Stable constitutive expression of glycoprotein B (gpUL55) of human cytomegalovirus in permissive astrocytoma cells

Reis, Bernardo Sgarbi; Bogner, Elke; Reschke, M.; Richter, Arndt; Mockenhaupt, T.; Radsak, K.

doi:10.1099/0022-1317-74-7-1371

Cited by 23 publications

(17 citation statements)

References 39 publications

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“…The anti-myc antibodies detected only the uncleaved precursor form of gB, suggesting that introduction of the myc tag on the C terminus of this glycoprotein could have altered normal intracellular trafficking of gB or alternatively that a majority of gB following transient expression in these cells was found as the uncleaved form as reported by other investigators (Fig. 4A) (15,33). In this experiment, the anti-myc antibodies detected a protein from cells cotransfected with the gM-and gN-expressing plasmids that migrated between 80 and 90 kDa, a mass consistent with an aggregated form of gM or perhaps, an incompletely reduced gM/gN complex that could contain an incompletely modified form(s) of gN (5,23,24).…”

Section: Resultssupporting

confidence: 55%

Human Cytomegalovirus Infection Elicits a Glycoprotein M (gM)/gN-Specific Virus-Neutralizing Antibody Response

Shimamura

Mach

Britt

2006

J Virol

105

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Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that infects 40 to 90% of adult human populations. HCMV infections are often asymptomatic in healthy individuals but can cause severe organ and life-threatening disease in immunocompromised patients. The antiviral antibody response to HCMV infection is complex and is known to include virus-neutralizing antibody production against surface glycoproteins encoded by HCMV. We have investigated the human antibody response to a complex of HCMV surface glycoproteins composed of glycoprotein M (gM)/gN, the gene products of the UL100 and UL73 open reading frames. Mouse monoclonal antibodies generated against gM/gN have previously been shown to neutralize HCMV infection of human fibroblasts in vitro. To determine whether human antibodies reactive with the gM/gN complex possess virus-neutralizing properties, we isolated human antibodies reactive with gM/gN from pooled human HCMV hyperimmune globulin by affinity purification using recombinant gM/gN. The affinitypurified human anti-gM/gN antibodies reacted specifically by immunofluorescence with HCMV-infected human fibroblasts and with cells transiently expressing gM/gN, but not with cells transfected with plasmids encoding other immunogenic HCMV proteins. The anti-gM/gN antibodies also reacted specifically only with gM/gN in immunoblot assays using lysates of transfected cells expressing specific HCMV proteins. Last, human anti-gM/gN antibodies efficiently neutralized infectious HCMV in vitro with a capacity comparable to that of human anti-gB antibodies. These data indicated that gM/gN can elicit a virus-neutralizing antibody response in humans infected with HCMV and therefore should be considered a potential candidate for inclusion in prophylactic CMV vaccines.

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Section: Resultssupporting

confidence: 55%

Human Cytomegalovirus Infection Elicits a Glycoprotein M (gM)/gN-Specific Virus-Neutralizing Antibody Response

Shimamura

Mach

Britt

2006

J Virol

105

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“…To our knowledge, the only other cell line capable of full permissive infection by HCMV is the human glioblastoma-astrocytoma cell line U373 MG cells (43), which are no longer available from the ATCC, as there are questions about their authenticity (21). Using the homogenous population of Kasumi-3 cells, we were able to infect over 11% of viable cells, which, when cultured long-term, were capable of maintaining the viral genome in an environment of repressed viral gene transcription.…”

Section: Discussionmentioning

confidence: 99%

A Myeloid Progenitor Cell Line Capable of Supporting Human Cytomegalovirus Latency and Reactivation, Resulting in Infectious Progeny

O’Connor

Murphy

2012

J Virol

115

159

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Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractablein vitromodel system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractablein vitromodel system with which to study HCMV latency and reactivation.

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“…Construction of expression plasmid pRC/CMV-gB coding for HCMV gB (25) and pRC/CMV-gB(Del3) coding for HCMV gB lacking residues 865-906 (26) has been described previously. The constructs pCMUIV-CD8 coding for wild-type CD8 and pCMUIV-CD8/E19 coding for the fusion protein CD8/E19 were generously provided by T. Nilsson (Heidelberg, Germany) (27).…”

Section: Dna Constructsmentioning

confidence: 99%

“…For this purpose, parallel cultures of COS7 cells were transfected with expression plasmids pRC/CMV-gB encoding INM-bound authentic HCMV gB (25) or with pCMUIV-CD8/E19 encoding an ER-resident CD8 chimera (27) (Fig. 1A).…”

Section: Cell Fractionation Preparation Of Extracts and Immunoprecimentioning

confidence: 99%

Identification of a Novel Signal Sequence That Targets Transmembrane Proteins to the Nuclear Envelope Inner Membrane

Meyer

Radsak

2000

Journal of Biological Chemistry

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Herpesvirus maturation requires translocation of glycoprotein B homologue from the endoplasmic reticulum to the inner nuclear membrane. Glycoprotein B of human cytomegalovirus was used in this context as a model protein. To identify a specific signal sequence within human cytomegalovirus glycoprotein B acting in a modular fashion, coding sequences were recombined with reporter proteins. Immunofluorescence and cell fractionation demonstrated that a short sequence element within the cytoplasmic tail of human cytomegalovirus glycoprotein B was sufficient to translocate the membrane protein CD8 to the inner nuclear membrane. This carboxyl-terminal sequence had no detectable nuclear localization signal activity for soluble ␤-Galactosidase and could not be substituted by the nuclear localization signal of SV40 T antigen. For glycoprotein B of herpes simplex virus, a carboxyl-terminal element with comparable properties was found. Further experiments showed that the amino acid sequence DRLRHR of human cytomegalovirus glycoprotein B (amino acids 885-890) was sufficient for nuclear envelope translocation. Single residue mutations revealed that the arginine residues in positions 4 and 6 of the DRLRHR sequence were essential for its function. These results support the view that transmembrane protein transport to the inner nuclear membrane is controlled by a mechanism different from that of soluble proteins.The nuclear envelope consists of three morphologically distinct membrane domains (1, 2): (i) the outer nuclear membrane, which is contiguous with the membranes of the rough endoplasmic reticulum (RER), 1 (ii) the pore membranes, which connect outer and inner nuclear membranes and are associated with the nuclear pore complexes, and (iii) the inner nuclear membrane (INM), which is adjacent to the nuclear lamina, a mesh-work of intermediate filament proteins termed lamins (3). Inner and outer nuclear membranes enclose the perinuclear space that continues into that of the RER. While the outer nuclear membrane shares the membrane proteins of the RER, the INM contains a distinct set of integral proteins (4), including the lamin B receptor (LBR) (5) and the lamina-associated polypeptides 1 and 2 (LAP1 and LAP2) (6, 7).In cells that are virus-infected, the various cellular membranes are decorated with specific viral proteins in addition. In the case of herpesviruses that undergo a characteristic nuclear phase of maturation, viral transmembrane glycoproteins are also translocated from the site of their synthesis into the INM, e.g. the glycoprotein B (gB) homologues of Epstein-Barr virus (8, 9), herpes simplex virus type 1 (HSV-1) (10), and human cytomegalovirus (HCMV) (11). The specific localization in the INM not only suggests a role of these proteins in herpesvirus envelopment at the inner nuclear membrane (12) but also implies that specific mechanisms mediate their transport from the RER to the INM compartment. It is currently not precisely known how this apparently specific targeting of cellular and viral transmembrane protein...

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Stable constitutive expression of glycoprotein B (gpUL55) of human cytomegalovirus in permissive astrocytoma cells

Cited by 23 publications

References 39 publications

Human Cytomegalovirus Infection Elicits a Glycoprotein M (gM)/gN-Specific Virus-Neutralizing Antibody Response

Human Cytomegalovirus Infection Elicits a Glycoprotein M (gM)/gN-Specific Virus-Neutralizing Antibody Response

A Myeloid Progenitor Cell Line Capable of Supporting Human Cytomegalovirus Latency and Reactivation, Resulting in Infectious Progeny

Identification of a Novel Signal Sequence That Targets Transmembrane Proteins to the Nuclear Envelope Inner Membrane

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