Comparison of dot-blot DNA hybridisation and immediate early nuclear antigen production in cell culture for the rapid detection of human cytomegalovirus in urine

ISIS 2922 is a phosphorothioate oligonucleotide that is complementary to human cytomegalovirus (CMV) immediate-early (IE) RNA and that exhibits potent and specific antiviral activity against CMV in cell culture assays. Specific assay systems were developed to separately characterize the antisense and nonantisense components of the antiviral activity mediated by ISIS 2922. In U373 cells transformed with cDNA encoding the CMV IE 55-kDa (IE55) protein, expression was inhibited at nanomolar concentrations comparable to effective concentrations in antiviral assays. The specificity of inhibition was demonstrated by using control oligonucleotides incorporating progressive base changes to destabilize oligonucleotide-RNA base pairing and by showing a lack of inhibition of the CMV IE72 product expressed from the same promoter. Inhibition of IE55 protein expression correlated with a reduction in mRNA levels consistent with an RNase H-mediated termination event. Studies with virus-infected cells demonstrated that antisense and nonantisense mechanisms contribute to the antiviral activity of ISIS 2922. Base complementarity to target RNA was important for optimal activity in antiviral assays, but base changes affecting parameters other than hybridization affinity also influenced antiviral activity. Sequence-independent inhibition of virus adsorption to host cells by phosphorothioate oligonucleotides was also observed at high concentrations. Therefore, at least three different mechanisms may contribute to the antiviral activity of ISIS 2922 in cell culture: antisense-mediated inhibition of target gene expression; nonantisense, sequence-dependent inhibition of virus replication; and sequence-independent inhibition of virus adsorption to host cells.

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Rapid detection of cytomegalovirus in cell culture by indirect immunoperoxidase staining with monoclonal antibody to an early nuclear antigen.

Journal of Clinical Microbiology ◽

10.1128/jcm.21.5.669-673.1985 ◽

1985 ◽

Vol 21 (5) ◽

pp. 669-673 ◽

Cited By ~ 45

Author(s):

P D Swenson ◽

M H Kaplan

Keyword(s):

Cell Culture ◽

Monoclonal Antibody ◽

Rapid Detection ◽

Nuclear Antigen ◽

Immunoperoxidase Staining ◽

Indirect Immunoperoxidase ◽

Indirect Immunoperoxidase Staining

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Sumoylation of the major immediate-early IE2 protein of human cytomegalovirus Towne strain is not required for virus growth in cultured human fibroblasts

Journal of General Virology ◽

10.1099/vir.0.79954-0 ◽

2004 ◽

Vol 85 (8) ◽

pp. 2149-2154 ◽

Cited By ~ 17

Author(s):

Hye-Ra Lee ◽

Jin-Hyun Ahn

Keyword(s):

Human Cytomegalovirus ◽

Human Fibroblasts ◽

Artificial Chromosome ◽

Immediate Early ◽

Nuclear Antigen ◽

Virus Growth ◽

Cultured Human Fibroblasts ◽

Proliferating Cell ◽

Revertant Virus

Sumoylation of the major immediate-early IE2 protein of human cytomegalovirus has been shown to increase transactivation activity in target reporter gene assays. This study examined the role of IE2 sumoylation in viral infection. A Towne strain-based bacterial artificial chromosome clone was generated encoding a mutated form of the IE2 protein with Lys→Arg substitutions at positions 175 and 180, the two major sumoylation sites. When human fibroblast (HF) cells were infected with the reconstituted mutant virus, (i) viral growth kinetics, (ii) the accumulation of IE1 (UL123), IE2 (UL122), p52 (UL44) and pp65 (UL83) proteins and (iii) the relocalization of the cellular small ubiquitin-like modifier (SUMO)-1, p53 and proliferating cell nuclear antigen proteins into viral DNA replication compartments were comparable with those of the wild-type and the revertant virus. The data demonstrate that sumoylation of IE2 is not essential for virus growth in cultured HF cells.

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