Non-Catalytic Site HIV-1 Integrase Inhibitors Disrupt Core Maturation and Induce a Reverse Transcription Block in Target Cells

Mini Balakrishnan,Tomas Cihlar,Luong Tsai,Christopher O’Sullivan,Roman Sakowicz,Angela Tsai,Anita Niedziela-Majka,Stephen R Yant,Rujuta A Bam,Kirsten M Stray,David Harrich

doi:10.1371/journal.pone.0074163

Abstract

HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly.

Highlights

Advances in antiretroviral drug development have enabled effective long-term control of HIV-1 infection and the prevention of disease progression
When the NCINIs GS-A and GS-B were restricted to only the target cell infection phase, both compounds showed a marked reduction in potency (80- and 28-fold, respectively) relative to that observed in the full-cycle assay
NCINIs have been shown to inhibit integration in target cells [30]. This block was thought to occur via inhibition of the IN-LEDGF interactions, more recent data reveal that NCINIs promote and rigidify IN dimers in a manner that impedes the assembly of the IN-vDNA complex [31,32]

Summary

Introduction

Advances in antiretroviral drug development have enabled effective long-term control of HIV-1 infection and the prevention of disease progression. Integration is accomplished via two catalytic steps following the completion of reverse transcription: i) 39-processing that involves trimming the 39-ends of the vDNA and ii) DNA strand transfer, where IN inserts the vDNA into the host chromosome. Through these events, IN remains bound to the vDNA ends as part of a pre-integration complex (PIC) along with several viral and host proteins [2,3]. The host protein LEDGF/p75 is critical for vDNA integration because it mediates the tethering of the IN-DNA complex to the host chromatin [4,5]. Somatic knock-out of LEDGF was shown to severely attenuate the replication of laboratory-adapted HIV-1 strains and completely block the growth of clinical isolates [14]

Methods

Results

Conclusion