PNA Targeting the PBS and A-Loop Sequences of HIV-1 Genome Destabilizes Packaged tRNA 3Lys in the Virions and Inhibits HIV-1 Replication

Abstract

During assembly of the HIV-1 virions, cellular tRNA Lys 3 is packaged into the virion particles and is utilized as a primer for the initiation of reverse transcription. The 3′-terminal 18 nucleotides of the cellular tRNA Lys 3 are complementary to nucleotides 183–201 of the viral RNA genome, referred to as the primer binding sequence (PBS). Additional sequences (A-Loop) upstream of the PBS are essential for tRNA primer selection. We report here that a PNA targeted to PBS and A-Loop sequence (PNA PBS) exhibits high specificity for its target sequence and prevents tRNA Lys 3 priming on the viral genome. We also demonstrate that PNA PBS is able to invade the duplex region of the tRNA Lys 3–viral RNA complex and destabilize the priming process, thereby inhibiting the in vitro initiation of reverse transcription. The endogenously packaged tRNA Lys 3 bound to the PBS region of the viral RNA genome in the HIV-1 virion is efficiently competed out by PNA PBS, resulting in near complete inhibition of initiation of endogenous reverse transcription. Examination of the effect of PNA PBS on HIV-1 production in CEM cells infected with pseudotyped HIV-1 virions carrying luciferase reporter exhibited dramatic reduction of HIV-1 replication by nearly 99%. Analysis of the mechanism of PNA PBS-mediated inhibition indicated that PNA PBS interferes at the step of reverse transcription. These findings suggest the antiviral efficacy of PNA PBS in blocking the process of HIV-1 replication.