salsman et al, 2008]. In particular, our group has described accumulation in nucleoli of the major tegument protein pp65 of the incoming HCMV at very early stages of infection, also showing that a functional relationship is likely to exist between the nucleolar localization of pp65 and the correct development of HCMV lytic cycle [Arcangeletti et al, 2003, 2009]. In this respect, it is of importance that the nucleolus is involved in the control of the cell cycle in mammalian cells [Boisvert et al, 2007] also through its role in the biogenesis of ribosomes [Ruggero and Pandolfi, 2003; Pliss et al, 2005].…”

Dynamic and Nucleolin-Dependent Localization of Human Cytomegalovirus UL84 to the Periphery of Viral Replication Compartments and Nucleoli

“…How this occurs is unclear, but a direct interaction with nucleolin would provide a simple explanation. Alternatively, it has been reported that several viral proteins, including pp65, can localize to the nucleolus during infection (32)(33)(34)(35). UL84 and pp65 have been reported to interact within the infected cell (16), and thus, pp65 may recruit UL84 to the nucleolus or vice versa, or other viral or cellular factors may be involved.…”