[Inhibition of respiratory syncytial virus replication in cultured cells by RNA-cleaving DNAzyme].
OBJECTIVE
DNAzyme/Deoxyribozyme is another novel molecular biological tool following the ribozyme. DNAzyme consists of a 15-nucleotide (nt) internal loop as its catalytic domain and two flanking substrate-recognition domains of 7 to 8 nt each which is complementary to substrate. The RNA substrate is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. DNAzyme has been applied in fields such as viral infectious disease, tumor, cardiovascular disease and genetic disease. But there is no report about using DNAzyme for anti-respiratory syncytial virus purpose. To observe the inhibitory effects of RNA-cleaving DNAzymes on respiratory syncytial virus (RSV) replication in cultured cells.


METHODS
Anti-RSV RNA-cleaving DNAzyme DZ604 was designed to target the RSV genome at the start of the NS2 gene in an effort to inhibit the RNA replication. Microscope and electron microscope were used to observe the effects of anti-RSV genomic RNA DNAzyme on cytopathogenic effect (CPE) and ultrastructural change of 9HTE cell induced by RSV infection. Viral plaque forming reduction assay and MTT assay were used to detect the anti-RSV activity and protective function for RSV infected 9HTE cells of DNAzyme.


RESULTS
Anti-RSV genomic RNA DNAzyme (DZ604) significantly improved CPE of RSV-infected 9HTE cells. The time to appearance of CPE and of total CPE was delayed by using DZ604 in a dose-dependent manner. At a 5 micro mol/L concentration of DZ604, CPE of 9HTE cells induced by RSV infection at 10 and 1 multiplicity of infection (MOI) was not improved. At smaller MOI (0.1, 0.01, 0.001, 0.0001) of RSV infection, CPE of 9HTE cells was significantly lightened by DZ604 at the same concentration. DZ604 also significantly improved ultrastructural change of 9HTE cells at early stage of RSV infection. Reduction in RSV yield was 85.56% and 8.33% at concentrations of 5 micro mol/L and 0.25 micro mol/L of DZ604. DZ604 inhibited RSV yield in a dose-dependent manner (P < 0.05). Non-specific DNAzyme did not have anti-RSV activity (P > 0.05).


CONCLUSIONS
Anti-RSV genomic RNA DNAzyme designed and synthesized in our laboratory was capable of inhibiting respiratory syncytial virus replication specifically in cultured cells. Our data indicated that DNAzymes could be useful for the prevention against respiratory syncytial virus infection.
Zhao
Chang-an
CA
Microbiology & Immunology Lab, Children's Hospital, Chongqing University of Medical Sciences, Chongqing 400014, China.
Zhao
Xiao-dong
XD
Yu
Hai-guo
HG
Wu
Ya-ping
YP
Yang
Xi-qiang
XQ
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D021881:DNA, Catalytic / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008854:Microscopy, Electron; D020545:Respiratory Mucosa / Q000166:cytology / Q000648:ultrastructure / Q000821:virology; D012136:Respiratory Syncytial Viruses / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects*
D021881:DNA, Catalytic