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Spectroscopic and electrochemical characterization of CD4 binding site of HIV-1 exterior envelope GP120

Human immunodeficiency virus type 1 (HIV-1) is characterized by extensive genetic variability, as a consequence of high replication and mutation rates and frequent recombination [1,2]. HIV destroys the human immune system and causes lethal diseases such as acquired immune deficiency syndrome (AIDS) [3], one of the most devastating disease, attacking humanity today, affecting 65 million people with 11 000 new cases per day [4]. HIV-1 entry depends on the sequential interaction with the CD4 receptors on the CD4+ T cells and the members of the chemokine receptor family [5]. The entry of HIV-1 into cell is mainly mediated by the viral envelope glycoproteins [6], which are synthesized as an ~850- amino acid precursor. After trimerization and posttranslational modification by carbohydrate, 160-kDa glycoprotein (gp160) is formed. Proteolysis of gp160, processed in the Golgi apparatus, provides a creation of gp120 exterior envelope glycoprotein containing relatively conserved CD4 binding site and gp41 trans-membrane envelope glycoprotein [7-9]. In the mature HIV-1 envelope glycoprotein trimer, the three gp120 subunits are noncovalently bound to three membrane-anchored gp41 subunits [10], as it can be seen in Fig.

Overall scheme of HIV-1 virion with the expression of interaction between HIV-1 envelope glycoproteins with CD4 receptor binding site. One of the subunit of gp41 is depicted. Binding with CD4 results in the apposition of layer 1 and layer 2, the formation of the bridging sheet and the projection of the V3 loop away from the gp120 core towards coreceptor. This rearrangement of gp120 allows the gp41 ectodomain to undergo additional conformational changes, necessary for HIV-1 entry. The gp120 OD stays for outer domain of glycoprotein 120, gp120 ID stays for inner domain of glycoprotein 120, 1; 2; 3 stays for loops that form three topological layers and CCR5 stays for coreceptor (chemokine receptor).

For entry of HIV-1 into a host cell, the gp120 subunit associates with the CD4 receptor and the CCR5 coreceptor, and this induces series of conformational changes culminating in virus and host cell membrane fusion. Most primary HIV-1 strains use the chemokine receptor CCR5 as coreceptor in conjunction with CD4 for virus entry. However, some strains has evolved to use related receptors [11]. Binding of gp120 to CD4 causes conformational changes observable in variable loop regions V1/V2 and V3, causing the V3 loop to evaginate, thus becoming exposed to the co-receptors [12], which is shown in Fig. 1. The precise mechanisms of interaction between V1/V2, V3 and chemokine receptors are not well understood [13]. The final step of viral entry, fusion of the viral components with target membrane, is achieved by gp 41 [14,15]. After binding of gp120 to CD4 and coreceptors, conformational changes occur, leading to gp41 unfolding and the hydrophobic fusion peptide sequence extends towards the host cell membrane [16]. The insertion of the peptide leads to fold into a hairpin-like structure, believed to be responsible for the fusion of the HIV to the host cell [17].Due to the nature of fusion, there are several possible targets for the development of drugs with synergistic effects on inhibition of viral entry steps, at which the interference can be attempted. Generally, these targets may affect viral entry by the inhibition of CD4 binding due to a blocking of conservative CD4 binding site of gp120. Hence, glycoprotein cannot interact with receptors and coreceptors and process of conformational changes, whereas the triggering the fusion is stopped. Therefore, the aim of this study was a synthesis of CD4 binding site and its subsequent spectroscopic characterization. Moreover, electrochemical measurements were carried out to optimize the conditions for analyses serving for rapid and accurate monitoring of various peptide-peptide interactions helpful for development of new potential peptides with antiviral or other effects

Práce je spojená s projektem NanoBioMetalNet CZ.1.07/2.4.00/31.0023

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