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Robert F Siliciano

Publications and source records attributed to Robert F Siliciano.

At least 19 recordsLinked to original sources
PubMed2025

Decoding heterogeneous single-cell perturbation responses.

Understanding how cells respond differently to perturbation is crucial in cell biology, but existing methods often fail to accurately quantify and interpret heterogeneous single-cell responses. Here we introduce the perturbation-response score (PS), a method to quantify diverse perturbation responses at a single-cell level. Applied to single-cell perturbation datasets such as Perturb-seq, PS outperforms existing methods in quantifying partial gene perturbations. PS further enables single-cell dosage analysis without needing to titrate perturbations, and identifies 'buffered' and 'sensitive' response patterns of essential genes, depending on whether their moderate perturbations lead to strong downstream effects. PS reveals differential cellular responses on perturbing key genes in contexts such as T cell stimulation, latent HIV-1 expression and pancreatic differentiation. Notably, we identified a previously unknown role for the coiled-coil domain containing 6 (CCDC6) in regulating liver and pancreatic cell fate decisions. PS provides a powerful method for dose-to-function analysis, offering deeper insights from single-cell perturbation data.

Single-Cell Analysis↗
PubMed2007

Experimental approaches to the study of HIV-1 latency.

Viral latency is a reversibly non-productive state of infection that allows some viruses to evade host immune responses. As a consequence of its tropism for activated CD4(+) T cells, HIV-1 can establish latent infection in resting memory CD4(+) T cells, which are generated when activated CD4(+) T cells return to a quiescent state. Latent HIV-1 persists as a stably integrated but transcriptionally silent provirus. In this state, the virus is unaffected by immune responses or antiretroviral drugs, and this latent reservoir in resting CD4(+) T cells is a major barrier to curing the infection. Unfortunately, there is no simple assay to measure the number of latently infected cells in a patient, nor is there an entirely representative in vitro model in which to explore the molecular mechanisms of latency. This Review will consider current approaches to the analysis of HIV-1 latency both in vivo and in vitro.

CD4-Positive T-Lymphocytes↗
PubMed2006

Novel pathway for induction of latent virus from resting CD4(+) T cells in the simian immunodeficiency virus/macaque model of human immunodeficiency virus type 1 latency.

Although combination therapy allows the suppression of human immunodeficiency virus type 1 (HIV-1) viremia to undetectable levels, eradication has not been achieved because the virus persists in cellular reservoirs, particularly the latent reservoir in resting CD4(+) T lymphocytes. We previously established a simian immunodeficiency virus (SIV)/macaque model to study latency. We describe here a novel mechanism for the induction of SIV from latently infected resting CD4(+) T cells. Several human cell lines including CEMx174 and Epstein-Barr virus-transformed human B-lymphoblastoid cell lines mediated contact-dependent activation of resting macaque T cells and induction of latent SIV. Antibody-blocking assays showed that interactions between the costimulatory molecule CD2 and its ligand CD58 were involved, whereas soluble factors and interactions between T-cell receptors and major histocompatibility complex class II were not. Combinations of specific antibodies to CD2 also induced T-cell activation and virus induction in human resting CD4(+) T cells carrying latent HIV-1. This is the first demonstration that costimulatory signals can induce latent virus without the coengagement of the T-cell receptor, and this study might provide insights into potential pathways to target latent HIV-1.

Animals↗
PubMed2006

Isolation and characterization of replication-competent human immunodeficiency virus type 1 from a subset of elite suppressors.

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who control viremia to levels below the limit of detection of current assays. The mechanisms involved in this control have not been fully elucidated. Several studies have demonstrated that some ES are infected with defective viruses, but it remains unclear whether others are infected with replication-competent HIV-1. To answer this question, we used a sensitive coculture assay in an attempt to isolate replication-competent virus from a cohort of 10 ES. We successfully cultured six replication-competent isolates from 4 of the 10 ES. The frequency of latently infected cells in these patients was more than a log lower than that seen in patients on highly active antiretroviral therapy with undetectable viral loads. Full-length sequencing of all six isolates revealed no large deletions in any of the genes. A few mutations and small insertions and deletions were found in some isolates, but phenotypic analysis of the affected genes suggested that their function remained intact. Furthermore, all six isolates replicated as well as standard laboratory strains in vitro. The results suggest that some ES are infected with HIV-1 isolates that are fully replication competent and that long-term immunologic control of replication-competent HIV-1 is possible.

Base Sequence↗
PubMed2006

Maintenance of viral suppression in HIV-1-infected HLA-B*57+ elite suppressors despite CTL escape mutations.

Rare human immunodeficiency virus 1-infected individuals, termed elite suppressors (ES), maintain plasma virus levels of <50 copies/ml and normal CD4 counts without therapy. The major histocompatibility complex class I allele group human histocompatibility leukocyte antigen (HLA)-B*57 is overrepresented in this population. Mutations in HLA-B*57-restricted epitopes have been observed in ES, but their significance has remained unclear. Here we investigate the extent and impact of cytotoxic T lymphocyte (CTL) escape mutations in HLA-B*57+ ES. We provide the first direct evidence that most ES experience chronic low level viremia. Sequencing revealed a striking discordance between the genotypes of plasma virus and archived provirus in resting CD4+ T cells. Mutations in HLA-B*57-restricted Gag epitopes were present in all viruses from plasma but were rare in proviruses, suggesting powerful selective pressure acting at these epitopes. Surprisingly, strong CD8+ T cell interferon-gamma responses were detected against some mutant epitopes found in plasma virus, suggesting the development of de novo responses to viral variants. In some individuals, relative CD8+ T cell interleukin-2 responses showed better correlation with the selection observed in vivo. Thus, analysis of low level viremia reveals an unexpectedly high level of CTL escape mutations reflecting selective pressure acting at HLA-B*57-restricted epitopes in ES. Continued viral suppression probably reflects CTL responses against unmutated epitopes and residual or de novo responses against epitopes with escape mutations.

CD4 Lymphocyte Count↗
PubMed2006

HIV-1 viral load blips are of limited clinical significance.

Many patients on highly active antiretroviral therapy (HAART) who achieve undetectable HIV-1 RNA levels experience transient episodes of detectable viraemia or blips, suggesting there is incomplete suppression of viral replication. This raises concern that drug resistance mutations could develop and cause eventual treatment failure. However, data from recent studies indicate that most blips are actually random biological and statistical variations around a mean viral load below detectable levels (<50 copies/mL) or due to false elevations of viral load from laboratory processing artefacts. Blips are not typically associated with the development of resistance mutations and most importantly are not associated with virological or clinical failure of previously adequate HAART.

Antiretroviral Therapy, Highly Active↗
PubMed2006

Marked intraindividual variability in antiretroviral concentrations may limit the utility of therapeutic drug monitoring.

BACKGROUND: Effective therapeutic drug monitoring for antiretrovirals requires a better understanding of intraindividual variability in pharmacokinetics. METHODS: We determined concentrations of human immunodeficiency virus (HIV) protease and nonnucleoside reverse-transcriptase inhibitors for 10 patients with undetectable plasma HIV RNA levels who had been receiving stable regimens for > or = 11 months. Plasma samples were collected at the same time of day 3 times per week for up to 4 months. Patients were instructed to take their antiretrovirals at the same time every day. Plasma protease and nonnucleoside reverse-transcriptase inhibitor concentrations were determined using high-performance liquid chromatographic methods. Pharmacokinetic variability was expressed as intraindividual percentage coefficient of variation (ICV), which was calculated as the patient's standard deviation divided by the mean drug concentration for that patient. RESULTS: ICV was determined for 6 drugs for 10 patients, for a total of 17 different patient-drug combinations, using 600 total samples. ICV was unexpectedly high for most patients who were receiving protease inhibitors (ICVs for individual patients taking lopinavir/ritonavir were 24%, 33%, 51%, and 92%; for patients taking nelfinavir/M8 metabolite, they were 30%/44% and 39%/54%; for patients taking ritonavir, they were 34% and 43%; for patients taking saquinavir, they were 52% and 55%). ICVs for patients receiving nonnucleoside reverse-transcriptase inhibitors were lower (for patients receiving efavirenz, they were 7%, 13%, 29%, and 51%; for a patient receiving nevirapine, it was 25%). The median ICV for all patients receiving protease inhibitors (n = 12) was 43.5%, and for all patients receiving nonnucleoside reverse-transcriptase inhibitors (n = 5), the median ICV was 25%. CONCLUSIONS: Intraindividual variability in concentrations of antiretrovirals was surprisingly high in virologically suppressed patients. Possible contributors include food effects, concomitant use of prescription and herbal medications, assay variability, or medication timing, which was assessed by self-report. High intraindividual pharmacokinetic variability may limit the utility of single measurements in therapeutic drug monitoring for some antiretroviral agents.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Residual human immunodeficiency virus type 1 viremia in some patients on antiretroviral therapy is dominated by a small number of invariant clones rarely found in circulating CD4+ T cells.

Antiretroviral therapy can reduce human immunodeficiency virus type 1 (HIV-1) viremia to below the detection limit of ultrasensitive clinical assays (50 copies of HIV-1 RNA/ml). However, latent HIV-1 persists in resting CD4+ T cells, and low residual levels of free virus are found in the plasma. Limited characterization of this residual viremia has been done because of the low number of virions per sample. Using intensive sampling, we analyzed residual viremia and compared these viruses to latent proviruses in resting CD4+ T cells in peripheral blood. For each patient, we found some viruses in the plasma that were identical to viruses in resting CD4+ T cells by pol gene sequencing. However, in a majority of patients, the most common viruses in the plasma were rarely found in resting CD4+ T cells even when the resting cell compartment was analyzed with assays that detect replication-competent viruses. Despite the large diversity of pol sequences in resting CD4+ T cells, the residual viremia was dominated by a homogeneous population of viruses with identical pol sequences. In the most extensively studied case, a predominant plasma sequence was also found in analysis of the env gene, and linkage by long-distance reverse transcriptase PCR established that these predominant plasma sequences represented a single predominant plasma virus clone. The predominant plasma clones were released for months to years without evident sequence change. Thus, in some patients on antiretroviral therapy, the major mechanism for residual viremia involves prolonged production of a small number of viral clones without evident evolution, possibly by cells other than circulating CD4+ T cells. The sequences have been deposited in GenBank. The accession numbers are DQ 391282 to DQ 391351 (for env) and DQ 391352 to DQ 392955 (for RT).

Antiretroviral Therapy, Highly Active↗
PubMed2006

Neutralizing antibodies do not mediate suppression of human immunodeficiency virus type 1 in elite suppressors or selection of plasma virus variants in patients on highly active antiretroviral therapy.

Neutralizing antibodies (NAb) against autologous virus can reach high titers in human immunodeficiency virus type 1 (HIV-1)-infected patients with progressive disease. Less is known about the role of NAb in HIV-1-infected patients with viral loads of <50 copies/ml of plasma, including patients on effective highly active antiretroviral therapy (HAART) and elite suppressors, who control HIV-1 replication without antiretroviral therapy. In this study, we analyzed full-length env sequences from plasma viruses and proviruses in resting CD4(+) T cells of HAART-treated patients, elite suppressors, and untreated HIV-1-infected patients with progressive disease. For each patient group, we assessed plasma virus neutralization by autologous, contemporaneous plasma. The degree of env diversity, the number of N-linked glycosylation sites, and the lengths of variable loops were all lower in elite suppressors than in HAART-treated and untreated viremic patients. Both elite suppressors and HAART-treated patients had lower titers of NAb against HIV-1 lab strains than those of untreated viremic patients. Surprisingly, titers of NAb against autologous, contemporaneous plasma viruses were similarly low in chronic progressors, elite suppressors, and HAART-treated patients. In elite suppressors and HAART-treated patients, titers of NAb against autologous plasma viruses also did not differ significantly from titers against autologous proviruses from resting CD4(+) T cells. These results suggest that high-titer NAb are not required for maintenance of viral suppression in elite suppressors and that NAb do not select plasma virus variants in most HAART-treated patients. Both drug-mediated and natural suppression of HIV-1 replication to levels below 50 copies/ml may limit the stimulation and maintenance of effective NAb responses.

Antiretroviral Therapy, Highly Active↗
PubMed2006

Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

Acquired Immunodeficiency Syndrome↗
PubMed2005

HIV-1-induced depletion of CD4+ T cells in the gut: mechanism and therapeutic implications.

The mechanism of CD4+ T cell depletion remains a central unresolved issue in AIDS research. Recent studies have examined the massive depletion of CD4+ T cells observed in macaque models of acute HIV infection. A key question is whether the observed CD4+ T cell death is due to direct consequences of viral infection and to indirect mechanisms including increased expression of mediators of T-cell apoptosis. The therapeutic implications of the early CD4+ T cell loss are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Intermittent HIV-1 viremia (Blips) and drug resistance in patients receiving HAART.

CONTEXT: Many patients infected with human immunodeficiency virus type 1 (HIV-1) and receiving highly active antiretroviral therapy experience intermittent episodes of detectable viremia ("blips"), which may raise concerns about drug resistance, lead to costly repeat measurements of viral RNA, and sometimes trigger alterations in therapy. OBJECTIVE: To test the hypothesis that blips represent random biological and statistical variation around mean steady-state HIV-1 RNA levels slightly below 50 copies/mL rather than biologically significant elevations in viremia. DESIGN, SETTING, AND PATIENTS: Between June 19, 2003, and February 9, 2004, patients receiving therapy underwent intensive sampling (every 2-3 days) over 3 to 4 months to define the frequency, magnitude, and duration of blips and their association with drug levels and other clinical variables. Blips were defined as HIV-1 RNA measurements greater than or equal to 50 copies/mL preceded and followed by measurements less than 50 copies/mL without a change in treatment. To determine whether blips result from or lead to drug resistance, an ultrasensitive genotyping assay was used to detect drug resistance mutations before, during, and after blips. Patients were 10 HIV-1-infected asymptomatic adults recruited by clinicians and followed up in the Moore Clinic at the Johns Hopkins Hospital. Patients had suppression of viremia to below 50 copies/mL while receiving a stable antiretroviral regimen for 6 months or longer. MAIN OUTCOME MEASURES: At each time point, plasma HIV-1 RNA levels were measured in 2 independent laboratories and drug resistance mutations were analyzed by clonal sequencing. RESULTS: With the intensive sampling, blips were detected in 9 of 10 patients. Statistical analysis was consistent with random assay variation around a mean viral load below 50 copies/mL. Blips were not concordant on independent testing and had a short duration (median, <3 days) and low magnitude (median, 79 copies/mL). Blip frequency was not associated with demographic, clinical, or treatment variables. Blips did not occur in relation to illness, vaccination, or directly measured antiretroviral drug concentrations. Blips were marginally associated (P = .08) with reported episodes of nonadherence. Most importantly, in approximately 1000 independent clones sequenced for both protease and reverse transcriptase, no new resistance mutations were seen before, during, or shortly after blips. CONCLUSION: Most blips in this population appear to represent random biological and statistical variation around mean HIV-1 levels below 50 copies/mL rather than clinically significant elevations in viremia.

Adult↗
PubMed2005

SIV-specific T lymphocyte responses in PBMC and lymphoid tissues of SIV-infected pigtailed macaques during suppressive combination antiretroviral therapy.

There is currently no SIV macaque model in which the effects of combination antiretroviral therapy on tissue immune responses and latent reservoirs have been measured. This study was performed to define the impact of combination therapy on the specificity and distribution of the T lymphocyte response in multiple tissue compartments. Pigtailed macaques (Macaca nemestrina) were infected with SIV/17E-Fr and treated with combination antiretroviral therapy consisting of 9-R-(2-phosphonomethoxypropyl)adenine (PMPA) and beta-2',3'-dideoxy-3'-thia-5-fluorocytidine (FTC). The SIV-specific T lymphocyte response was measured in peripheral blood, spleen and several lymph nodes at necropsy by IFN-gamma Elispot analysis. Two animals (one treated, one untreated) had high acute peak viremia, which was associated with lower SIV-specific T lymphocyte responses in the peripheral blood and lymphoid tissues. In the treated animal, viremia was controlled to low or undetectable for the study duration, and virus-specific responses remained low. The untreated animal remained viremic throughout the study and developed clinical symptoms of AIDS. In contrast, the two animals that had lower acute peak viremia (one treated, one untreated) had more robust T lymphocyte responses, and controlled viral replication. Virus-specific responses were detected in the treated animal despite 6 months of suppressive therapy. These data suggest that in this model, in the context of acute peak viremia and weak T cell responses, combination therapy may be essential to control virus replication and disease progression. Conversely, in the setting of low initial viremia and robust T lymphocyte responses, treatment does not have a detrimental effect on the immune response.

Adenine↗
PubMed2005

Contribution of virus-like particles to the immunogenicity of human immunodeficiency virus type 1 Gag-derived vaccines in mice.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is a major target antigen for cytotoxic-T-lymphocyte-based vaccine strategies because of its high level of conservation. The murine model has been used extensively to evaluate potential HIV-1 vaccines. However, the biology of HIV-1 Gag is somewhat different in human and murine tissues. The ability of HIV-1 Gag to form virus-like particles (VLPs) in human cells is severely curtailed in murine cells. Hence, it is not known whether immunizing mice with expression vectors encoding HIV-1 Gag provides an accurate assessment of the immunogenicity of these candidate vaccines in primates. In this report, we made use of a chimeric Moloney murine leukemia virus (MMLV)-HIV-1 Gag in which the p17 matrix domain of HIV-1 was replaced with the p15 matrix and p12 domains from MMLV. Murine cells expressing this construct released significant amounts of VLPs. The construct preserved H-2d-restricted antigenic determinants in the remaining portion of HIV-1 Gag, allowing immunogenicity studies to be performed with mice. We demonstrated that immunizing mice with plasmid DNA or adenoviral vectors encoding this chimeric Gag did not significantly increase the HIV-1 Gag-specific cellular or humoral immune response when compared to immunization with a myristoylation-incompetent version of the construct. Thus, the release of VLPs formed in vivo may not play a major role in the immunogenicity of vectors expressing HIV-1 Gag constructs.

AIDS Vaccines↗
PubMed2005

G-->A hypermutation in protease and reverse transcriptase regions of human immunodeficiency virus type 1 residing in resting CD4+ T cells in vivo.

In vitro studies have shown that the host cytidine deaminase APOBEC3G causes lethal hypermutation in human immunodeficiency virus type 1 reverse transcripts unless its incorporation into virions is blocked by Vif. By examining stably archived sequences in resting CD4+ T cells, we show that hypermutation occurs in most if not all infected individuals. Hypermutated sequences comprised >9% of archived species in resting CD4+ T cells but were not found in plasma virus. Mutations occurred in predicted contexts, with notable hotspots. Thus, defects in Vif function in vivo give rise to hypermutated viral genomes that can be integrated but do not produce progeny viruses.

Antiretroviral Therapy, Highly Active↗
PubMed2005

Kinetics of human immunodeficiency virus type 1 decay following entry into resting CD4+ T cells.

In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4(+) T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4(+) cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4(+) T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4(+) T cells. Decay processes dominate in resting CD4(+) T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4(+) T cells is highly labile.

CD4-Positive T-Lymphocytes↗