Inhibition of Human Cytomegalovirus Replication in a Human Retinal Epithelial Cell Model by Antisense Oligonucleotides
The antiviral activity of first and second generation antisense oligonucleotides on human cytomegalovirus (CMV) replication was evaluated in two cell systems, the traditional system on human fibroblasts and on human retinal pigment epithelial (HRPE) cell culture system. To evaluate CMV replication s...
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Published in | Investigative ophthalmology & visual science Vol. 42; no. 1; pp. 163 - 169 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Rockville, MD
ARVO
01.01.2001
Association for Research in Vision and Ophtalmology |
Subjects | |
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Abstract | The antiviral activity of first and second generation antisense oligonucleotides on human cytomegalovirus (CMV) replication was evaluated in two cell systems, the traditional system on human fibroblasts and on human retinal pigment epithelial (HRPE) cell culture system.
To evaluate CMV replication strategies within the retina, an HRPE cell system permissive to CMV replication was developed. In this study, the antiviral activity of the antisense oligonucleotides, ISIS 2922 (Vitraven) and ISIS 13312, was evaluated in the traditional fibroblast antiviral assay and in the HRPE cell system. Antiviral activity was measured by evaluating inhibition of virus induced cytopathic effect, virus plaque formation, and virus gene expression.
Both oligonucleotides produced concentration-dependent inhibition of CMV cytopathic effect and CMV plaque formation in both human RPE cells and a human fibroblast cell line, MRC-5. The oligonucleotide, ISIS 2922, demonstrated a mean 50% inhibitory concentration (IC(50)) of 0.04 and 0.24 microM in HRPE and MRC-5 cells, respectively. The second-generation oligonucleotide, ISIS 13312, yielded similar results with IC(50) levels of 0.05 and 0.3 microM in HRPE and MRC-5 cells, respectively. Similar findings were obtained with a CMV clinical isolate. In addition, initiation of effective oligonucleotide treatment could be introduced 6 days after CMV infection in HRPE cells, whereas, in the fibroblast cell line, oligonucleotide treatment was only effective up to 3 days after infection. Semiquantitative RT-PCR analysis demonstrated significant inhibition of CMV intermediate early and late mRNAs by both oligonucleotides.
These studies demonstrate that HRPE cells were significantly more sensitive than fibroblasts to the antiviral actions of ISIS 2922 and ISIS 13312. Moreover, the data indicate that the anti-CMV potency of the two oligonucleotides was similar. The enhanced potency of these oligonucleotides in HRPE cells may be associated with a delay in viral gene transcription and slow viral replication and spread in these cells. |
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AbstractList | PURPOSEThe antiviral activity of first and second generation antisense oligonucleotides on human cytomegalovirus (CMV) replication was evaluated in two cell systems, the traditional system on human fibroblasts and on human retinal pigment epithelial (HRPE) cell culture system.METHODSTo evaluate CMV replication strategies within the retina, an HRPE cell system permissive to CMV replication was developed. In this study, the antiviral activity of the antisense oligonucleotides, ISIS 2922 (Vitraven) and ISIS 13312, was evaluated in the traditional fibroblast antiviral assay and in the HRPE cell system. Antiviral activity was measured by evaluating inhibition of virus induced cytopathic effect, virus plaque formation, and virus gene expression.RESULTSBoth oligonucleotides produced concentration-dependent inhibition of CMV cytopathic effect and CMV plaque formation in both human RPE cells and a human fibroblast cell line, MRC-5. The oligonucleotide, ISIS 2922, demonstrated a mean 50% inhibitory concentration (IC(50)) of 0.04 and 0.24 microM in HRPE and MRC-5 cells, respectively. The second-generation oligonucleotide, ISIS 13312, yielded similar results with IC(50) levels of 0.05 and 0.3 microM in HRPE and MRC-5 cells, respectively. Similar findings were obtained with a CMV clinical isolate. In addition, initiation of effective oligonucleotide treatment could be introduced 6 days after CMV infection in HRPE cells, whereas, in the fibroblast cell line, oligonucleotide treatment was only effective up to 3 days after infection. Semiquantitative RT-PCR analysis demonstrated significant inhibition of CMV intermediate early and late mRNAs by both oligonucleotides.CONCLUSIONSThese studies demonstrate that HRPE cells were significantly more sensitive than fibroblasts to the antiviral actions of ISIS 2922 and ISIS 13312. Moreover, the data indicate that the anti-CMV potency of the two oligonucleotides was similar. The enhanced potency of these oligonucleotides in HRPE cells may be associated with a delay in viral gene transcription and slow viral replication and spread in these cells. The antiviral activity of first and second generation antisense oligonucleotides on human cytomegalovirus (CMV) replication was evaluated in two cell systems, the traditional system on human fibroblasts and on human retinal pigment epithelial (HRPE) cell culture system. To evaluate CMV replication strategies within the retina, an HRPE cell system permissive to CMV replication was developed. In this study, the antiviral activity of the antisense oligonucleotides, ISIS 2922 (Vitraven) and ISIS 13312, was evaluated in the traditional fibroblast antiviral assay and in the HRPE cell system. Antiviral activity was measured by evaluating inhibition of virus induced cytopathic effect, virus plaque formation, and virus gene expression. Both oligonucleotides produced concentration-dependent inhibition of CMV cytopathic effect and CMV plaque formation in both human RPE cells and a human fibroblast cell line, MRC-5. The oligonucleotide, ISIS 2922, demonstrated a mean 50% inhibitory concentration (IC(50)) of 0.04 and 0.24 microM in HRPE and MRC-5 cells, respectively. The second-generation oligonucleotide, ISIS 13312, yielded similar results with IC(50) levels of 0.05 and 0.3 microM in HRPE and MRC-5 cells, respectively. Similar findings were obtained with a CMV clinical isolate. In addition, initiation of effective oligonucleotide treatment could be introduced 6 days after CMV infection in HRPE cells, whereas, in the fibroblast cell line, oligonucleotide treatment was only effective up to 3 days after infection. Semiquantitative RT-PCR analysis demonstrated significant inhibition of CMV intermediate early and late mRNAs by both oligonucleotides. These studies demonstrate that HRPE cells were significantly more sensitive than fibroblasts to the antiviral actions of ISIS 2922 and ISIS 13312. Moreover, the data indicate that the anti-CMV potency of the two oligonucleotides was similar. The enhanced potency of these oligonucleotides in HRPE cells may be associated with a delay in viral gene transcription and slow viral replication and spread in these cells. |
Author | Hooks, John J Grillone, Lisa R Detrick, Barbara Nagineni, Chandrasekharam N Henry, Scott P Anderson, Kevin P |
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Keywords | Human Cell culture Cytomegalovirus Herpesviridae Pigment epithelium Retina Antisense oligonucleotide Betaherpesvirinae Biological activity Virus Eye Antiviral Epithelial cell Replication Fibroblast Retinal |
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Snippet | The antiviral activity of first and second generation antisense oligonucleotides on human cytomegalovirus (CMV) replication was evaluated in two cell systems,... PURPOSEThe antiviral activity of first and second generation antisense oligonucleotides on human cytomegalovirus (CMV) replication was evaluated in two cell... |
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SubjectTerms | Antiviral Agents - pharmacology Biological and medical sciences Blotting, Southern Cells, Cultured Cytomegalovirus - drug effects Cytomegalovirus - genetics Cytomegalovirus - growth & development Cytopathogenic Effect, Viral - drug effects Dose-Response Relationship, Drug Eye Fibroblasts - virology Humans Medical sciences Oligonucleotides, Antisense - pharmacology Pharmacology. Drug treatments Pigment Epithelium of Eye - drug effects Pigment Epithelium of Eye - pathology Pigment Epithelium of Eye - virology Reverse Transcriptase Polymerase Chain Reaction RNA, Viral - analysis Thionucleotides - pharmacology Viral Plaque Assay Virus Replication - drug effects |
Title | Inhibition of Human Cytomegalovirus Replication in a Human Retinal Epithelial Cell Model by Antisense Oligonucleotides |
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