The effect of murine cytomegalovirus IE-3 specific shRNA is dependent on intragenic target site due to multiple transcription initiation sites

Hdl Handle:
http://hdl.handle.net/10675.2/680
Title:
The effect of murine cytomegalovirus IE-3 specific shRNA is dependent on intragenic target site due to multiple transcription initiation sites
Authors:
Marshall, Brendan; Zhang, Ming; Atherton, Sally S
Abstract:
Background: Murine cytomegalovirus (MCMV) is closely related to human cytomegalovirus (HCMV) which is responsible for a variety of diseases, including retinitis, in immunocompromised individuals. Small inhibitory RNA molecules directed against essential viral regulatory genes may prove clinically useful.; Methods: Small hairpin RNAs (shRNAs) directed against the essential MCMV immediate early-3 gene (IE-3) were designed and tested in vitro at m.o.i.'s of 2 and 0.2 to determine if virus replication could be inhibited.; Results: At m.o.i. = 2, a MCMV IE-3 specific shRNA specific for sequences at the beginning of exon 5 inhibited virus replication with a maximum decrease in virus titer of approximately two logs at day 5 p.i. Surprisingly, however, at m.o.i. = 0.2, the same shRNA enhanced virus replication. In the latter case, the main IE-3 product observed in infected cells was not the expected 88 kd full length IE-3 protein observed at high m.o.i. but rather a truncated 45 kd form of this protein. Rapid analysis of 5' cDNA ends (5' RACE) indicated that substantial differences exist in the transcript profile produced by the IE-3 gene at low and high m.o.i. early after infection and that multiple transcripts are produced under both conditions. One such transcript, which originated in exon 5 of the IE-3 gene, was located outside the region targeted by our shRNA and was the major transcript produced at low m.o.i. Targeting of this exon 5 transcript with a second shRNA resulted in inhibition of virus replication at both low and high m.o.i.; Conclusions: These studies indicate that IE-3 has a complex transcriptional profile and that shRNA targeting of this and other viral regulatory genes which produce multiple transcripts may have unexpected effects on virus replication.
Citation:
Herpesviridae. 2011 Sep 18; 2:9
Issue Date:
18-Sep-2011
URI:
http://hdl.handle.net/10675.2/680
DOI:
10.1186/2042-4280-2-9
PubMed ID:
21923934
PubMed Central ID:
PMC3192721
Type:
Article
ISSN:
2042-4280
Appears in Collections:
Department of Cellular Biology and Anatomy: Faculty Research and Presentations

Full metadata record

DC FieldValue Language
dc.contributor.authorMarshall, Brendanen_US
dc.contributor.authorZhang, Mingen_US
dc.contributor.authorAtherton, Sally Sen_US
dc.date.accessioned2012-10-26T16:29:31Z-
dc.date.available2012-10-26T16:29:31Z-
dc.date.issued2011-09-18en_US
dc.identifier.citationHerpesviridae. 2011 Sep 18; 2:9en_US
dc.identifier.issn2042-4280en_US
dc.identifier.pmid21923934en_US
dc.identifier.doi10.1186/2042-4280-2-9en_US
dc.identifier.urihttp://hdl.handle.net/10675.2/680-
dc.description.abstractBackground: Murine cytomegalovirus (MCMV) is closely related to human cytomegalovirus (HCMV) which is responsible for a variety of diseases, including retinitis, in immunocompromised individuals. Small inhibitory RNA molecules directed against essential viral regulatory genes may prove clinically useful.en_US
dc.description.abstractMethods: Small hairpin RNAs (shRNAs) directed against the essential MCMV immediate early-3 gene (IE-3) were designed and tested in vitro at m.o.i.'s of 2 and 0.2 to determine if virus replication could be inhibited.en_US
dc.description.abstractResults: At m.o.i. = 2, a MCMV IE-3 specific shRNA specific for sequences at the beginning of exon 5 inhibited virus replication with a maximum decrease in virus titer of approximately two logs at day 5 p.i. Surprisingly, however, at m.o.i. = 0.2, the same shRNA enhanced virus replication. In the latter case, the main IE-3 product observed in infected cells was not the expected 88 kd full length IE-3 protein observed at high m.o.i. but rather a truncated 45 kd form of this protein. Rapid analysis of 5' cDNA ends (5' RACE) indicated that substantial differences exist in the transcript profile produced by the IE-3 gene at low and high m.o.i. early after infection and that multiple transcripts are produced under both conditions. One such transcript, which originated in exon 5 of the IE-3 gene, was located outside the region targeted by our shRNA and was the major transcript produced at low m.o.i. Targeting of this exon 5 transcript with a second shRNA resulted in inhibition of virus replication at both low and high m.o.i.en_US
dc.description.abstractConclusions: These studies indicate that IE-3 has a complex transcriptional profile and that shRNA targeting of this and other viral regulatory genes which produce multiple transcripts may have unexpected effects on virus replication.en_US
dc.rightsCopyright ©2011 Marshall et al; licensee BioMed Central Ltd.en_US
dc.titleThe effect of murine cytomegalovirus IE-3 specific shRNA is dependent on intragenic target site due to multiple transcription initiation sitesen_US
dc.typeArticleen_US
dc.identifier.pmcidPMC3192721en_US
dc.contributor.corporatenameDepartment of Cellular Biology and Anatomy-
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