Tomonori Ueno

Enhancement of procollagen biosynthesis by p180 through augmented ribosome association on the endoplasmic reticulum in response to stimulated secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

Nucleolin and the Packaging Signal, ψ, Promote the Budding of Human Immunodeficiency Virus Type-1 (HIV-1)

Gag proteins of human immunodeficiency virus type 1 (HIV‐1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA‐binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV‐1 gag proteins, the packaging signal, ψ and nucleolin affect the budding of HIV‐1. Here we report that nucleolin enhances the release of HIV‐1 virions which contain ψ. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV‐1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV‐1.

Regulation of polysome assembly on the endoplasmic reticulum by a coiled-coil protein, p180

A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.

Expansion of the trans-Golgi network following activated collagen secretion is supported by a coiled–coil microtubule-bundling protein, p180, on the ER

A coiled-coil endoplasmic reticulum (ER) protein, p180, was originally reported as a ribosome-binding receptor on the rough ER and is highly expressed in secretory tissues. Recently, we reported new functions of p180 as a microtubule-bundling protein on the ER. Here, we investigated the specific roles of p180 in the Golgi complex organization following stimulated collagen secretion. Targeted depletion of p180 by siRNA transfection caused marked reduction of TGN, while other marker levels for the cis or medial Golgi were not markedly changed. Ascorbate stimulation resulted in trans-Golgi network (TGN) expansion to the periphery in control cells that is characterized by both increased membrane amounts and extended shape. In contrast, loss of p180 resulted in retraction of the TGN regardless of ascorbate stimulation. The TGN developed to the periphery along stabilized microtubule bundles, and overexpression of MTB-1 fragment caused dominant-negative phenotypes. Once disorganized, the retracted TGN did not recover in the absence of p180 despite elevated acetylated tubulin levels. TGN46 and p180 were co-distributed in epithelial basal layer cells of human mucosal and gastrointestinal tissues. Taken together, we propose a novel function of p180-abundant ER on the TGN expansion, both of which are highly developed in various professional secretory cells.

Human topoisomerase I promotes HIV-1 proviral DNA synthesis: Implications for the species specificity and cellular tropism of HIV-1 infection

Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10–15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.

Use of a GFP-PML-expressing cell line as a biosensor for human cytomegalovirus infection.

Human cytomegalovirus (HCMV) infection has a marked effect on promyelocytic leukemia (PML) bodies. Here, we describe a novel real-time monitoring system for HCMV-infected cells in vitro using a newly established cell line that stably expresses GFP-PML protein. Upon infection, HCMV causes specific dispersion of GFP-PML bodies, thereby allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Quantitative protocols using either an NPB fluorescence assay or a GFP-PML imaging assay are also described. The NPB fluorescence assay is rapid, sensitive, and sufficiently simple for screening of inhibitory reagents, while the GFP-PML imaging assay is highly sensitive and applicable to drug susceptibility testing of low-titer clinical isolates.

Detection of active human cytomegalovirus by the promyelocytic leukemia body assay in cultures of PBMCs from patients undergoing hematopoietic stem cell transplantation

A novel detection system was established previously for cells infected with the human cytomegalovirus (HCMV) in vitro that utilizes the unique IE1‐dependent nuclear dispersion of promyelocytic leukemia (PML) bodies early in the HCMV replication cycle. This assay system, designated “the PML assay,” makes use of the GFP‐PML‐expressing cell line SE/15, and allows real‐time monitoring of infected cells by fluorescence microscopy without any staining procedures. A rapid and quantitative drug susceptibility testing was developed for low‐titer clinical isolates propagated in fibroblasts in vitro. The present study sought to exploit the PML assay for evaluating in vivo status of HCMV without virus isolation. Progeny viruses were detected directly from peripheral blood mononuclear cells (PBMCs) infected in vivo obtained from hematopoietic stem cell transplantation recipients. The overall positivity of the PML assay tended to correlate with the levels of genomic DNA. Direct phenotypic susceptibility testing detected one ganciclovir (GCV)‐resistant case among 19 samples, which was confirmed further by genomic and plaque reduction assays. However, in another patient with the sequence‐proven mutant confirmed by sequencing, the progeny viruses exhibiting GCV‐resistance were not detected. Studies on the isolated virus from the latter patient suggested the possibility that replication efficiency may differ between PBMCs and lesions infected in vivo, which may hamper the detection of GCV‐resistant viruses by the PML assay, at least in this case. Taken together, the PML assay is sufficiently sensitive to monitor replication‐competent HCMV directly from PBMCs infected in vivo, and provides a novel tool for comparing the characteristics of HCMV strains infected in vivo. J. Med. Virol. 84:479–486, 2012.