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

Arcangeletti, Maria Cristina; Rodighiero, Isabella; Gatti, Rita; Orlandini, Guido; Ferraglia, F.; Motta, Federica; Covan, Silvia; Razin, Sergey V.; Dettori, G.; Chezzi, Carlo

doi:10.1002/jcb.22268

Cited by 8 publications

(11 citation statements)

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“…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).…”

Section: Resultssupporting

confidence: 92%

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

Cristea

Moorman

Terhune

et al. 2010

J Virol

145

181

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The human cytomegalovirus (HCMV) virion protein pUL83 (also termed pp65) inhibits the expression of interferon-inducible cellular genes. In this work we demonstrate that pUL83 is also important for efficient induction of transcription from the viral major immediate-early promoter. Infection with a mutant virus containing a premature translation termination codon in the UL83 open reading frame (ORF) (UL83Stop) resulted in decreased transcription from the major immediate-early promoter in a time-and multiplicitydependent manner. Expression of pUL83 alone is capable of transactivating the promoter in a reporter assay, and pUL83 associates with the promoter in infected cells. To investigate the mechanism by which the protein regulates the major immediate-early promoter, we utilized a mutant virus expressing an epitope-tagged pUL83 from its own promoter to identify protein binding partners for pUL83 during infection. We identified and confirmed the interaction of pUL83 with cellular IFI16 family members throughout the course of HCMV infection. pUL83 recruits IFI16 to the major immediate-early promoter, and IFI16 binding at the promoter is dependent upon the presence of pUL83. Consistent with the results obtained with the UL83Stop virus, infection of IFI16 knockdown cells with wild-type virus resulted in decreased levels of immediate-early transcripts compared to those of control cells. These data identify a previously unknown role for pUL83 in the initiation of the human cytomegalovirus gene expression cascade.Viral infection is marked by a race between the competing interests of the virus and the host cell. Efficient initiation of viral gene expression is critical to circumvent host defenses aimed at blocking viral gene expression. Human cytomegalovirus (HCMV), a betaherpesvirus encoding nearly 200 predicted proteins (57, 59), has evolved multiple means to evade the initial host cell response to infection. The first viral proteins expressed, the immediate-early proteins, play an important role in this process. Immediate-early proteins are detected in fibroblasts within 4 h of infection and thus are available to function at very early stages in the viral life cycle to block antiviral signaling events. For example, the IE1 protein binds to and inhibits STAT1 and STAT2 (64), two host cell proteins critical for the activation of interferon-inducible gene expression; and IE2 has also been implicated in regulation of transcription of antiviral genes (80). pTRS1 blocks the activation of protein kinase R (PKR), an important regulator of protein translation in response to innate immune signals (16,33,54,85), and pUS3 inhibits antigen presentation by infected cells by sequestering and degrading the major histocompatibility complex (MHC) class I heavy-chain complex (39,48,55).In addition to their role in subverting the host response to viral infection, immediate-early proteins are critical for the induction of viral gene expression. IE1 binds to and inhibits histone deacetylases (HDACs) to ensure a chromatin structure on viral...

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Section: Resultssupporting

confidence: 92%

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

Cristea

Moorman

Terhune

et al. 2010

J Virol

145

181

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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].…”

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confidence: 99%

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

Arcangeletti

Rodighiero

Mirandola

et al. 2011

J of Cellular Biochemistry

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The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, 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 support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Bender

Coen

Strang

2014

J Virol

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Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAGtagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. IMPORTANCEThe protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular proteins required for HCMV genome replication and replicating viral DNA. We found that UL84 localizes with viral proteins, viral DNA, and the cellular nucleolar protein nucleolin in the subnuclear replication compartments in which viral DNA replication occurs. Unexpectedly, we also found localization of UL84 with nucleolin in nucleoli and showed that the presence of nucleolin is involved in localization of UL84 to the nucleus. These results add to previous work showing the importance of nucleolin in replication compartment architecture and viral DNA synthesis and are relevant to understanding UL84 function.

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

Cited by 8 publications

References 50 publications

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

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

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

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

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