Abstract

Inhibition of HIV-1 replication by shRNAs against strategic targets of the nef viral gene

L. Alvarez-Rozo¹, M.E. Cardona^1,2, A.J. Mohamed², C.I.E. Smith², J. Hinkula^3,4, H.J. Arteaga<sup>1,2

1</sup>Universidad Industrial de Santander, Department of Basic Sciences, Bucaramanga, Colombia, ²Karolinska Institute, Department of Laboratory Medicine, Huddinge, Sweden, ³Swedish Institute for Infectious Disease Control, Department of Virology, Stockholm, Sweden, ⁴Karolinska Institute, Department of Microbiology, Tumor and Cell Biology, Stockholm, Sweden

Background: RNA interference (RNAi) is a conserved antiviral and gene-expression regulation system. RNAi gene silencing is mediated by double stranded small interfering RNA (siRNA) oligonucleotides, which cleavage only exact complementary mRNA. Several HIV-1 genes have been efficiently targeted by artificial siRNAs. However, due to the high mutation rate of the HIV-1 genome, viral escape mutants are rapidly induced. Essential proteins for viral replication should work at a wide extent of strongly selected mutated variants. Conversely, siRNAs targeting of DNA coding for proteins conferring viral virulence but not essential for replication, could induce low virulence escape mutants which are under lower selective pressure. Strategic selection of targets may attenuate this phenomenon or produce disabled, mutated HIV-1 strains.
Nef is a viral virulence protein, non-essential for replication. In this work strategic siRNA targets, defined by conservation and/or functionality of the domains coded by the target sequence of the nef gene were identified in the polypurine tract, the myristoylation signal, the proline-rich motif and the dimerization region.
Methods: Nine siRNAs against these nef regions were chosen and designed by hand due to restrictions imposed by the computational algorithms. Oligonucleotides coding for shRNA sequences were cloned into a Poll III promoter-containing plasmid and transiently co-transfected into HeLa T4 cells with a Nef-dEGFP reporter vector. The siRNA efficiency was evaluated by flow cytometry, Western blot and qRT-PCR.
Results: Highly efficient shRNAs against the proline-rich motif (two shRNAs with more than 95% of inhibition efficiency), myristoylation signal (1 with 95%), dimerization region (two with 95%) and the Polypurine tract (1with 85%) were identified.
Conclusions: In short, it is possible to design efficient siRNA against short strategic regions of the HIV-1 genome, which apparently are not suitable according computational algorithms. Our plasmids expressing shRNA against HIV-1 generated by this approach may be improved candidates for avoiding induction of escape mutants.