Molecular strategies to inhibit HIV-1 replication

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In addition to RANTES and its derivatives, the chemokine ligands macrophage inflammatory proteins 1alpha/beta (MIP-1alpha and MIP-1beta) also show an inhibiting effect by mediating a receptor blockade [8,11-13]. Examples of chemokine ligands that inhibit infection of X4 isolates include stromal cell-derived factor-1alpha (SDF-1alpha) and its derivatives that inhibit HIV-1 fusion and entry by minimizing the accessibility to the co-receptor on the cell surface and by inhibiting the SU-CXCR4 interaction [9,11-13].
The CCR5 amino-terminal domain is thought to play an important role in virus fusion and entry. This knowledge has been utilized in the development of anti-CCR5 Mabs whose epitopes include residues in the amino-terminal domain. Mabs of this kind strongly inhibit SU binding to CCR5 but only moderately inhibit HIV-1 fusion and entry [12]. Another type of Mab, the anti-ECL2 Mab whose epitopes include residues from one of the extracellular loops on CCR5 (ECL2), potently inhibits HIV-1 fusion and entry, but only moderately inhibits SU binding [12]. PRO 140, also an anti-CCR5 Mab, inhibits viral fusion with the cell membrane at concentrations that do not prevent the CCR5 chemokine receptor activity. It binds a complex epitope spanning multiple extracellular domains on CCR5, and although it acts as a weak antagonist it does not induce signaling or downregulation of CCR5. It is thought that the antiviral effect is exerted through a mechanism involving receptor blockade [14]. Mab 12G5 is a monoclonal antibody that recognizes an epitope on CXCR4. This epitope is also present in ECL2, and binding inhibits HIV-1 fusion [12,15]. A potential disadvantage of this strategy is that binding of the antibody to a receptor may trigger unwanted signal transduction [14,16].
Peptides, resembling the CCR5 transmembrane helices, inhibit HIV-1 replication and chemokine signaling by disrupting helix-helix interactions, which may influence the CCR5 structure [12]. T22 is a positively charged cyclic 18-mer antimicrobial peptide, which presumably inhibits SU-CXCR4 interaction by associating with the negatively charged surface of CXCR4 [8,12]. A truncated form of SDF-1alpha, consisting of the 16 amino-terminal residues of SDF-1alpha, also seems to possess such a blocking effect [12].
Recently, a new kind of CCR5 antagonist has been discovered in a protozoan parasite, Toxoplasma gondii [17]. This protein, cyclophilin-18 (C-18), has several potential antiviral properties including CCR5 binding, induction of the production of interleukin-12 (IL-12) from murine dendritic cells, inhibition of fusion and infectivity of R5 isolates by co-receptor antagonism and blocking of syncytia formation.
Small organic molecules, such as AMD3100, potently inhibit HIV-1 replication by an interaction with residues present on one of the CXCR4 extracellular loops, ECL2, and residues within a transmembrane helix, TM4. Upon binding to these residues AMD3100 spans the main ligand-binding cavity of CXCR4, which probably constrains the co-receptor in an inactive conformation [12].
Individuals with a homozygous deletion in the gene encoding CCR5 are healthy and protected against HIV-1 transmission, which suggests that down regulation may not pose any clinical side effects. This knowledge has led to the development of strategies that directly target the mRNA encoding CCR5 or CXC4, either by ribozymes [6,18], anti-sense RNA [6,18,19] or RNAi [20]. The latter strategy, the siRNA approach, has led to successful blocking of HIV-1 entry, protection of cells from infection and delay of virus replication [21-24]. Interestingly, it is thought that single-stranded siRNAs (the anti-sense strand of a siRNA duplex) act through the same RNAi pathway, but at a later stage than double-stranded siRNA, thereby requiring less time to exert their antiviral activity [21,25].
The CD4-SU interaction
Soluble CD4 (sCD4) is an anti-HIV-1 protein, which can be expressed and secreted from genetically engineered cells. It is a truncated form of the CD4 receptor, composed of the ectodomain that inhibits laboratory-adapted strains of HIV-1. sCD4 probably prevents the binding of the virus to the cell, by binding directly to Env, or indirectly by inducing or repressing cellular factors that influence the viral gene expression [18,19].
When sCD4 binds to SU it acts by extending the distance to TM, which inhibits the fusion. But when used against primary isolates, sCD4 was much less successful because of a lower affinity for sCD4. Surprisingly, some isolates became more infectious upon sCD4 treatment. An explanation for this may be that an interaction between the SU protein and sCD4 induces changes in SU, allowing it to bind the co-receptor with higher affinity or increased kinetics. In addition this interaction can eventually facilitate the fusion of HIV-1 with CD4- cells expressing the co-receptor [13]. This has led to the development of a tetrameric version of sCD4, PRO542, in which the SU-binding region of CD4 is fused to the conserved region of human immunoglobulin IgG2. This fusion protein has a high affinity for the SU protein and has shown promising results in phase I clinical trials [8,13].
siRNA-directed silencing of CD4 mRNA expression has been shown to specifically inhibit HIV-1 entry and thus HIV-1 replication [23,24]. However, CD4 silencing in vivo may interfere with its role in normal immune function. Thus an approach targeting the CD4-binding domain of the SU protein would be more relevant. This has successfully been achieved by expressing a 0.5 kb dsRNA containing the major CD4-binding domain of the SU protein, as the target of the env gene. By this approach it has been possible to significantly suppress the expression of the HIV-1 CA-p24 antigen in human peripheral blood mononuclear cells (PBMCs) and in HeLa-CD4+ for a relatively long period of time [26].
Strategies based on the intracellular expression of antibodies specific for the HIV-1 envelope (anti-SU) have also been shown to inhibit virus replication. This strategy is based on the usage of sFvs, containing the smallest structural domain that still possesses complete antigen and binding-site specificity of the parental antibody. They are secreted into the medium where they probably act as inhibitors by direct interaction with the viral proteins [18] to neutralize the virus [19].
Cyanovirin (CV-N), an 11 kDa protein originally isolated from cyanobacteria, potently inactivates diverse strains of HIV-1. It has a high affinity for the SU protein, and when bound it inhibits the SU-CD4 interaction. CV-N possesses the advantage that even high concentrations are non-toxic and it is an extremely stable protein. CV-N has also been coupled to a cytotoxin (Pseudomonas exotoxin), thereby selectively killing HIV-1 infected SU-expressing cells [15].
The TM-mediated virus-cell membrane fusion
As the SU protein binds to CD4, it initiates conformational changes in SU, making the interaction between the SU protein and the co-receptors more favorable. After attachment to the co-receptor further conformational changes occur in both the SU and TM proteins, thus weakening their interaction. During this process a transitory pre-hairpin intermediate of the TM protein is created, freeing the previously buried fusion peptide to interact with the host-cell membrane. This exposes the N-peptide and the C-peptide regions on the pre-hairpin intermediate that have been targets for several inhibiting strategies including synthetic C-peptides, N-peptides and sFvs.
C-peptides are based on the C terminal end of the fusion peptide, and mimics this part of the fusion peptide when it has its correct fusogenic conformation. T-20, a 36-amino acid C-peptide, is a potent inhibitor of HIV-1 infection. It acts through a dominant negative mechanism and interacts by binding to a conserved domain on the N-peptide present in the pre-hairpin intermediate. The function of this domain is to mediate a structural change, which allows the pre-hairpin intermediate to form a fusogenic hairpin state. Binding of T-20 inhibits this process and thereby impedes fusion. Disadvantages of the C-peptide strategy are the cost of C-peptide synthesis and the relatively large amounts necessary for an antiviral effect. In addition, their size makes them non-amenable to oral routes of entry and they must be injected instead [13,27,28].
The 5-Helix is a 25 amino acid N-peptide consisting of five of the six helixes constituting the C-peptide. The peptide is presumed to inhibit fusion, through binding with high affinity to the C-peptide. However because N-peptides have a strong tendency to aggregate the inhibition could also be due to their intercalation into the TM amino-terminal coiled coil [27,28].
A third kind of peptides named D-peptides have also proven effective. These peptides are small 16?18 D-amino acids residues that specifically bind to three hydrophobic pockets present at the end of the N-peptide. Since such peptides are unnatural, they are resistant to proteolytic degradation, which makes them attractive for clinical use [13,27].
Recently, a non-neutralizing antibody directed against epitopes exposed on the fusion peptide has been reported to possess antiviral effect [29]. This antibody does not neutralize HIV-1 entry when produced as a soluble protein. However, when expressed on the cell surface as a membrane-bound sFv, it is turned into a neutralizing antibody, which markedly inhibits HIV-1 replication and cell-cell fusion by a mechanism that is thought to involve an interaction with the exposed fusion peptide. This results in inhibition of the subsequent fusion process. In the same study, this sFv was targeted into the ER and trans-Golgi network of HIV-1 susceptible cell lines where it was found to significantly block the maturation process of the viral Env protein resulting in an impairment of viral assembly.
Reverse transcription and proviral integration
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