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  • Aurora A Inhibitor I The HCV NS A protease consists of two s

    2022-08-03

    The HCV NS3/4A protease consists of two subunits, NS3 and NS4A. NS3 comprises an N-terminal serine protease domain and a C-terminal RNA helicase domain [12]. NS4A acts as a cofactor, which ties the holoenzyme complex to an intracellular membrane compartment. The presence of the central region of NS4A (aa 21–32) allows for proper folding of NS3. There are two helices involved in NS3/4A membrane association and consequent structural organization: one helix is an in-plane amphipathic α-helix in the protease domain of NS3 and the second is a transmembrane α-helix, formed by the N-terminal 21 Aurora A Inhibitor I of NS4A [13]. The structural organization of NS3/4A results in the positioning of the serine protease active site in strictly defined topology with respect to the membrane. The NS3/4A membrane association affects HCV replication, proteolytic cleavage of host factors, and possibly also drug design. The N terminal protease domain of NS3 processes the non-structural region of the viral polyprotein and some host cellular proteins [14]. This domain plays a central role in the RNA replication of HCV and virus proliferation. The C terminal helicase assists RNA in reaching and maintaining its functional conformational state. Although each of the domains can function independently, the helicase has been shown to function more efficiently within the full-length polypeptide. Additionally, molecular dynamics simulations indicated allosteric coupling between the helicase and protease activities [[15], [16], [17]]. The NS3 protease is recognized by the innate immune response (IIR) [18]. HCV RNA is detected via Toll-like receptors, which detect viral RNA during entry and uncoating of the virion. This requires the Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF). The TRIF cascade is essential for an inflammatory immune response. Cleavage of TRIF by NS3 occurs after Cys-372, separating two major binding domains [12], one of which plays a fundamental role in IFN-β production. HCV NS3 also binds the kinase TBK1, notably via its helicase domain and not the protease domain [19], leading to inhibition of IFN production. In addition to interaction Aurora A Inhibitor I with cell surface receptors, interaction between the pathogen and intracellular receptors, such as retinoic acid inducible gene (RIG-I) [20], activates the IIR [21]. One of the steps of this immune response activation includes the induction and release of IFN-β, activating IFN-stimulated genes, which affect the IIR [22]. The second mechanism of IIR activation involves RIG-I detection of cytosolic HCV RNA and its replication products. After the virus is detected, RIG-I binds to the membrane-associated Mitochondrial Antiviral Signalling protein (MAVS) [23], inducing production of type 1 interferon. It was found that NS3 cleaves MAVS at Cys-508, resulting in dissociation of the N-terminus from the mitochondria [24]. Cleavage by the NS3 protease of both the proteins TRIF and MAVS thus leads to blockage of IFN-β activation [[25], [26]]. In addition, NS3/4A is involved in the activation of epidermal growth factor (EGF), an extensively studied receptor tyrosine kinase RTK [27]. This activation occurs via cleavage of T cell protein tyrosine phosphatase (TC-PTP) by NS3/4A at two Cys-(Ser/Ala) peptide bonds. The TC-PTP downregulation leads to enhanced EGF-induced phosphorylation of the epidermal growth factor receptor (EGFR), and results in an increase of EGF-induced Akt activation. Akt activation has been shown to promote HCV replication [28]. The multifunctionality of HCV NS3 has made it a popular target of antiviral therapy. To date, while 215 proteins are known to interact with NS3, very few known partners are membrane proteins [[29], [30], [31]]. As viral processes such as infectivity, assembly and budding are governed by membrane proteins, it is likely that many more interactions exist. The lack of known NS3-membrane protein interactions is attributed to the limited compatibility of current methods for determining protein–protein interactions (PPIs) with membrane proteins, which require membranes for correct folding and functionality. Therefore many of these interactions remain unrevealed. In addition, while several PPIs occur between NS3 and host proteins, few proteins are known to be cleaved by the NS3 protease. In order to gain a deeper understanding of interactions between NS3 and host membrane proteins, a high throughput PPI study would be a first step in defining the cellular pathways with which NS3 interferes.