Herpesviridae
Department of Cellular Biology and Anatomy, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA.
Published: September 2011
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.
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http://dx.doi.org/10.1186/2042-4280-2-9 | DOI Listing |
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College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, Jiangsu Province, P.R. China.
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