Marshall Brendan, Zhang Ming, Atherton Sally S
Department of Cellular Biology and Anatomy, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA.
Herpesviridae. 2011 Sep 18;2(1):9. doi: 10.1186/2042-4280-2-9.
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.
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.
鼠巨细胞病毒（MCMV）与人类巨细胞病毒（HCMV）密切相关，HCMV可导致免疫功能低下个体出现多种疾病，包括视网膜炎。针对病毒必需调控基因的小干扰RNA分子可能具有临床应用价值。
设计针对MCMV即刻早期3基因（IE-3）的小发夹RNA（shRNA），并在感染复数为2和0.2时进行体外测试，以确定是否能抑制病毒复制。
在感染复数为2时，一种针对外显子5起始序列的MCMV IE-3特异性shRNA可抑制病毒复制，在感染后第5天病毒滴度最大降低约两个对数。然而，令人惊讶的是，在感染复数为0.2时，相同的shRNA却增强了病毒复制。在后一种情况下，感染细胞中观察到的主要IE-3产物不是在高感染复数时预期的88kd全长IE-3蛋白，而是该蛋白的截短形式45kd。5' cDNA末端快速分析（5' RACE）表明，感染后早期IE-3基因在低感染复数和高感染复数时产生的转录本谱存在显著差异，且在两种情况下都会产生多种转录本。其中一种转录本起源于IE-3基因的外显子5，位于我们的shRNA靶向区域之外，是低感染复数时产生的主要转录本。用第二种shRNA靶向该外显子5转录本可在低感染复数和高感染复数时均抑制病毒复制。
这些研究表明，IE-3具有复杂的转录谱，针对该基因及其他产生多种转录本的病毒调控基因的shRNA可能对病毒复制产生意想不到的影响。
