Biomedical subjects

K Radsak

Publications and source records attributed to K Radsak.

At least 37 records · Page 2Linked to original sources

PubMed1995

Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediated by the human endoprotease furin.

Inhibition of endoproteolytic cleavage of glycoprotein B (gB; gpUL55) of human cytomegalovirus was achieved by treatment of infected fibroblasts with decanoyl peptidyl chloromethyl ketone (decRVKR-CMK), which inhibits the action of cellular subtilisin-like endoproteases with the amino acid recognition motif R x K/R R. Uncleaved gB precursor molecules of 160 kDa that were accumulated were endoglycosidase H resistant, suggesting that correct cellular transport occurred in the presence of the drug. The inhibitor also prevented endoproteolytic gB processing in CV-1 cells infected with a recombinant vaccinia virus-gB construct (VVgB). Evidence for direct involvement of the ubiquitous subtilisin-like endoprotease furin in gB cleavage was obtained from the observation that coinfection of CV-1 cells with WgB and a recombinant vaccinia-human furin construct reestablished endoproteolytic activity which was normally absent late after infection with WgB alone.

Cells, Cultured↗

PubMed1995

Constitutive expression of human cytomegalovirus glycoprotein B (gpUL55) with mutagenized carboxy-terminal hydrophobic domains.

Stable transfectants were selected from human astrocytoma cells (U373) after transfection with recombinant expression vectors carrying the human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) gene with alternative deletions of hydrophobic domain segment 1 (hd1) or segment 2 (hd2) of the carboxy-terminal potential bipartite membrane anchor domain. Comparative analysis of HCMV gB forms from cell lines gB(Mhd1) and gB(Mhd2), expressing mutagenized gB, and those from cells expressing authentic gB showed that deletion of hd1, but not that of hd2, interfered with efficient proteolytic cleavage of the gB precursor. Both mutagenized gB forms exhibited correct transport to the cell surface. Deletion of hd2, but not that of hd1, caused loss of membrane anchoring of the gB molecule, resulting in secretion of the respective gB form into the culture medium. The carboxy-terminal cleavage product of the soluble gB molecule, which migrated more slowly than its authentic counterpart, was modified by complex carbohydrate side chains and formed disulphide-linked complexes. Our observations indicate that hd2 is essential as well as sufficient for membrane anchoring of the HCMV gB molecule. For hd1, a potential fusogenic role is suggested by the conserved positional pattern of glycine residues, which is comparable to that of known fusion peptides of other viruses.

Amino Acid Sequence↗

PubMed1994

Structural analysis of the US-segment of a viable temperature sensitive human cytomegalovirus mutant.

Structural analysis of the US-segment of a viable temperature sensitive human cytomegalovirus mutant (ts9) by the use of restriction enzymes, specific amplifications by the polymerase chain reaction and DNA sequencing, revealed deletions of open reading frames (orf) US14 and part of US15 in addition to that concerning US1 through US13 as reported previously [9]. It was further verified that the Hind III H-fragment and a major portion of the Hind III W-fragment were duplicated in inverted orientation. By the use of a monospecific antibody prepared against a procaryotic recombinant US11-specific product it was shown that the ts9 mutant was in fact deficient in a 30 kDa polypeptide, the gene product of orf US11.

Base Sequence↗

PubMed1994

Replication of human cytomegalovirus in a rhabdomyosarcoma cell line depends on the state of differentiation of the cells.

The replication of human cytomegalovirus (HCMV) was investigated in a new human rhabdomyosarcoma cell line (KFR) with morphological and biochemical characteristics of fetal striated muscle precursors (rhabdomyoblasts). KFR cells exhibited the unique property for spontaneous morphological transformation from a poorly-differentiated state into well-differentiated (myotube-like) rhabdomyoblasts. The poorly-differentiated rhabdomyoblasts promoted both complete viral gene expression and the production of infectious virus. In contrast, in well-differentiated rhabdomyoblasts HCMV infection was abortive. The results showed that replication of HCMV in this human rhabdomyosarcoma cell line depended on the state of cellular differentiation.

Antigens, Viral↗

PubMed1993

Identification of the gene product encoded by ORF UL56 of the human cytomegalovirus genome.

Experiments were undertaken to identify the product of open reading frame (orf) UL56 of human cytomegalovirus (HCMV), the putative homolog of infected cell protein (ICP) 18.5 of herpes simplex virus type 1 (HSV-1) that is thought to be involved in viral nucleocapsid maturation. Northern blotting using a unique fragment of orf UL56 revealed a specific transcript of about 3.0 kb in HCMV-infected fibroblasts early and late postinfection (p.i.). Two overlapping fragments of UL56 were subcloned for procaryotic expression and the recombinant proteins were used for affinity purification of an antibody fraction from human convalescent serum (pab UL56). The affinity purified antibody recognized a polypeptide with an apparent molecular weight of about 130 kDa (p 130) in immunoblots of virions and of infected cell extracts late p.i. Upon treatment of isolated virions with nonionic detergent the p 130 polypeptide separated with the nucleocapsid/tegument fraction. The amino acid sequence of p 130 deduced from the nucleotide sequence exhibited a high homology to ICP 18.5 of HSV-1.

Blotting, Northern↗

PubMed1993

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

Permanent cell lines showing homogeneous constitutive expression of glycoprotein B (gpUL55; gB) of human cytomegalovirus (HCMV) were selected, in the presence of geneticin, from human astrocytoma cells (U373) after transfection with recombinant pRC/CMV-gB carrying the complete coding sequence for HCMV gB and for aminoglycoside phosphotransferase. The biosynthesis and processing including specific proteolytic cleavage, formation of disulphide-linked oligomers as well as transport of recombinant gB in three of four established transformed cell lines essentially resembled that found in infected parental U373 except for eventual degradation after 2 h of gB synthesis. Analysis of the fourth transformant expressing uncleaved gB suggested that proteolytic cleavage is not required for normal intracellular transport. The stable transformants retained permissiveness for productive superinfection with HCMV. The application of cell lines transformed with mutagenized HCMV gB for the rescue of genetically engineered HCMV mutants is discussed.

Astrocytoma↗

PubMed1993

Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes.

We have investigated by electron microscopy the envelopment of progeny human cytomegalovirus particles and vaccinia virus particles. As host cells we used primary human foreskin fibroblasts, in which both viruses replicate, and also AtT20 cells in which vaccinia virus, but not human cytomegalovirus, replicates. As we show here, primary human foreskin fibroblasts contain tubular early endosomes like those in AtT20 cells and many other cells in culture. Our aim was to ascertain whether or not the cisternae which wrap the maturing progeny viral particles are related to tubular endosomes. When infected cells were incubated with the fluid phase endocytic tracer horseradish peroxidase (HRP) at appropriate times after infection (5-8 h post vaccinia infection and 72 h or 96 h post cytomegalovirus infection), we found that many of the partially or fully enveloped vaccinia and human cytomegalovirus particles in the cytoplasm had HRP reaction product in the lumen of their cisternal envelope. Examination of single and serial sections indicated that the cisternal envelopes of the progeny virions were derived from tubular endosomes. Pulse-chase experiments with HRP showed that the viral particles with envelopes derived from tubular endosomes were not directed to late endosomes and autophagic digestion but were released from the cells. Experiments with Brefeldin A established that the envelopment of viral particles by endosomal cisternae proceeded for at least 2 h in the absence of a Golgi apparatus. Our data indicate that vaccinia virus and human cytomegalovirus (and presumably other pox and herpes viruses) have evolved to utilize early endocytic compartments to achieve their egress from host cells. We also found that in cells infected by vaccinia virus the delivery of the endocytic tracer to the Golgi apparatus is greatly enhanced over that in controls or cells infected with human cytomegalovirus. The cytopathic effects of vaccinia virus therefore include perturbation of membrane traffic between endocytic and exocytic compartments.

Brefeldin A↗

PubMed1992

Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus.

Infected cell proteins immunoprecipitated from human cytomegalovirus (HCMV)-infected fibroblasts with glycoprotein H (gH)-specific conformation-dependent monoclonal antibody (mab 14-4 b) were found to consist of three components of 86 kDa, 89 kDa, and 125 kDa (gp 86, 89, and 125). Affinity purified antibodies from human convalescent serum reactive with an NH2-terminal epitope of gH recognized three polypeptides of comparable size in immunoblots, suggesting antigenic relatedness of these three components of the gH-complex. Using subcellular fractions for immunoblotting, gp 86 was identified as an endoglycosidase H (endo H)-sensitive gH-form present in the nuclear fraction whereas gp 89 and gp 125 were endo H-resistant and present in the membrane fraction or in virions. Incomplete endo H-digestion suggested that four of six predicted N-glycosylation sites of the gH molecule were occupied by carbohydrate side chains. Analysis under nonreducing conditions revealed that the compartmentalized as well as virion-associated gH analogs form high molecular weight complexes. The relation of the recognized gH analogs to the processing pathway of gH is discussed.

Cells, Cultured↗

PubMed1992

Inhibition of human cytomegalovirus maturation by brefeldin A.

Brefeldin A (BFA) was found to interfere with specific events of human cytomegalovirus (HCMV) maturation in human fibroblasts. Ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to BFA during the late infectious cycle primarily prevented Golgi-dependent processes, e.g. envelopment of naked cytoplasmic nucleocapsids in the trans-Golgi network (TGN) and normal processing of glycoprotein B. In contrast, the nuclear phase of viral morphogenesis, e.g. transport budding at the nuclear envelope, was not impaired. These observations were compatible with the interpretation that HCMV morphogenesis may involve sequential budding events at the nuclear envelope and at cisternae of the TGN. BFA treatment during the early infectious cycle efficiently inhibited HCMV-DNA synthesis and thus late viral functions, preventing production of viral progeny. Cytotoxicity was excluded as a cause for these findings.

Antiviral Agents↗

PubMed1991

Analyses of the effect of monensin on glycosphingolipid metabolism.

The effect of monensin on glycosphingolipid metabolism was reinvestigated. It was found that monensin increased the uptake of [3H]galactose by human fibroblasts, causing an enhanced metabolic labelling of glycosphingolipids. The preferential incorporation of radioactivity into ceramide monosaccharide in the presence of monensin was accompanied by an equally increased rate of degradation. However, using radiolabelled precursors of the ceramide moiety, a generally diminished uptake of radioactivity into all glycosphingolipids was observed under the influence of monensin, with the exception of mono- and di-hexosylceramide. The enhanced incorporation of radiolabel by these two glycolipids, as well as their increased cellular chemical content, may reflect early synthesis in a monensin-insensitive pre-Golgi compartment after disruption of the cellular Golgi transport system by monensin.

Cells, Cultured↗

PubMed1991

A 15-kilobase-pair region of the human cytomegalovirus genome which includes US1 through US13 is dispensable for growth in cell culture.

The genome of a temperature-sensitive, DNA-negative mutant of human cytomegalovirus was cloned in cosmids and analyzed by restriction endonuclease mapping and Southern blotting. The data presented show that in the mutant genome, nearly half of the short segment was deleted (14.3 to 15.1 kb; map position, 0.83 to 0.9), including the genes for a potential immediate early protein (US3) and a structural glycoprotein of 47 to 52 kDa (US6 through US11). The deleted DNA region was replaced by a 20.8- to 21.6-kb fragment that represented an inverted repetition of the retained portion of the short segment (map position, 0.92 to 1.0), suggesting that US20 through US36 were duplicated in the mutant. Northern (RNA) blots with appropriate probes of total cell RNA extracted from mutant-infected cells confirmed the absence of mRNAs originating from US3 or from US8 through US11. It is concluded that the deleted genes are dispensable for human cytomegalovirus replication in cell culture.

Base Composition↗

PubMed1990

Inhibition of endoproteolytic cleavage of cytomegalovirus (HCMV) glycoprotein B by palmitoyl-peptidyl-chloromethyl ketone.

Endoproteolytic cleavage of glycoprotein B (gB) of human cytomegalovirus (HCMV) is inhibited by palmitoylated peptidyl-chloromethyl ketone (palFAKR-CEK) at concentrations above 30 microM. Inhibitor treatment of HCMV-infected human fibroblasts neither interfered with exposure of gB on the plasma membrane, detected by surface membrane immunostaining, nor reduced production of intracellular infectious viral progeny. Release of infectious virus, on the other hand, was impaired.

Amino Acid Chloromethyl Ketones↗

PubMed1990

Nuclear compartmentation of glycoprotein B of human cytomegalovirus.

Maturation of glycoprotein B (gB) of human cytomegalovirus (HCMV) includes a series of sequential glycosylation steps followed by proteolytic cleavage of the precursor protein. Inhibitors of glycosylation and glycoprotein processing, including tunicamycin, monensin, and bromoconduritol, were used to define further the processing pathway of HCMV gB. The results of these studies indicated that cotranslational glycosylation and intracellular transport are essential for subsequent cleavage of the precursor; early trimming in the endoplasmic reticulum is not a prerequisite but facilitates this processing event. Analysis of purified nuclei with gB-specific monoclonal antibody suggested that the mannose-rich gB-precursor intermediate(s) is (are) compartmentalized in the nuclear fraction. Immunoelectron microscopy revealed that HCMV gB was localized in the outer as well as in the inner nuclear membranes of HCMV-infected fibroblasts.

Acetylglucosaminidase↗

PubMed1989

Alteration of nuclear lamina protein in human fibroblasts infected with cytomegalovirus (HCMV).

Immunoblotting with monoclonal as well as polyclonal lamin antibodies revealed that nuclear lamina proteins of human foreskin fibroblasts (HFF) were differentially affected after infection with human cytomegalovirus (HCMV). Lamin A immunoreactivity was progressively lost during the course of the infectious cycle whereas that of lamin C was comparatively stable. This process was not observed in herpes simplex virus-infected HFF. On the other hand, noninfected arrested HFF stimulated by serum to enter S-phase also exhibited loss of lamin A immunoreactivity. Selective in vivo proteolysis of lamin A is suggested as the possible underlying mechanism.

Cells, Cultured↗

PubMed1989

Induction by sodium butyrate of cytomegalovirus replication in human endothelial cells.

Cultured endothelial cells are shown to be induced in regard to permissiveness to human cytomegalovirus by temporary treatment postinfection with sodium butyrate (1-2 mM). Drug-treated cells are demonstrated to exhibit expression of immediate early and early viral antigens, synthesis of viral DNA and viral structural glycoprotein B. Progeny virus could be visualized by electron microscopy.

Antigens, Viral↗

PubMed1988

Polypeptide synthesis in human fibroblasts infected with DNA-negative mutants of cytomegalovirus.

Two DNA-negative temperature-sensitive mutants of human cytomegalovirus strain AD169 were shown to be deficient in induction of several prominent viral polypeptides when grown in human foreskin fibroblasts under nonpermissive conditions. The use of a monospecific polyclonal antiserum allowed recognition of a further defect in processing of an 'early' viral polypeptide of 135 kD.

Cells, Cultured↗

PubMed1987

Retinoic acid alters the metabolic 3H-labelling of glycosphingolipids.

Retinoic acid increases the incorporation of radioactivity from a mixture of [3H]-galactose and [3H]-glucosamine into glycosphingolipids of serum-starved quiescent human foreskin fibroblasts with a preferential labelling of ceramide mono- and dihexoside as compared to ceramide tri- and tetrahexoside. Under the conditions used, no similar change in the specific labelling of glycoprotein is observed. Alteration in [3H]-precursor uptake into glycolipids comparable to that seen under the influence of retinoic acid does not occur in the presence of phorbolester, colchicine, butyrate or after infection with cytomegalovirus.

Butyrates↗

PubMed1987

Inhibition by monensin of human cytomegalovirus DNA replication.

Monensin, at concentrations which depended on the multiplicity of infection, was found to prevent DNA replication of human cytomegalovirus (HCMV) as well as production of viral progeny in human foreskin fibroblasts. The drug did not affect DNA replication of herpes simplex virus. Inhibition of consecutive HCMV DNA synthesis was also observed following delayed addition of the drug within 12-24 hours postinfection, but was fully reversible upon its removal. Viral replication proceeded, however, without impairment in cultures treated with monensin prior to infection. Induction of viral DNA polymerase activity was not impeded by the inhibitor. Analysis of protein- and glycoprotein synthesis revealed that monensin interfered with the production of a number of HCMV-specific polypeptides. Furthermore, evidence was obtained that the drug may hinder intracellular transport of a 135 kd glycopolypeptide.

Biological Transport↗