Jason B. Dinoso

In HIV-1-infected individuals on currently recommended antiretroviral therapy (ART), viremia is reduced to <50 copies of HIV-1 RNA per milliliter, but low-level residual viremia appears to persist over the lifetimes of most infected individuals. There is controversy over whether the residual viremia results from ongoing cycles of viral replication. To address this question, we conducted 2 prospective studies to assess the effect of ART intensification with an additional potent drug on residual viremia in 9 HIV-1-infected individuals on successful ART. By using an HIV-1 RNA assay with single-copy sensitivity, we found that levels of viremia were not reduced by ART intensification with any of 3 different antiretroviral drugs (efavirenz, lopinavir/ritonavir, or atazanavir/ritonavir). The lack of response was not associated with the presence of drug-resistant virus or suboptimal drug concentrations. Our results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.

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-kappaB 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.

The treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART), a combination of three or more antiretroviral drugs, suppresses viremia below the clinical limit of detection (50 HIV-1 RNA copies/ml), but latently infected resting CD4(+) T cells serve as lifelong reservoirs, and low-level viremia can be detected with special assays. Recent studies have provided evidence for additional reservoirs that contribute to residual viremia but are not present in circulating cells. Identification of all the sources of residual viremia in humans may be difficult. These discoveries highlight the need for a tractable model system to identify additional viral reservoirs that could represent barriers to eradication. In this study, simian immunodeficiency virus (SIV)-infected pig-tailed macaques (Macaca nemestrina) were treated with four antiretroviral drugs to develop an animal model for viral suppression during effective HAART. Treatment led to a biphasic decay in viremia and a significant rise in levels of circulating CD4(+) T cells. At terminal infection time points, the frequency of circulating resting CD4(+) T cells harboring replication-competent virus was reduced to a low steady-state level similar to that observed for HIV-infected patients on HAART. The frequencies of resting CD4(+) T cells harboring replication-competent virus in the pooled head lymph nodes, gut lymph nodes, spleen, and peripheral blood were reduced relative to those for untreated SIV-infected animals. These observations closely parallel findings for HIV-infected humans on suppressive HAART and demonstrate the value of this animal model to identify and characterize viral reservoirs persisting in the setting of suppressive antiretroviral drugs.

The time to suppression of HIV-1 viremia to below the limit of detection of standard clinical assays is an important prognostic indicator for patients on highly active antiretroviral therapy (HAART). Recent clinical trials of the integrase inhibitor raltegravir have demonstrated more rapid viral decay than previously seen with reverse transcriptase (RT) or protease inhibitor-based regimens. Because of the therapeutic importance of drugs that target different steps in the virus life cycle, it is imperative to consider whether viral dynamics are affected by the stage of the viral life cycle at which an antiretroviral drug acts. We use a mathematical model to investigate the effects of various drug classes on the dynamics of HIV-1 decay and show that the stage at which a drug acts affects the dynamics of viral decay. We find that the drug class acting latest in the viral life cycle dictates the dynamics of HIV-1 decay. In general, we find that the later in the life cycle an inhibitor acts, the more rapid the decay in viremia, and we illustrate this by comparing the effect of RT and integrase inhibitors on viral dynamics. We conclude that the rapid decay observed in patients on integrase-inhibitor-containing regimens is not necessarily an indication of greater drug efficacy but rather an expected consequence of the fact that this drug acts later in the life cycle. We propose that clinically observed viral decay rates for HAART regimens should be evaluated in the context of the drug classes that are represented.

Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein mu NS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with mu NS. We found that all three of these proteins--lambda 1, lambda 2, and sigma 2--localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with mu NS, on the other hand, each core surface protein colocalized with mu NS in globular inclusions, supporting the initial hypothesis. We also found that lambda 1, lambda 2, and sigma 2 each localized to filamentous inclusions formed upon the coexpression of mu NS and mu 2, a structurally minor core protein that associates with microtubules. The first 40 residues of mu NS, which are required for association with mu 2 and the RNA-binding nonstructural protein sigma NS, were not required for association with any of the three core surface proteins. When coexpressed with mu 2 in the absence of mu NS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with mu 2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of mu NS that had been preformed in those cells, providing evidence that mu NS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins mu NS and mu 2 in this process.