Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.

Human cytomegalovirus (HCMV) causes severe sequelae in immunocompromised hosts. Current antiviral therapies have serious adverse effects, with treatment in many clinical settings problematic, making new therapeutic approaches necessary. We examined the in vitro efficacy of small interfering RNAs (si...

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Autores principales:	Stuart T Hamilton, Jens Milbradt, Manfred Marschall, William D Rawlinson
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c88c754fd4094e3195f941f2eec85799
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spelling	oai:doaj.org-article:c88c754fd4094e3195f941f2eec857992021-11-18T08:17:39ZHuman cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.1932-620310.1371/journal.pone.0097231https://doaj.org/article/c88c754fd4094e3195f941f2eec857992014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24887060/?tool=EBIhttps://doaj.org/toc/1932-6203Human cytomegalovirus (HCMV) causes severe sequelae in immunocompromised hosts. Current antiviral therapies have serious adverse effects, with treatment in many clinical settings problematic, making new therapeutic approaches necessary. We examined the in vitro efficacy of small interfering RNAs (siRNAs) targeting the HCMV gene transcripts UL54 (DNA polymerase), UL97 (protein kinase) and UL122/123 (immediate-early proteins) as inhibitors of viral protein expression and virus replication in cell cultures. Two siRNAs for each HCMV target (designated A and B) were assessed for inhibition efficacy using western blot and standard plaque assays. Continuous human embryonic kidney 293T cells were treated with HCMV or non-specific scrambled (siSc) siRNA followed by transfection with plasmids expressing the target transcripts. Human MRC-5 fibroblasts were HCMV-siRNA or siSc treated, infected with HCMV strain AD169 (1 pfu/cell) and HCMV immediate-early (IE1p72 and IE2p86), early (pp65), early-late (pUL97) and true late (MCP) protein and virus progeny production measured during a single round of replication. Concordant results showed siUL54B, siUL97A and siUL122B displayed the most potent inhibitory effects with a reduction of 92.7%, 99.6% and 93.7% in plasmid protein expression, 65.9%, 58.1% and 64.8% in total HCMV protein expression and 97.2%, 96.2% and 94.3% (p<0.0001) in viral progeny production respectively. Analysing the siRNA inhibitory effects during multiple rounds of HCMV replication at a multiplicity of infection of 0.001 pfu/cell, siUL54B, siUL97A and siUL122B treatment resulted in a reduction of 80.0%, 59.6% and 84.5% in total HCMV protein expression, 52.9%, 49.2% and 58.3% in number of cells infected and 98.5%, 91.4% and 99.1% (p<0.0001) in viral progeny production at 7 dpi respectively. These results suggest potential in vivo siRNA therapies targeting the HCMV gene transcripts UL54, UL97 and UL122/123 would be highly effective, however, the antiviral efficacy of siRNAs targeting UL97 may be more highly dependent on viral load and methods of administration.Stuart T HamiltonJens MilbradtManfred MarschallWilliam D RawlinsonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 6, p e97231 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Stuart T Hamilton Jens Milbradt Manfred Marschall William D Rawlinson Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.
description	Human cytomegalovirus (HCMV) causes severe sequelae in immunocompromised hosts. Current antiviral therapies have serious adverse effects, with treatment in many clinical settings problematic, making new therapeutic approaches necessary. We examined the in vitro efficacy of small interfering RNAs (siRNAs) targeting the HCMV gene transcripts UL54 (DNA polymerase), UL97 (protein kinase) and UL122/123 (immediate-early proteins) as inhibitors of viral protein expression and virus replication in cell cultures. Two siRNAs for each HCMV target (designated A and B) were assessed for inhibition efficacy using western blot and standard plaque assays. Continuous human embryonic kidney 293T cells were treated with HCMV or non-specific scrambled (siSc) siRNA followed by transfection with plasmids expressing the target transcripts. Human MRC-5 fibroblasts were HCMV-siRNA or siSc treated, infected with HCMV strain AD169 (1 pfu/cell) and HCMV immediate-early (IE1p72 and IE2p86), early (pp65), early-late (pUL97) and true late (MCP) protein and virus progeny production measured during a single round of replication. Concordant results showed siUL54B, siUL97A and siUL122B displayed the most potent inhibitory effects with a reduction of 92.7%, 99.6% and 93.7% in plasmid protein expression, 65.9%, 58.1% and 64.8% in total HCMV protein expression and 97.2%, 96.2% and 94.3% (p<0.0001) in viral progeny production respectively. Analysing the siRNA inhibitory effects during multiple rounds of HCMV replication at a multiplicity of infection of 0.001 pfu/cell, siUL54B, siUL97A and siUL122B treatment resulted in a reduction of 80.0%, 59.6% and 84.5% in total HCMV protein expression, 52.9%, 49.2% and 58.3% in number of cells infected and 98.5%, 91.4% and 99.1% (p<0.0001) in viral progeny production at 7 dpi respectively. These results suggest potential in vivo siRNA therapies targeting the HCMV gene transcripts UL54, UL97 and UL122/123 would be highly effective, however, the antiviral efficacy of siRNAs targeting UL97 may be more highly dependent on viral load and methods of administration.
format	article
author	Stuart T Hamilton Jens Milbradt Manfred Marschall William D Rawlinson
author_facet	Stuart T Hamilton Jens Milbradt Manfred Marschall William D Rawlinson
author_sort	Stuart T Hamilton
title	Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.
title_short	Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.
title_full	Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.
title_fullStr	Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.
title_full_unstemmed	Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.
title_sort	human cytomegalovirus replication is strictly inhibited by sirnas targeting ul54, ul97 or ul122/123 gene transcripts.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/c88c754fd4094e3195f941f2eec85799
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Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.

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