Inhibition of intra‐ and extra‐cellular Tat function and HIV expression by pertussis toxin B‐oligomer

Abstract

HIV‐1‐transactivating factor Tat contributes to virus replication and to the onset of AIDS‐associated pathologies by targeting different infected and uninfected cell types. We previously demonstrated that the B‐oligomer of pertussis toxin (PTX‐B) inhibits HIV infection and replication in primary T cells and macrophages and Tat‐dependent HIV‐1 long terminal repeat (LTR) transactivation inT lymphoid Jurkat cells. Here we demonstrate that PTX‐B inhibits Tat‐dependent NF‐κB activation and HIV‐1 LTR‐transactivation in non‐permissive epithelial HL3T1 cells in a phosphatidylinositol 3′‐kinase‐dependent way. PTX‐B exerts its inhibition both when Tat is produced endogenously in transfected cells and in cells incubated with the extracellular Tat protein. In this latter case, PTX‐B does not interfere with extracellular Tat uptake by cells. PTX‐B inhibited also interleukin‐8 secretion and virus expression stimulated in chronically infected U1 promonocytic cells by intra‐ and/or extracellular Tat. The genetically modified holotoxin PT‐9 K/129G retains the capacity to inhibit Tat transactivating activity and HIV replication in both HIV‐permissive and non‐permissive cells. Inconclusion, PTX‐B acts as a "pleiotropic" inhibitor of Tat, and this may significantly contribute to the broad spectrum of anti‐HIV‐1 effects exerted by PTX‐B in different cell types, and suggests PTX‐B and its derivatives as prototypic for the development of anti‐Tat drugs.

Pertussis Toxin B-Oligomer Suppresses IL-6 Induced HIV-1 and Chemokine Expression in Chronically Infected U1 Cells via Inhibition of Activator Protein 1

“…These results support the hypothesis that PTX-B or related molecules, such as the genetically modified PT-9K/129G (83), which retains all the HIV inhibitory features of PTX-B (9,26,84), could represent potent new pharmacological agents against HIV infection, as we have also recently demonstrated in infected lymphoid histocultures (85) and hu-PBL SCID mice infected with HIV (86).…”

“…Its state of relative viral latency is mostly dependent upon a defective Tat/Tat region interaction (24,25). Indeed, transfection of a Tat-expressing plasmid (10,22,24) or incubation of U1 cells with exogenous Tat protein (25,26) rescues viral production. Minimal to undetectable levels of HIV are produced by unstimulated U1 cells, although robust virion release can be promptly induced in these cells upon stimulation with phorbol esters, such as PMA (27), and several proinflammatory cytokines (28,29).…”

Activation of the Adenosine A1Receptor Inhibits HIV-1 Tat-Induced Apoptosis by Reducing Nuclear Factor-κB Activation and Inducible Nitric-Oxide Synthase

“…Because this response was inhibited by pertussis toxin, an inhibitor of the guanine nucleotide regulatory protein G i , the investigators (Haughey et al, 1999) concluded that the increase in intracellular Ca 2ϩ indicates that Tat interacts with a plasma membrane site coupled to G i and activation of phospholipase C. However, recent evidence indicates that inhibition of Tat intra-and extracellular functions, such as activation of NF-B, HIV-1 long terminal repeat transactivation, and transforming growth factor-␤ production, are inhibited by the B-oligomer of pertussis toxin, which is devoid of ADP ribosyltransferase activity (Rizzi et al, 2004;Zocchi et al, 2005). This suggests that the conclusion of an association of the Tat plasma membrane "receptor" with G i may be incorrect.…”

BSA conjugates bearing multiple copies of the basic domain of HIV-1 Tat: Prototype for the development of multitarget inhibitors of extracellular Tat

“…To determine whether BSA-Tat-NLS inhibits Tat transactivating activity by interfering with these intracellular processes, HL3T1 cells were transiently transfected with an expression vector harboring the HIV-1 tat cDNA, obtaining an intracellular overexpression of Tat. This experimental model has been already used to bypass the process of extracellular Tat internalization (Tyagi et al, 2001) and to discriminate between intra-or extracellular inhibitors of Tat (Rizzi et al, 2004). As shown in Fig.…”

“…In these experimental conditions, the extracellular Tat-antagonist heparin (Rusnati et al, 1997a) behaves exactly as BSA-Tat-NLS. Relevant to this point, a 3-h incubation allows the internalization of an amount of Tat that is sufficient to exert an optimal LTR-transactivation (Tyagi et al, 2001) and that becomes inaccessible to extracellular inhibitors remaining, however, sensible to intracellular inhibitors (Rizzi et al, 2004). • C. At the end of incubations, cell extracts were assayed for the CAT levels.…”