Binding of human cytomegalovirus to sulfated glucuronyl glycosphingolipids and their inhibitory effects on the infection.

Ogawa-Goto, Kiyoko; Arao, Yujiro; Ito, Yukishige; Ogawa, Tomoya; Abe, Toshiaki; Kurata, Takeshi; Irie, Shinkichi; Akanuma, Hiroshi

doi:10.1099/0022-1317-79-10-2533

Cited by 8 publications

(8 citation statements)

References 35 publications

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“…HCMV strains (Towne, Davis, and other clinical isolates) were propagated and titrated in human embryonic lung (HEL) fibroblasts as described previously (26). For cell-associated clinical isolates, the number of plaque-forming cells (PFCs) present in an infected culture was estimated according to previously described methods (19).…”

Section: Methodsmentioning

confidence: 99%

Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Ueno

Eizuru

Katano

et al. 2006

Antimicrob Agents Chemother

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Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

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Section: Methodsmentioning

confidence: 99%

Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Ueno

Eizuru

Katano

et al. 2006

Antimicrob Agents Chemother

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“…Similarly, a peptide generated against 3-OS HS using phage display library screening blocked HCMV entry (26). In addition, several sulfated polysaccharides (dextran sulfate, pentosan polysulfate, and heparin), copolymers of acrylic acid with vinyl alcohol sulfate, and 3-O-sulfated saccharide have proved to be potent inhibitors of HCMV infectivity in vitro (17,27). In this study, we investigated the susceptibility of HIS cells to HCMV infection and the role of 3-OS HS during this process.…”

mentioning

confidence: 99%

A Role for 3- O -Sulfated Heparan Sulfate in Promoting Human Cytomegalovirus Infection in Human Iris Cells

Baldwin

Maus

Zanotti

et al. 2015

J Virol

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H uman cytomegalovirus (HCMV), a member of the betaherpesvirus family, is the leading cause of congenital neurological complications in neonates as a result of maternal infection (1-4). Among immunocompromised patients-especially those with an organ transplant, chemotherapy, or AIDS-the reactivation of virus causes life-threatening diseases, such as gastroenteritis, encephalitis, pneumonitis, and graft rejection (5-7). In addition, HCMV infection is also implicated in widespread ocular damage and potential vision loss from retinitis, anterior uveitis, and corneal endotheliitis (8-11). Because HCMV infection of the iris is a risk factor during anterior uveitis, understanding the dynamics of viral infection at the molecular level becomes relevant for understanding the viral pathogenesis in general and developing novel strategies to prevent blindness. Therefore, we used primary cultures of human iris stromal (HIS) cells as a model to determine the susceptibility and role of 3-O-sulfated heparan sulfate (3-OS HS) during HCMV uveitis.Although HCMV entry into host cells is poorly understood (12, 13), it is clearly a multistep process that requires complex interactions between viral envelope glycoproteins and the host cell receptors (12). It has been suggested that HCMV glycoprotein B (gB) binds to heparan sulfate (HS) during viral attachment, resulting in a high virion concentration at the cell surface and further binding to the cellular receptor (14-17). This interaction has been proposed to modulate immune responses (14, 18). To date, three receptors, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor ␣ (PDGFR␣), and cellular integrins (␣2␤1, ␣6␤1, and ␣v␤3), have been implicated during HCMV entry (19)(20)(21)(22)(23)(24). In addition, recent studies have suggested the potential role of a sulfated form of HS during HCMV entry. For instance, a library of peptides derived from human hemofiltrate and screened for inhibitory effects on HCMV infection implicated the role of 6-O-sulfated HS (6-OS HS) (25) in HCMV entry. Similarly, a peptide generated against 3-OS HS using phage display library screening blocked HCMV entry (26). In addition, several sulfated polysaccharides (dextran sulfate, pentosan polysulfate, and heparin), copolymers of acrylic acid with vinyl alcohol sulfate, and 3-O-sulfated saccharide have proved to be potent inhibitors of HCMV infectivity in vitro (17,27). In this study, we investigated the susceptibility of HIS cells to HCMV infection and the role of 3-OS HS during this process. Our data demonstrate the significance of 3-OS HS during HCMV entry, and we propose a unique in vitro model for future studies related to 3-OS HS-dependent induction of proinflammatory cytokines and virus tropism.Susceptibility of primary cultures of HIS cells to HCMV infection. The HIS cultures were prepared in accordance with institutional review board-approved protocols and were isolated from anonymously donated human eyes (provided by the Illinois Eye Bank, Chicago, IL) via sterile dissecti...

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“…Human embryonic lung (HEL) fibroblasts between passages 15 and 25 were inoculated with HCMV (Towne strain) as previously described (16). Prior to inoculation, the cells grown on 6-cm-diameter dishes were pretreated in the medium containing drugs (10 M nocodazole, 10 M Taxol, 0.5 M cytochalasin B, and 5 M colchicines; all purchased from Sigma, St. Louis, Mo.)…”

mentioning

confidence: 99%

Microtubule Network Facilitates Nuclear Targeting of Human Cytomegalovirus Capsid

Ogawa-Goto

Tanaka

Gibson

et al. 2003

J Virol

105

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We assessed the requirement of the host cytoskeleton for the intracytosolic transport of incoming human cytomegalovirus (HCMV) capsids. Treatments with microtubule (MT)-depolymerizing drugs nocodazole and colchicine led to a drastic decrease in levels of IE1 antigen, whereas cytochalasin B had no effect on the level of IE1 as determined by Western blot analyses. Sequential treatment including nocodazole washout and removal of cell surface virion revealed that HCMV entry into the cells occurred normally in the absence of the MT network. This finding was also supported by data obtained by monitoring pUL83 signals with an immunofluorescent assay (IFA). Furthermore, we demonstrated a close association of incoming HCMV capsids with MTs by IFA and ultrastructural analyses. In the absence of the MT network, the capsids which had entered the cytoplasm did not move to close proximity of the nucleus. These data suggest that HCMV capsids associate with the MT network to facilitate their own movement to the nucleus before the onset of immediate-early (IE) gene expression and that this association is required to start efficient IE gene expression.Human cytomegalovirus (HCMV) is a prevalent pathogen responsible for significant morbidity and mortality in immunosuppressed individuals. It is a member of the betaherpesvirus family containing a double-stranded 230-kbp DNA genome, which is predicted to encode over 200 open reading frames (15). Like other herpesviruses, HCMV comprises three structural elements, an icosahedral capsid containing the DNA genome, a tegument, and an envelope. The dense tegument layer is distinguished from those of other herpesvirus families and is composed of a large number of proteins, but little is known about its structure or function (9). The infectious cycle of HCMV begins with multistep binding of virus to the cell surface, followed by penetration. Although release and dissociation of some tegument components occur upon entry (19), unlike those of alphaherpesviruses, the capsids are assumed to retain abundant structural tegument proteins and supposed to move towards the nuclear envelope (NE). Among the many unresolved processes, the mechanism of HCMV capsid transport in the cytoplasm remains unknown. Since movement of virus through the cytosol is not likely to occur by free diffusion but rather by a cellular transport system, it is conceivable that interactions of viral tegument proteins with the host machinery involved in cellular transport systems permits an active process (22). It has been reported that the efficient transport of herpes simplex virus type 1 (HSV-1) within cells in culture requires the microtubule (MT)-dependent motor proteins (8, 23), and interaction of tegument components with the motor proteins has been reported (7). MTs are organized in a polarized fashion with minus ends at the MT organization center (MTOC) and plus ends at the cell periphery; therefore, an interaction with MTs could facilitate proper movement of the cytosolic capsids to the NE, as has been shown for other v...

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Binding of human cytomegalovirus to sulfated glucuronyl glycosphingolipids and their inhibitory effects on the infection.

Cited by 8 publications

References 35 publications

Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

A Role for 3- O -Sulfated Heparan Sulfate in Promoting Human Cytomegalovirus Infection in Human Iris Cells

Microtubule Network Facilitates Nuclear Targeting of Human Cytomegalovirus Capsid

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