Highly active antiretroviral therapy (HAART) can control HIV-1 replication, but suboptimal treatment allows for the evolution of resistance and rebound viremia. A comparative measure of antiviral activity under clinically relevant conditions would guide drug development and the selection of regimens that maximally suppress replication. Here we show that current measures of antiviral activity, including IC(50) and inhibitory quotient, neglect a key dimension, the dose-response curve slope. Using infectivity assays with wide dynamic range, we show that this slope has noteworthy effects on antiviral activity. Slope values are class specific for antiviral drugs and define intrinsic limitations on antiviral activity for some classes. Nucleoside reverse transcriptase inhibitors and integrase inhibitors have slopes of approximately 1, characteristic of noncooperative reactions, whereas non-nucleoside reverse transcriptase inhibitors, protease inhibitors and fusion inhibitors unexpectedly show slopes >1. Instantaneous inhibitory potential (IIP), the log reduction in single-round infectivity at clinical drug concentrations, is strongly influenced by slope and varies by >8 logs for anti-HIV drugs. IIP provides a more accurate measure of antiviral activity and in general correlates with clinical outcomes. Only agents with slopes >1 achieve high-level inhibition of single-round infectivity, a finding with profound implications for drug and vaccine development.

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4 + T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4 + T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4 + T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-κB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.

Most current evidence suggests that the infection of resting CD4؉ T cells by human immunodeficiency virus type 1 (HIV-1) is not productive due to partial or complete blocks in the viral life cycle at steps prior to integration of the viral genome into the host cell chromosome. However, stimulation of an infected resting T cell by antigen, cytokines, or microenvironmental factors can overcome these blocks and allow for the production of progeny virions. In this study, we sought to understand the structure and fate of the virus in unstimulated resting CD4 ؉ T cells. Using a novel linker-mediated PCR assay designed to detect and characterize linear unintegrated forms of the HIV-1 genome, we demonstrate that reverse transcription can proceed to completion following the infection of resting T cells, generating the substrate for the retroviral integration reaction. However, reverse transcription in resting T cells is far slower than in activated T cells, requiring 2 to 3 days to complete. The delay in completing reverse transcription may make the viral DNA genome more susceptible to competing decay processes. To explore the relationship between the formation of the linear viral genome and the stability of the preintegration state, we employed a recombinant HIV-1 virus expressing the enhanced green fluorescent protein to measure the rate at which HIV-1 decays in the preintegration state. Our results demonstrate that the preintegration state is labile and decays rapidly (half-life ‫؍‬ 1 day) following the entry of HIV-1 into a resting T cell, with significant decay occurring during the slow process of reverse transcription.