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Human immunodeficiency virus type one (HIV-1) infection is considered to be one of the most destructive pandemics in recorded history (UNAIDS, 2009). It is known to be the cause of the acquired immunodeficiency syndrome (AIDS) and is responsible for the majority of global infections. UNAIDS and the WHO have estimated that AIDS has killed over 25 million people since it was first recognized in 1981 and the majority of these deaths are taking place in Sub-Saharan Africa, slowing down economic growth and increasing poverty (WHO 2009, UNAIDS 2009, Greener, 2002). Prevention and anti-viral drugs are the only existing strategy to reduce virus transmission and replication as there are no effective vaccines currently available. So far, antiretroviral medication has been successful in reducing the viral load in HIV infected patients. Improved access to antiretroviral drugs amongst developing countries has seen a decline in the number of AIDS related deaths. However, routine access to such antiretroviral treatment is not available in all countries (Palella et al, 1998; Mathers and Loncar, 2006; Braitstein et al, 2006). Advances in the treatment of HIV-1 infection, such as administration of highly active antiretroviral therapy (HAART) using the combination of inhibitors that target viral protease and reverse transcriptase, often together with inhibitors of viral entry, has led to dramatic reduction in HIV-1-related morbidity and mortality (Palella et al., 1998).
Although current therapy regimens have been successful, their long-term efficacy is jeopardized by the rapid generation of multi-drug-resistant mutations of HIV-1, long-term persistence of drug-resistant viral reservoirs and severe side-effects of current therapeutics (Li et al, 2007, Hue et al., 2009; Shafer and Schapiro, 2008, Smith et
al., 2010)). Drug resistance creates a major barrier to clinical efficacy of antiretroviral therapy (Gotte et al, 1999). hence, there is a demand for developing new drugs that target cellular factors essential for virus replication as they might be less susceptible to virus mutation than the current antiviral drugs (Coley et al, 2009; Kellam, 2006). This approach depends on the identification of cellular factors involved in HIV-1 replication. In fact, a number of studies have recently been successful in identifying HIV-1 host dependency factors, which might be targeted in the future (Brass et al., 2008; Konig et al., 2008; Bushman et al., 2009).
HIV-1 contains only 9000 bases of RNA and only nine genes that code for 15 proteins which must exploit multiple host cell functions for successful infection (Cohen, 2008). integration of the reverse transcribed cDNA of HIV-1 into the genome of a newly infected cell to generate the provirus is an essential step of HIV-1 replication cycle (Nisole and Saib, 2004; Cannon et al, 1994; Engelman et al 1995). The main protein required to achieve this reaction is the virally encoded integrase which is able to catalyze two reactions of the integration process: the 3' processing and then the strand transfer. Several studies have indicated that the in vivo integration process involves cellular proteins and host factors, aiding the integration of the virus in the cellular genome. during different steps of the integration process these cellular protein can have a key function in for example nuclear import, integration site selection, integrase catalysis, and DNA gap repair (Engrud et al, 1995; LaFemina, 1992; Sakai et al, 1993). One of the major host co-factor involved in promoting HIV-1 integration is LEDGF, which binds to integrase and helps tether the viral complex to chromatin. Recently, inhibitors of LEDGF have been developed and shown to be effective in vitro (Christ et al., 2010). Together with the CCR5 antagonist Maraviroc (Dorr et al., 2005; Yost et al., 2009), these novel compounds demonstrate the feasibility of targeting selected host cell factors to inhibit HIV-1 replication.
Several studies in recent times have performed whole-genome surveys to determine the entire set of cellular genes that are critical for HIV-1 infection (Brass et al., 2008; König et al., 2008; Zhou et al., 2008). Transportin 3 (TNPO3 or TRN-SR2), an importin-beta family protein that imports serine/arginine-rich proteins (SR proteins) into the nucleus (Kataoka et al, 1999), has been identified to have a role in HIV-1 integration and has generated particular interest lately. Using yeast two-hybrid and pull-down assays, researchers found TNPO3 as an HIV-1 IN (integrase) binding protein and highlighted the role of integrase in mediating the requirement for TNPO3 during HIV-1 infection (Christ et al, 2008). Furthermore, in two independent genome-wide siRNA screens TNPO3 was identified as host factors required by HIV-1 for infection (Brass et al, 2008; Konig at al., 2008). The two screens highlighted the role of TNPO3 in nuclear import of HIV-1 preintegration complex (PIC). Depletion of TNPO3 inhibited HIV-1 and HIV-2, but not MLV infection, demonstrating a potential role as a lentiviral-specific host cofactor (Christ et al., 2008; Brass et al, 2008; Konig at al., 2008). In addition, the block to HIV-1 infection was identified at PIC nuclear import. It was also found that the recombinant TNPO3 protein binds to HIV-1 IN but not Murine leukemia virus (MLV) IN in vitro, indicating that the TNPO3-IN interaction may possibly promote lentiviral-specific PIC nuclear import (Christ et al, 2008).
However, a recent study has failed to support an important role for integrase binding in influencing TNPO3 dependency during HIV-1 infection (Krishnan et al, 2010). the authors correlated the infectivities of an expanded set of retroviral vectors, including simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), Rous sarcoma virus (RSV), in TNPO3 knockdown cells with relative IN-TNPO3 binding affinities. TNPO3 was identified as a fairly prolific IN binding protein, showing affinity for the majority of tested IN proteins, including good affinity for MLV IN. However, no correlation could be found between the protein binding profiles and the requirement for the host factor during virus infection. In addition to integrase, capsid has been highlighted as a retroviral nuclear import determinant. Consequently, analysis of MLV/HIV-1 chimera viruses identified the genetic determinant of sensitization to TNPO3 KD to the HIV-1 capsid protein. Therefore capsid, not integrase, was identified as the dominant viral factor that dictates TNPO3 dependency during HIV-1 infection (Krishnan et al, 2010).
During the assembly stage of HIV-1 life cycle, numerous copies of the viral Gag polyprotein assemble at the cell membrane and bud to form a non-infectious, immature virion (von Shwedler et al., 2003). Subsequently, Gag is cleaved by the protease, which releases the capsid proteins for assembly into the capsid shell of the mature HIV-1. Approxiamately 1200 molecules of the capsid protein p24 form the inner shell around the viral RNA load (Burner et al., 1999; Li et al, 2007). Therefore, p24 interactions with itself and with neighbouring structural proteins certainly play an essential role in capsid assembly and the maturation steps leading to infectious virus particles, both during and after budding from the cell membrane. Since the HIV-1 capsid protein p24 plays an important role in virus assembly, maturation and disassembly, the compounds that directly bind to the p24 may inhibit virus infection and could be a novel class of antiviral drugs (Li et al, 2007, Tian et al., 2009; Tang et al., 2003).
Recently Fassati et al. have used a chemical genetic approach and screened several well-characterised small compounds targeting DNA-dependent motor proteins with the basis that they might inhibit HIV-1 nuclear trafficking or integration (Vozzolo et al., submitted). They found that inhibitors of Topo1 and DNA helicases were either too toxic or were weak inhibitors of HIV-1 infection, however an antibiotic called Coumermycin A1 (C-A1) showed potent antiretroviral activity. C-A1 consists of two noviose sugars, two Y-5-methyI-2-pyrrole moieties and two 4-hydroxy-8-methylcoumarin cores which are connected by a pyrrole (Fig. 1) (Lewis et al., 1996). C-A1 is a coumarin antibiotic that inhibits bacterial gyrase B and mammalian Topoisomerase II by competing for ATP binding on the N-terminus of the molecule. Successful binding of C-A1 results in inhibition of ATPase activity of gyrase B and rapid arrest of DNA replication (Gellert et al., 1976; Lewis et al., 1996; Baba et al., 1989)
They also found that C-A1 inhibits two steps of the HIV-1 life cycle. It impairs both HIV-1 integration and gene expression and but the two blocks map to distinct targets (Vozzolo et al., submitted). Target discovery identified Hsp90 as the target for C-A1-mediated inhibition of gene expression. However, the Hsp90 inhibition mediated by C-A1 did not affect HIV-1 integration; in fact, the block to HIV-1integration mediated by C-A1 mapped to the capsid protein p24. A virus mutant that escaped C-A1 had a point mutation at position 105 in capsid. When the A105S capsid mutation was introduced into a wild type HIV-1 backbone, the resulting mutant virus was resistant to CA1 and the A105S mutation conferred resistance to the integration block imposed by CA-1. Moreover, recent molecular docking analysis (in collaboration with David Selwood and Paul Gane, Department of Medicinal Chemistry, UCL) has revealed a novel coumermycin-binding pocket in the N-terminus domain of capsid protein, which includes the Alanine 105, making the capsid pocket a potentially attractive antiviral target. Importantly, the very same capsid mutation (A105S) made the virus independent of TNPO3 for replication, suggesting a possible link between TNPO3 and C-A1 (Vozzolo et al., submitted). These results demonstrated a previously unknown function of capsid in HIV-1 integration.
Therefore, in light of these findings, here we investigated the influence of capsid on HIV-1 integration by testing the hypothesis that C-A1 and TNPO3 disrupted the same pathway required for HIV-1 infection and that C-A1 blocked the interaction between capsid and TNPO3, leading to inhibition of HIV-1 integration. One way to test this hypothesis is to examine if depletion of TNPO3 sensitizes or de-sensitizes cells to the antiretroviral activity of C-A1. If indeed the interaction between capsid and TNPO3 is the target, a profound depletion of TNPO3 should de-sensitize cells to C-A1 since only very small amounts of TNPO3 would be left available for the drug to exert its effect. In other words, addition of C-A1 should not block HIV-1 infection above the levels seen in TNPO3-depleted cells. In addition, mutations in capsid that confer independence from TNPO3 for infection should also be insensitive to C-A1. Our approach highlighted a functional connection between capsid, TNPO3 and integration and also a potential connection between C-A1 and TNPO3 function which might be subject to inhibition by C-A1 and so represents a promising therapeutic target.