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| Dharmacon in the Real World |
Dharmacon SMARTpool® siRNA - identification of
genes critical to HIV replication
Allison O'Brien, PhD
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Recently, two independent studies demonstrated, in a clinically relevant setting,
the utility of siRNA-mediated silencing in the identification of novel targets for
alternative HIV therapies. Both the National Center for HIV, STD, and TB Prevention
at the Center for Disease Control (CDC) and the Genomics Institute of the Novartis
Research Foundation (GNF) used Dharmacon's SMARTselection™ and
SMARTpool® technologies to identify genes involved in HIV replication.
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Since its discovery in 1981, HIV (Human Immunodeficiency Virus) has led to the
deaths of 25 million people worldwide, and for the last several years has taken
the lives of 3 million people per year (1). Although there have been great
improvements in the treatment of HIV, AIDS (autoimmune deficiency syndrome), the
disease caused by HIV, still continues to be a worldwide pandemic. Initial studies
of the HIV life cycle (Figure 1) led to treatments targeting viral proteins important
for viral replication. These drugs fall under the categories Protease Inhibitors
(PIs), Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs), and
Non-Nucleoside Reverse and Transcriptase Inhibitors (NNRTIs).
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Click to enlarge
Figure 1. Illustration of the Life Cycle of HIV
(Human Immunodeficiency Virus) |
Despite the initial success of these therapies, an increasing problem is the
emergence of drug resistant strains of HIV that arise due to the high viral mutation
rate. Thus, to create new HIV treatments, researchers continue to seek new targets
to impede HIV infection. Factors that are important for viral entry into the host
cell, but non-essential to host survival, are potentially good targets to inhibit the
HIV life cycle (Figure 1). As the host cell genome is more stable than the viral
genome, this approach may lead to drugs that better evade viral resistance. Host cell
membrane receptors are prime targets as they facilitate viral entry. Initial efforts
toward inhibiting host factors for HIV treatment focused on the membrane receptor,
CD4, and the co-receptors, CCR5 and CXCR4, which are utilized by HIV to gain entry
into the host cell. Figure 1 diagrams the HIV life cycle, where the virus binds to
the membrane receptors, fuses with the host cell membrane, and the viral RNA enters
and infects the host cell. There are several drugs in clinical trials to inhibit these
membrane receptors and fall under the category of Entry Inhibitors (including Fusion
Inhibitors) (2). However, there are concerns with these drugs accelerating the disease,
as CCR5 inhibitors might drive HIV to utilize other receptors, such as the co-receptor
CXCR4, for entry into the host cell, creating a more rapid decline in host cells (3).
Consequently, the discovery of novel targets and host factors involved in HIV infection
remains a very active area of research.
With the goal of identifying novel host cell factors, the Genomics Institute of the Novartis
Research Foundation (GNF) performed an siRNA screen targeting 5,000 genes for potential
therapeutic targets (4). The screen revealed that the knockdown of the novel host factor
PAK3 (p21-Activated Kinase) negatively affected HIV infection. While it was previously known
that a member of the PAK family was involved in the viral life cycle, the exact member was
controversial and most research supported involvement of PAK2 (4). To more clearly define the
individual roles of the group I PAK genes, Dharmacon SMARTpool siRNA reagents were
utilized to silence each of the three family members, PAK1, PAK2, and PAK3. The siRNA
reagents effectively reduced the protein level of each target however, only the siRNA against
PAK1 and PAK3 led to a significant reduction in HIV infection. Further analysis demonstrated
that the knockdown of Pak1 decreased the number of integrated proviruses. (A provirus is a
retrovirus that has integrated into the DNA of a host cell, as shown in Figure 1.) This
conclusion was significant as siRNA-mediated gene silencing determined the exact member of
the PAK family involved in the viral life cycle, a result that previous efforts were unable
to ascertain. Finally, GNF researchers found that the overexpression of PAK1 enhanced
HIV-IIIb infection twofold over the negative control, further reinforcing its role in HIV
infection. Thus, due to the strong dependence of HIV infection on PAK1 expression, PAK1 was
found to be a viable host cell target for HIV drug treatments.
In a different study, the National Center for HIV, STD, and TB Prevention at the Center for
Disease Control (CDC), having previously identified 200 candidate host genes that potentially
play a critical role in the life cycle of HIV, focused their study on the Rab proteins (5).
Rab proteins regulate vesicular transport pathways, facilitating the entry and exit of material
from the cell. Dharmacon SMARTpool siRNA reagents were used to silence Rab9 and Rab11A,
as well as other genes involved in the same transport pathway including TIP47, p40, and PIKfyve.
The CDC researchers found that Rab9 siRNA caused a 90% reduction in Rab9 mRNA, and greatly
reduced HIV-1 replication by 90%. To further characterize cellular transport mechanisms, cells
were transfected individually with either Rab9, TIP47, p40, PIKfyve or Rab11A SMARTpool
siRNAs and then infected with HIV. For all five targets, 80% mRNA knockdown was assessed using
RT-PCR. Moreover, HIV replication decreased by 75-90%, confirming that HIV utilizes TIP47 and
that HIV replication depends on the expression of the Rab9 effector p40, Rab11A, and PIKfyve
(which promotes p40 membrane attachment). This inhibition of HIV replication further supports
the dependence of the vesicular transport pathway on Rab9. Furthermore, the inhibition of HIV
by all five siRNAs indicates a potential pathway of viral egress and implicates Rab9 as an
important host cell target for HIV treatment.
In conclusion, these studies demonstrate the utility of siRNA-mediated silencing as a robust
tool to identify genes that regulate cellular pathways. Furthermore, RNA interference is a
powerful approach for discovering new genes that may serve as targets for therapeutics and
for gaining insight into viral replication mechanisms.
References
1. UNAIDS/WHO "AIDS Epidemic Update: December 2005.
2005. United Nations Programme on AIDS. [Available here]
2. Entry Inhibitors (including Fusion Inhibitors).
2006. Aids Meds. [Available here]
3. Investigational Drugs: Entry and Fusion Inhibitors.
2006. Aids Info. [Available here]
4. Nguyen, D. G., et al. ""UnPAKing" human immunodeficiency virus (HIV)
replication: using small interfering RNA screening to identify novel cofactors
and elucidate the role of group I PAKs in HIV infection."
J Virol 80.1 (2006): 130-7.
5. Murray, J. L., et al. "Rab9 GTPase is required for replication of human
immunodeficiency virus type 1, filoviruses, and measles virus."
J Virol 79.18 (2005): 11742-51.
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| Dharmacon in the Real World |
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