Prediction and detection of a viral microRNA in AgMNPV infected High Five cells

REYES CARINA, FERRELLI MARÍA LETICIA; GARCÍA MARÍA LAURA; GHIRINGHELLI PABLO DANIEL; ROMANOWSKI VÍCTOR

Buenos Aires

Congreso; International Congress on Invertebrate Pathology and Microbial Control & 45th Annual Meeting of the Society for Invertebrate Pathology; 2012

Society for Invertebrate Pathology

MicroRNAs (miRNAs) have emerged as key players in host?pathogen interaction. Although many virus encoded miRNAs have been identified from different mammalian species, the large family of insect viruses Baculoviridae has hardly been investigated for elucidating the role of miRNAs in host?pathogen interaction. Baculovirus encoded miRNAs were found to be evolutionarily conserved among some closely related members of the group I in the Alphabaculovirus genus. In this study, we have searched for Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) encoded miRNAs in silico through the comparison with the previously identified miRNAs encoded by Bombyx mori nucleopolyhedrovirus (BmNPV). lef-8 had appeared as a putative bmnpv-mir-4 target. Complementary miR-4 seed region was fully conserved between lef-8 from AgMNPV and BmNPV even though identity percentage in the gene sequence is around 75%. Sinteny analysis allowed us to select regions to look for the miR-4 coding gene in AgMNPV through seed region searching. A number of putative agmnpv-miR-4 sequences were found. In order to find the pre-miRNA, several flanking sequences (~100 nt) of putative mature agmnpv-miR-4 were selected and submitted to Mfold program for predicting their secondary structure. A single pre-miR-4 structure was selected with a free energy change (deltaG) of -25.6 kcal/mole. Northern blot hybridization was carried out using RNAs from both AgMNPV infected and uninfected Hi5 cells and an antisense end-labeled oligonucleotide specific to the predicted agmnpv-miR-4 as a probe. In a preliminary assay, specific signal was detected in samples of RNA extracted 144 hours post infection.