Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production

Abstract

The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNATAR), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNATAR-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNATAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNATAR-transportan as a potential anti-HIV agent

Borna Disease Virus Glycoprotein Is Required for Viral Dissemination in Neurons

“…5) is unknown. PNA interference with human immunodeficiency virus infection has already proved to effectively inhibit viral replication in vitro (22,25,28), and this study underlines the therapeutic promise of antisense therapy by the use of cell-penetrating agents.…”

Pharmacokinetic Analysis of Polyamide Nucleic-Acid-Cell Penetrating Peptide Conjugates Targeted against HIV-1 Transactivation Response Element

“…We have shown that PNAs targeted to the primer binding site and A-loop sequences in the U5-PBS region of the HIV-1 genome block the initiation of reverse transcription (Lee et al, 1998), strongly destabilize the priming of natural tRNA 3 Lys primer with the viral genome, and inhibit HIV-1 replication (Kaushik and Pandey, 2002). We have further demonstrated that anti-HIV-1 PNA targeted to the transactivation response (TAR) element of HIV-1 LTR when transfected into HIV-1-infected cells prevents Tat-TAR interaction and blocks not only Tat-mediated transactivation of HIV-1 LTR transcription (Mayhood et al, 2000), but also HIV-1 production (Kaushik et al, 2002a(Kaushik et al, , 2002b). These studies demonstrate the therapeutic potential of PNA for intervention in HIV-1 replication and production by targeting multiple critical regions of the viral genome.…”

Inhibition of HIV-1 Replication by Oligonucleotide Analogues Directed to the Packaging Signal and Trans-Activating Response Region

“…HIV has been a frequent target for both RNAdegrading and steric-block approaches (Arzumanov et al, 2001b;Boulme et al, 1998;Darfeuille et al, 2002a;Darfeuille et al, 2004;Hiratou et al, 2001;Ittig et al, 2004;Kaushik et al, 2002;Park et al, 2000;Sei et al, 2000;Suzuki et al, 2002;Yamaguchi et al, 1997). For antisense technologies to become drug candidates, sequence specificity is essential.…”

“…ONs may act by inducing Ribonuclease H cleavage of a DNA-RNA hybrid, or by steric blocking. Steric-block ONs do not cause degradation of the target RNA, but bind to the target RNA at a high affinity, resulting in a gene expression block through mechanisms such as inhibiting elongation of translation (Michel et al, 2003), altering splicing pathways (Mercatante et al, 2002), and inhibiting RNA-protein interactions (Arzumanov et al, 2001b;Boulme et al, 1998;Darfeuille et al, 2002b;Darfeuille et al, 2004;Kaushik et al, 2002). These have been used to investigate gene function and have been explored as therapeutics against a variety of diseases, such as cytomegalovirus (CMV) retinitis (Leeds et al, 1997) and a range of cancers (Elayadi et al, 2002;Mercatante et al, 2002;Zhang et al, 2003).…”

“…This is present at the 5′-end of all viral transcripts. Steric block 2′-O-methyl (2′Ome; Arzumanov et al, 2001b;Boulme et al, 1998), 2′Ome/locked nucleic acid (LNA) mixmer (Arzumanov et al, 2001b), 2′ Ome G-clamp and peptide nucleic acid (Arzumanov et al, 2001b;Kaushik et al, 2002). ONs targeted to TAR displayed efficient steric block of the transactivator (Tat)-TAR interaction in vitro These ONs also inhibited Tat-dependent transcription from the viral long terminal repeats, and a 2′Ome/LNA ON had high-level activity in a HeLa-cell-based reporter assay of Tat-dependent trans-activation (Arzumanov et al, 2001b).…”