Jason B. Dinoso

Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy

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.

Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation

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.

A Simian Immunodeficiency Virus-Infected Macaque Model To Study Viral Reservoirs That Persist during Highly Active Antiretroviral Therapy

ABSTRACT 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.

Decay dynamics of HIV-1 depend on the inhibited stages of the viral life cycle

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.

Reovirus Nonstructural Protein μNS Recruits Viral Core Surface Proteins and Entering Core Particles to Factory-Like Inclusions

ABSTRACT Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein μNS 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 μNS. We found that all three of these proteins—λ1, λ2, and σ2—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 μNS, on the other hand, each core surface protein colocalized with μNS in globular inclusions, supporting the initial hypothesis. We also found that λ1, λ2, and σ2 each localized to filamentous inclusions formed upon the coexpression of μNS and μ2, a structurally minor core protein that associates with microtubules. The first 40 residues of μNS, which are required for association with μ2 and the RNA-binding nonstructural protein σNS, were not required for association with any of the three core surface proteins. When coexpressed with μ2 in the absence of μNS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with μ2 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 μNS that had been preformed in those cells, providing evidence that μNS 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 μNS and μ2 in this process.

T Cell Dynamics and the Response to HAART in a Cohort of HIV-1-Infected Elite Suppressors

Elite controllers or suppressors are untreated human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain undetectable viral loads. In this study, we show that most elite suppressors do not experience significant changes in T cell counts over a 10-year period. Interestingly, treatment of an elite suppressor with highly active antiretroviral therapy (HAART) led to a marked decrease in immune activation.

A Comparison of Viral Loads between HIV-1–Infected Elite Suppressors and Individuals Who Receive Suppressive Highly Active Antiretroviral Therapy

Elite suppressors are untreated individuals with human immunodeficiency virus type 1 (HIV-1) infection who maintain viral loads <50 copies/mL. Using a single-copy assay, we show that there is no statistically significant difference between the proportions of elite suppressors and patients receiving suppressive highly active antiretroviral therapy who have viral loads of <1 copy/mL.

Replication-Competent Simian Immunodeficiency Virus (SIV) Gag Escape Mutations Archived in Latent Reservoirs during Antiretroviral Treatment of SIV-Infected Macaques

ABSTRACT In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8+ T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8+ T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4+ T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8+ T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted.

Increased Ubiquitination and Other Covariant Phenotypes Attributed to a Strain- and Temperature-Dependent Defect of Reovirus Core Protein μ2

ABSTRACT Reovirus replication and assembly are thought to occur within cytoplasmic inclusion bodies, which we call viral factories. A strain-dependent difference in the morphology of these structures reflects more effective microtubule association by the μ2 core proteins of some viral strains, which form filamentous factories, than by those of others, which form globular factories. For this report, we identified and characterized another strain-dependent attribute of the factories, namely, the extent to which they colocalized with conjugated ubiquitin (cUb). Among 16 laboratory strains and field isolates, the extent of factory costaining for cUb paralleled factory morphology, with globular strains exhibiting higher levels by far. In reassortant viruses, factory costaining for cUb mapped primarily to the μ2-encoding M1 genome segment, although contributions by the λ3- and λ2-encoding L1 and L2 genome segments were also evident. Immunoprecipitations revealed that cells infected with globular strains contained higher levels of ubiquitinated μ2 (Ub-μ2). In M1-transfected cells, cUb commonly colocalized with aggregates formed by μ2 from globular strains but not with microtubules coated by μ2 from filamentous strains, and immunoprecipitations revealed that μ2 from globular strains displayed higher levels of Ub-μ2. Allelic changes at μ2 residue 208 determined these differences. Nocodazole treatment of cells infected with filamentous strains resulted in globular factories that still showed low levels of costaining for cUb, indicating that higher levels of costaining were not a direct result of decreased microtubule association. The factories of globular strains, or their μ2 proteins expressed in transfected cells, were furthermore shown to gain microtubule association and to lose colocalization with cUb when cells were grown at reduced temperature. From the sum of these findings, we propose that μ2 from globular strains is more prone to temperature-dependent misfolding and as a result displays increased aggregation, increased levels of Ub-μ2, and decreased association with microtubules. Because so few of the viral strains formed factories that were regularly associated with ubiquitinated proteins, we conclude that reovirus factories are generally distinct from cellular aggresomes.

Sustained elite suppression of replication competent HIV-1 in a patient treated with rituximab based chemotherapy.

The mechanism of elite control of HIV-1 replication is not fully understood. While immunosuppression due to rituximab based chemotherapy has been associated with increased replication of HBV, CMV, and HIV-1, control of replication-competent HIV-1 was maintained in an elite controller/suppressor treated with a regimen that included vincristine, cyclophosphamide, prednisone, four rounds of plasmapheresis and ten cycles of rituximab. The data suggests that de-novo antibody responses do not play a significant role in the control of viral replication in these patients.