Engineered external guide sequences are highly effective in inducing RNase P for inhibition of gene expression and replication of human cytomegalovirus.
External guide sequences (EGSs), which are RNA molecules derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. Using an in vitro selection procedure, we have previously generated EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the overlapping region of the mRNAs encoding human cytomegalovirus (HCMV) essential transcription regulatory factors IE1 and IE2. The EGS variant was approximately 25-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 93% in IE1/IE2 gene expression and a reduction of 3000-fold in viral growth were observed in HCMV-infected cells that expressed the variant, while cells expressing the tRNA-derived EGS exhibited a reduction of 80% in IE1/IE2 expression and an inhibition of 150-fold in viral growth. Our results provide the first direct evidence that EGS variant is highly effective in blocking HCMV gene expression and growth and furthermore, demonstrate the feasibility of developing effective EGS RNA variants for anti-HCMV applications by using in vitro selection procedures.
Yang
Yong-Hua
YH
Program in Infectious Diseases and Immunity, Program in Comparative Biochemistry, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720, USA.
Li
Hongjian
H
Zhou
Tianhong
T
Kim
Kihoon
K
Liu
Fenyong
F
D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D017874:Immediate-Early Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry; D012367:RNA, Viral / Q000378:metabolism*; D043262:Ribonuclease P / Q000378:metabolism*; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D058727:RNA, Small Untranslated
C088645:IE1 protein, cytomegalovirus; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D015534:Trans-Activators; D014764:Viral Proteins; D012343:RNA, Transfer; D043262:Ribonuclease P; D058727:RNA, Small Untranslated