Intracranial Administration of P Gene siRNA Protects Mice from Lethal Chandipura Virus Encephalitis

Kumar, Satyendra; Arankalle, Vidya A.

To deliver the siRNA in shRNA format and to compare efficiency of the siRNA and shRNA, the sequence of the P-2 siRNA was subjected to the link www.ambion.com/techlib/misc/ psilencer_converter.html. DNA oligos were synthesized and named as pH1-P2 or pCMV-P2 depending upon the plasmid promoter used for the expression of shRNA. Following a report documenting hypersusceptibility of Dicer-1-deficient mice to VSV infection as a result of impaired miR-24 and miR-93 expression [26] , we examined the contribution of miR-93 in the replication of CHPV. Use of bioinformatics tools, RNA22 (http://cbcsrv. watson.ibm.com/rna22_targets.html) and RNAhybrid (http:// bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html) revealed that CHPV P gene has two potential binding site for hsa-miR-93. After confirmation, the role of miR-93 in the CHPV replication was assessed by using the stem and loop of miR-93 in different combinations using pSilencer 2.0 H1 neo vector (Ambion, Applied Biosystems International, Foster City, CA) and named pH1-mir-93 (hsa-miR-93 precursor sequence), pH1-miR-93-P-2 (stem of miR-93 was replaced with P-2 siRNA) and pH1-mir-93-P-2-30 (stem and loop of miR-93 was replaced with P-2 siRNA and miR-30 respectively) was used. All the oligos were synthesized by IDT, USA (Table 3) . Sense and antisense oligos were annealed and ligated to either pSilencer 3.1 H1 neo or pSilencer 4.1 CMV neo vectors (Ambion, Applied Biosystems International, Foster City, CA) as recommended by the manufacturer. All the clones were screened by restriction digestion and confirmed by sequencing. Endofree maxi preps were made and quantitated. Different shRNA expressing plasmids (5 mg) were cotransfected individually with plasmid pAcGFP1N1-CHPV-P (500 ng). 24 hr post transfection, cells were harvested, RNA isolated and real time one step RT-PCR and FACS analysis were carried out. Different shRNA expressing plasmids were individually transfected 24 hr at post transfection, cells were infected with 36 PFU/ml of CHPV. At 12 and 24 hr post PI, supernatants were assayed for the presence of the CHPV by plaque assay and real time one step RT-PCR.