Inhibition of HIV-1 replication by
shRNAs against strategic targets of the nef viral gene
L. Alvarez-Rozo1, M.E. Cardona1,2, A.J. Mohamed2, C.I.E. Smith2, J. Hinkula3,4, H.J. Arteaga1,2
1Universidad 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
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.
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.
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.
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