Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors
Introduction
The epidemic of human immunodeficiency virus type 1 (HIV-1) still causes millions of new infections and deaths annually. The current therapy against HIV-1, combined antiretroviral therapy or cART, targets the virus with multiple inhibitors to prevent the selection of resistant viral strains. This means that infected persons have to adhere to a strict drug regimen, frequently leading to severe side-effects. It has been proposed that a durable gene therapy based on RNA interference (RNAi) could provide an answer to these problems (Arhel and Kirchhoff, 2009, Adamson and Freed, 2009).
RNAi is an evolutionary conserved mechanism induced by double-stranded RNA (dsRNA) that triggers sequence-specific gene silencing at the post-transcriptional level. The dsRNA-inducer molecule consists of 19–23 nucleotides with one strand complementary to the target mRNA (Hannon, 2002, Fire et al., 1998). RNAi has been shown to effectively inhibit the replication of different viruses, such as poliovirus, hepatitis A, B and C virus, enterovirus, coxsackievirus, rhinovirus and influenza virus (Gitlin et al., 2005, Kanda et al., 2005, Moore et al., 2005, Wilson and Richardson, 2005, Tan et al., 2008, Schubert et al., 2005, Phipps et al., 2004, Ge et al., 2003). Intracellularly expressed short hairpin RNAs (shRNAs) as well as transfected small interfering RNAs (siRNAs) have been successfully used against target sequences in the HIV-1 RNA genome (Coburn and Cullen, 2002, Lee et al., 2002). In cell lines that constitutively express antiviral shRNAs, HIV-1 replication could be inhibited for several weeks, but the virus eventually escaped from the RNAi-induced pressure (ter Brake et al., 2006). Sequencing of the target sequences of the viral escape variants allowed the identification of several escape mechanisms. First, a point mutation in the target sequence can reduce the complementarity with the shRNA inhibitor and thereby abolish the RNAi-suppression (Das et al., 2004). Second, the complete or part of the target region could be deleted, especially when non-essential viral genes are targeted (Das et al., 2004). Indeed, no such deletion-based escape was observed when essential and well-conserved viral sequences were targeted (von Eije et al., 2008). Third, resistance-causing mutations were infrequently observed outside the target region. These mutations elicit a structural change in the HIV-1 mRNA, thus making the target sequence inaccessible for the RNAi-machinery (Westerhout et al., 2005). These results demonstrate that the viral ability to escape from therapy is driven by its high mutation rate. However, HIV-1 is not able to escape when four shRNAs were used simultaneously, similar to the therapeutic success of cART (ter Brake et al., 2008).
Targeting of cellular co-factors, host proteins on which HIV-1 relies to complete its replication cycle, could present an alternative anti-escape approach. By targeting cellular co-factors rather than viral components two main problems concerning drug resistance might be solved. First, therapeutics directed against a viral RNA target have to act on all HIV-1 variants that circulate in the patient and in the epidemic, whereas a cellular mRNA target is constant. Second, it is expected that by targeting cellular co-factors the virus can only escape by evolving to use a different cellular co-factor (Nair et al., 2005, Zhou et al., 2004). Depending on the targeted co-factor, such an escape route may be impossible, although this idea has not been validated experimentally. An obvious disadvantage of co-factor suppression is the possibility of adverse effects on cell metabolism and the host organism. A promising co-factor for therapeutic intervention is the CCR5 molecule, which is one of the receptors for HIV-1 entry. A proportion of the human population carries a 21-base pair deletion in the CCR5 gene and does not express this viral receptor without any physiological problems, but these individuals cannot be infected with a CCR5-using HIV-1 strain (Huang et al., 1996, Liu et al., 1996). This result suggests that other co-factors could exist that are vital for HIV-1 replication, but whose depletion would not necessarily reduce host viability.
In recent years, much effort has been devoted to the identification of novel cellular co-factors that, directly or indirectly, contribute to the viral replication cycle. RNAi played an important role in these studies, as the effect of host protein knockdown on viral replication was assessed in large scale RNAi gene knockdown experiments. Hundreds of novel cellular co-factors for HIV-1 replication were recently identified (Rato et al., 2010, Zhou et al., 2008, Brass et al., 2008, Konig et al., 2008, Yeung et al., 2009, Kok et al., 2009). Proteomics analysis of HIV-1 infected cells also revealed numerous host proteins that could participate in viral replication (Toro-Nieves et al., 2009, Ringrose et al., 2008, Chan et al., 2007, Chan et al., 2009, Wang et al., 2008). However, there are some serious drawbacks to these screens, which used HEK293T or HeLa cells instead of T cells and laboratory-adapted HIV-1 variants or pseudo-typed virus. All these studies are transient in nature, based on the transfection of siRNAs rather than intracellularly expressed shRNAs. Thus, long-term effects on cell toxicity and viral replication could not be analyzed. In this study, we selected thirty candidate co-factors for stable knockdown in a human T cell line to test the impact on cell viability and HIV-1 replication. Co-factors were chosen based on their suggested importance in the viral replication cycle, although for some (like IPO7) this is not without discussion (Zielske and Stevenson, 2005, Ao et al., 2007). The thirty co-factors that we selected are distributed along all steps of the HIV-1 replication cycle, as it is not clear whether targeting a specific step, e.g. early or late, has an advantage. We also tested the concept that targeting of cellular co-factors prevents viral escape. We observed durable inhibition of viral replication upon knockdown of three co-factors (ALIX, TRBP and ATG16), without detecting viral escape.
Section snippets
shRNA constructs
pLKO.1 constructs expressing shRNA candidates and with the puromycin-resistance marker were from the MISSION™ TRC-Hs 1.0 library (Root et al., 2006). Constructs, including the negative control constructs SHC001 and SHC002 (hereafter named SHC1 and SHC2), were obtained from Sigma–Aldrich (St. Louis, MO) as bacterial clones. Plasmid DNA was extracted using the Nucleobond Midiprep columns according to the manufacturer's instructions (Macherey-Nagel, Düren, Germany). For every target gene, 4–5
HIV-1 co-factor screen
We selected thirty cellular co-factors that have been implicated in HIV-1 replication for stable RNAi-mediated knockdown in the human T cell line SupT1. These co-factors facilitate virus replication steps from entry to budding (Fig. 1). Among others, we targeted co-factors that facilitate entry into the target cell (e.g. receptors CD4 and CXCR4), integration of the proviral DNA in the host chromosome (e.g. ATM kinase and INI1), viral transcription (e.g. TRBP), virion assembly (e.g. ABCE1) and
Discussion
We tested whether we could inhibit HIV-1 replication in a human T cell line with a stable RNAi-mediated knockdown of a cellular protein that has been implicated in HIV-1 replication. Thirty cellular co-factors were chosen, distributed along the viral replication cycle. We tested 4 or 5 shRNA inhibitors per co-factor, generating over 140 stable T cell lines. The cell lines were first monitored for an impact on cell growth and cell lines with severe growth problems were excluded from the study.
Acknowledgments
This research was supported by the Dutch AIDS fund (Grant Nos. 2006006 and 2007028). We thank Stef Heynen for performing CA-p24 ELISA experiments. We also thank the Belgian Federal Government for financial support through the Inter-University Attraction Pole Grant No. P6/41.
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