[Antisense oligonucleotide inhibition of coxsackievirus B3 gene expression in HeLa cells and dose-response experiments].
OBJECTIVE
In this study, the authors investigated inhibition of coxsackievirus B (CVB) gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon and structural protein coding sequences and also observed the dose-response of the sequence specific inhibition of CVB plaque formation by antisense oligonucleotides.


METHODS
Antiviral activities of these oligonucleotides were evaluated by using plaque reduction assay, yield reduction assay, cytopathic effect (CPE) and Western blot analysis. The cells were treated with random oligonucleotides as a specificity control.


RESULTS
At a screening concentration of 5 micromole, 6 of the phosphorothioate oligonucleotide demonstrated some reduction of virus replication relative to untreated cells. 70%-90% inhibition of virus at 0.1 MOI (multiplicity of infection), 50% inhibition of virus infection at 10 MOI. The levels of the VP1 were reduced in CVB-infected cells treated with Scb561 and Scb733. VP1 was significantly reduced after treatment with 0.625 micromole Scb561 and almost undetectable in cells treated with 2.5 micromole Scb561. Dose response experiments implied that sequence specific oligonucleotide doses were related to effect on inhibition of CVB3 infection. When oligonucleotide doses were increased from 1.25 to 5 micromole, 75% to 90% inhibition were observed with Scb561 and 65% to 80% inhibition with Scb733, whereas random control failed to inhibit CVB replication (8% inhibition for each). CONCLUSION The present studies showed that antisense oligonucleotides against internal ribosome entry site (IRES) and translation initiation codon were capable of specifically inhibiting the synthesis of viral protein and subsequent productive CVB replication.The selective inhibition using antisense oligonucleotide might lead to development of an effective antiviral agent for future clinical evaluation.
Liu
Zhe-wei
ZW
Beijing Central Laboratory of Infection and Immunity, Capital Institute of Pediatrics, Beijing 100020, China.
Sun
Hong-mei
HM
Xiao
Zong-hui
ZH
Qiu
Nan
N
Zhang
Ting
T
Zhao
Jin-ming
JM
D020121:5' Untranslated Regions / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D029822:Enterovirus B, Human / Q000187:drug effects* / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012270:Ribosomes / Q000378:metabolism; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects
D020121:5' Untranslated Regions; D016376:Oligonucleotides, Antisense; D015678:Viral Structural Proteins