Abstract
Small
interfering RNAs (siRNAs) can induce potent gene silencing by
degradation of cognate mRNA. However, in dividing cells, the silencing
lasts only 3 to 7 days, presumably because of siRNA dilution with cell
division. Here, we investigated if sustained siRNA-mediated silencing
of human immunodeficiency virus type 1 (HIV-1) is possible in
terminally differentiated macrophages, which constitute an important
reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in
macrophages, and the viral structural gene for p24 were targeted either
singly or in combination. When transfected 2 days prior to infection,
both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the
entire 15-day period of observation, and combined targeting of both
genes abolished infection. To investigate whether exogenously
introduced siRNA is maintained stably in macrophages, we tested the
kinetics of siRNA-mediated viral inhibition by initiating infections at
various times (2 to 15 days) after transfection with CCR5 and p24
siRNAs. HIV suppression mediated by viral p24 siRNA progressively
decreased and was lost by day 7 posttransfection. In contrast, viral
inhibition by cellular CCR5 knockdown was sustained even when
transfection preceded infection by 15 days, suggesting that the
continued presence of target RNA may be needed for persistence of
siRNA. The longer sustenance of CCR5 relative to p24 siRNA in
uninfected macrophages was also confirmed by detection of internalized
siRNA by modified Northern blot analysis. We also tested the potential
of p24 siRNA to stably silence HIV in the setting of an established
infection where the viral target gene is actively transcribed. Under
these circumstances, long-term suppression of HIV replication could be
achieved with p24 siRNA. Thus, siRNAs can induce potent and
long-lasting HIV inhibition in nondividing cells such as
macrophages.
