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

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.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.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.RNAi is widely used to selectively inhibit gene expression both in vivo and in vitro. The process utilizes small RNAs to interfere with gene expression at both transcriptional and post-transcriptional levels by targeting newly transcribed RNAs for nucleolytic attack and also by causing transcriptional silencing of particular chromosomal regions, such as those rich in heterochromatin. These RNAs are known as siRNAs [1-3]. Another class of small RNA molecules known as micro RNAs (miRNAs) may repress gene expression through inhibition of protein