Inhibition of Human Immunodeficiency Virus Type 1 by Triciribine Involves the Accessory Protein Nef

Abstract

Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1The virally encoded accessory proteins (Vpr, Vpu, Vif, Nef) are not required for replication in certain cell lines in vitro, but they are important factors causing the clinical manifestations of the pathogenesis of human immunodeficiency virus (HIV) (59). Viral protein R (Vpr) plays an important role in regulating nuclear import for the preintegration complex (PIC), is required for integration/replication in nondividing cells, induces cell cycle arrest in proliferating cells, stimulates viral transcription, and regulates apoptosis in infected cells (9). Viral protein U (Vpu) is an integral membrane phosphoprotein responsible for the degradation of CD4 in the endoplasmic reticulum and enhances virion release from the cell surface by antagonizing the cellular protein tetherin (41, 57). The viral infectivity factor (Vif) is required for replication in nonpermissive cell lines that express APOBE3G but is dispensable for replication in permissive cell lines that do not express APOBEC3G (26, 58). The "negative" factor (Nef) actually enhances viral replication and is necessary for pathogenesis to AIDS (32). Nef is the first viral protein to accumulate to detectable levels in cells following infection and is the most versatile of the accessory proteins (53). It interacts with several cellular pathways that influence HIV replication and immune evasion, resulting in the downregulation of CD4 and major histocompatibility complex class I molecules (25, 38, 51), promotion of the synthesis of FasL (a proapoptotic ligand that initiates apoptosis in nearby cells) (67), and the inhibition of both p53 (a regulator of apoptosis) and ASK-1 (the kinase involved in the initiation of apoptosis) (16,20,24). Nef also binds to several signaling proteins, including the Src family of tyrosine kinases (Erk-1, Raf1, and PKC theta), resulting in the activation of the cellular pathways responsible for proliferation and survival (22, 50). As important factors for the manifestation of HIV pathogenesis, these proteins are potential targets for antiretroviral therapy.Triciribine (TCN; Fig. 1) is a tricyclic nucleoside originally synthesized by Schram and Townsend as a potential anticancer drug (56). It is unique compared to naturally occurring purine nucleosides, in that it contains a tricyclic base instead of a bicyclic base. It has been studied broadly both as an antineoplastic agent and as an antiviral active against HIV (37, 65). Recent reports have provided new and unique insight into the antineoplastic activity of TCN. The studies described in those reports demonstrated that inhibition of the kinase activity and the level of phosphorylation of protein kinase B (Akt) are factors that result in the suppression of cell growth and the induction of apoptosis in human cancer cells that harbor constitutively overexpressed levels of Akt (68). Both the antineoplastic and antiviral activities of TCN require that the compound be phosphorylated to its cor...

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“…The 50% cytotoxic concentrations (CC 50 s) were defined as compound concentrations at which the fluorescence signals were reduced by 50% relative to that of the no-inhibitor (DMSO only) controls and were determined using GraphPad Prism 6 software. Cytotoxicity in PBMCs was determined as described previously (38).…”

“…At 4 days postinfection, the culture supernatants were subjected to RT assay to monitor viral replication as previously described (37). The 50% inhibitory concentrations (IC 50 s) were defined as compound concentrations that reduced RT levels to 50% of those measured in the absence of inhibitor (dimethyl sulfoxide [DMSO]-only controls) and were determined using GraphPad Prism 6 software (38). The IC 50 s in primary peripheral blood mononuclear cells (PBMCs) were determined by Southern Research Institute (Frederick, MD) for a multiclade panel of HIV-1 isolates, as reported previously (38).…”

“…The 50% inhibitory concentrations (IC 50 s) were defined as compound concentrations that reduced RT levels to 50% of those measured in the absence of inhibitor (dimethyl sulfoxide [DMSO]-only controls) and were determined using GraphPad Prism 6 software (38). The IC 50 s in primary peripheral blood mononuclear cells (PBMCs) were determined by Southern Research Institute (Frederick, MD) for a multiclade panel of HIV-1 isolates, as reported previously (38). PBMCs were purified from anonymized, healthy blood donors by Ficoll-Hypaque gradient centrifugation.…”

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