• 1-LTR
• Circular 1-LTR
• HIV
• Linear 1-LTR

• DNA, Circular/genetics
• DNA, Circular/metabolism
• Humans
• DNA, Viral/genetics
• DNA, Viral/metabolism
• Virus Integration
• HIV-1/genetics
• HIV-1/physiology
• Virus Replication
• HIV Long Terminal Repeat
• Cell Line
• Gene Expression

• National Cancer Institute (NCI)

• Biogenesis
• Cancer therapeutics
• Physiological response
• eccDNA classification
• eccDNAs

• R01 CA222214 NCI NIH HHS
• R01 CA226269 NCI NIH HHS
• R01 CA245395 NCI NIH HHS
• R21 CA256278 NCI NIH HHS

Infusion of broadly neutralizing antibodies (bNAbs) has shown promise as an alternative to anti-retroviral therapy against HIV. A key challenge is to suppress viral escape, which is more effectively achieved with a combination of bNAbs. Here, we propose a computational approach to predict the efficacy of a bNAb therapy based on the population genetics of HIV escape, which we parametrize using high-throughput HIV sequence data from bNAb-naive patients. By quantifying the mutational target size and the fitness cost of HIV-1 escape from bNAbs, we predict the distribution of rebound times in three clinical trials. We show that a cocktail of three bNAbs is necessary to effectively suppress viral escape, and predict the optimal composition of such bNAb cocktail. Our results offer a rational therapy design for HIV, and show how genetic data can be used to predict treatment outcomes and design new approaches to pathogenic control.

• CRISPR/Cas9
• HIV-1
• J-Lat
• Rev
• Tat
• endonuclease
• endonucleases
• gene editing
• gene therapy
• latent infection
• latent reservoir

• CRISPR-Associated Protein 9
• CRISPR-Cas Systems
• DNA, Circular
• Gene Editing
• Gene Expression Regulation, Viral
• Genetic Therapy
• HEK293 Cells
• HIV Infections/virology
• HIV-1/genetics
• Humans
• Proviruses/genetics
• RNA, Guide, CRISPR-Cas Systems/genetics
• Terminal Repeat Sequences

• 2017KM79NN Ministero Università e Ricerca, PRIN 2017
• 862714 EC | H2020 | H2020 Priority Excellent Science | H2020 Future and Emerging Technologies (FET)
• SENSOR Regione Toscana (Tuscany Region)

• aging
• immune checkpoint molecules
• virus suppression

• 2-LTR circles
• Aviremic patients
• Blood cells
• HIC
• HIV-1
• Reservoir
• Total HIV DNA
• Unintegrated HIV DNA
• Viremic patients
• qPCR

• Biomarkers
• DNA, Viral
• HIV Infections/drug therapy
• HIV-1/genetics
• Humans
• Pilot Projects
• Virus Replication

• Ministero della Salute
• FanoAteneo

Tat protein is essential to fully activate HIV transcription and processing of viral mRNA, and therefore determines virus expression in productive replication and the establishment and maintenance of latent infection. Here, we used thermodynamic and structure analyses to define a highly conserved sequence-structure in tat mRNA that functions as Tat IRES modulator of tat mRNA (TIM-TAM). By impeding cap-dependent ribosome progression during authentic spliced tat mRNA translation, TIM-TAM stable structure impacts on timing and level of Tat protein hence controlling HIV production and infectivity along with promoting latency. TIM-TAM also adopts a conformation that mediates Tat internal ribosome entry site (IRES)-dependent translation during the early phases of infection before provirus integration. Our results document the critical role of TIM-TAM in Tat expression to facilitate virus reactivation from latency, with implications for HIV treatment and drug development.

• HIV-1
• Immune activation
• Immune responses
• Latent viral infection
• T-cell activation
• Therapeutic vaccine

HIV controllers (HICs) are a rare group of HIV-1-infected individuals able to naturally control viral replication. Several studies have identified the occurrence of HIV dual infections in seropositive individuals leading to disease progression. In HICs, however, dual infections with divergent outcomes in pathogenesis have been described.

Here, we present a case report of a HIC diagnosed in late 1999 who displayed stable CD4⁺ T cell levels and low plasmatic viral load across 12 years of follow-up. In early 2013, the patient started to present an increase in viral load, reaching a peak of 10,000 copies/ml in early 2014, followed by an oscillation of viremia at moderate levels in the following years. The genetic diversity of env proviral quasispecies from peripheral blood mononuclear cells (PBMCs) was studied by single genome amplification (SGA) at six timepoints across 2009–2017. Phylogenetic analyses of env sequences from 2009 and 2010 samples showed the presence of a single subtype B variant (called B₁). Analyses of sequences from 2011 and after revealed an additional subtype B variant (called B₂) and a subsequent dominance shift in the proviral quasispecies frequencies, with the B₂ variant becoming the most frequent from 2014 onwards. Latent syphilis related to unprotected sexual intercourse was diagnosed a year before the first detection of B_2, evidencing risk behavior and supporting the superinfection hypothesis. Immunologic analyses revealed an increase in CD8⁺ and CD4⁺ T cell immune activation following viremia increase and minor T cell subset alterations during follow-up. HIV-specific T cell responses remained low throughout the follow-up period.

Altogether, these results show that loss of viremia control in the HIC was associated with superinfection. These data alert to the negative consequences of reinfection on HIV pathogenesis, even in patients with a long history of viremia control and an absence of disease progression, reinforcing the need for continued use of adequate prevention strategies.

• Dual infection
• HIV-1
• HIV-controllers
• LTNP
• Superinfection

• HIV reservoirs
• HIV-infected women
• drug resistance
• nearest-neighbor analysis
• phylogenetics
• viral rebound

• Cure
• DNA quantification
• HIV-1
• Reservoir
• Therapeutic vaccines

• CD4-Positive T-Lymphocytes/virology
• Cell Line
• DNA, Viral/analysis
• DNA, Viral/genetics
• Genetic Variation
• HIV Infections/virology
• HIV Long Terminal Repeat/genetics
• HIV-1/genetics
• HIV-1/isolation & purification
• Humans
• Polymerase Chain Reaction
• Viral Load/methods

Despite the low level of viral replication in HIV controllers (HICs), studies have reported viral mutations related to escape from cytotoxic T-lymphocyte (CTL) response in HIV-1 plasma sequences. Thus, evaluating the dynamics of the emergence of CTL-escape mutants in HICs reservoirs is important for understanding viremia control. To analyze the HIV-1 mutational profile and dynamics of CTL-escape mutants in HICs, we selected 11 long-term non-progressor individuals and divided them into the following groups: (1) viremic controllers (VCs; n = 5) and (2) elite controllers (ECs; n = 6). For each individual, we used HIV-1 proviral DNA from PBMCs related to earliest (V_E) and latest (V_L) visits to obtain gag and nef sequences using the Illumina HiSeq system. The consensus of each mapped gene was used to assess viral divergence, and next-generation sequencing data were employed to identify SNPs and variations within and flanking CTL epitopes.

Divergence analysis showed higher values for nef compared to gag among the HICs. EC and VC groups showed similar divergence rates for both genes. Analysis of the number of SNPs showed that VCs present more variability in both genes. Synonymous/non-synonymous mutation ratios were < 1 for gag among ECs and for nef among ECs and VCs, exhibiting a predominance of non-synonymous mutations. Such mutations were observed in regions encoding CTL-restricted epitopes in all individuals. All ECs presented non-synonymous mutations in CTL epitopes but generally at low frequency (< 1%); all VCs showed a high number of mutations, with significant frequency changes between V_E and V_L visits. A higher frequency of internal mutations was observed for gag epitopes, with significant changes across visits compared to Nef epitopes, indicating a pattern associated with differential genetic pressure.

The high genetic conservation of HIV-1 gag and nef among ECs indicates that the higher level of viremia control restricts the evolution of both genes. Although viral replication levels in HICs are low or undetectable, all individuals exhibited CTL epitope mutations in proviral gag and nef variants, indicating that potential CTL escape mutants are present in HIC reservoirs and that situations leading to a disequilibrium of the host-virus relationship can result in the spread of CTL-escape variants.

The online version of this article (10.1186/s12977-018-0444-z) contains supplementary material, which is available to authorized users.

• CTL epitope
• Escape mutant
• HIV controller
• HIV-1
• Next-generation sequencing
• Single-nucleotide polymorphism

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4⁺ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4 + T-cells in a single experiment. The scdPCR method was then applied to CD4⁺ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous—increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

•

A common approach to HIV cure involves reactivating HIV-infected cells.

We designed and implemented a microfluidic, single-cell assay to directly measure the number of individual cells from individuals on antiretroviral therapy that produce HIV-1 RNA. The assay also allows for single-cell quantification of human genomic DNA and messenger RNA following identification and isolation of actively HIV-1-infected cells. The ability to directly measure transcriptional activity of HIV-1 in individual cells followed by downstream characterization of human and viral genetic information within these cells has the potential to provide a greater mechanistic understanding of experimental strategies to purge residual HIV-1 reservoirs.

• Digital PCR
• HIV reactivation
• HIV reservoirs
• Histone deacetylase inhibitors
• Human Immunodeficiency Virus (HIV)
• Single cell quantification

Ongoing intra-host HIV-1 evolution has been shown in individuals that naturally suppress the viremia to low levels (HIV controllers) by the analysis of the RNA in plasma compartment. Detection of evolution at the DNA proviral compartment in HIV controllers, however, has been more challenging and the precise correlation between the systemic viral suppression level and rate of reservoir’s reseeding in those individuals is not fully understood. In this sense, we examined the proviral DNA quasispecies by single genome amplification of the env gene in a cohort of 23 HIV controllers from Brazil, divided in three groups, according to the level of systemic viral suppression: (1) elite controllers with persistent undetectable viral load (PEC, n = 6); (2) elite controllers with occasional episodes of transient (51–400 copies/mL) viremia (EEC, n = 7); and (3) viremic controllers with persistent low-level (80–2000 copies/mL) viremia (VC, n = 10).

The HIV-1 diversity of the PBMC-associated proviral quasispecies in EC was significantly (P < 0.01) lower than in VC, but not significantly different between PEC and EEC groups. We detected a considerable variation in the average pairwise nucleotide distance and proportion of unique sequences in the HIV-1 proviral quasispecies of PEC and EEC. Some PEC and EEC displayed highly homogenous proviral populations with large clusters of identical sequences, while others exhibited relatively diverse proviral populations with a high proportion of unique sequences comparable to VC subjects. The long-term (10–15 years) follow-up of the HIV-1 proviral populations revealed a complete evolutionary stasis in one PEC and measurable divergence rates in one EEC [3.1 (1.2–5.6) × 10⁻³ substitutions/site/year and one VC [2.9 (0.7–5.1) × 10⁻³ substitutions/site/year].

There is no simple relationship between systemic viral suppression and intra-host proviral diversity or rate of reservoir’s reseeding in chronically infected HIV controllers. Our results demonstrate that very divergent patterns of intra-host viral diversity and divergence could be detected in the setting of natural suppression of HIV-1 replication and that ongoing evolution and reseeding of the PBMC proviral reservoir occurs in some elite controllers.

The online version of this article (doi:10.1186/s12977-017-0354-5) contains supplementary material, which is available to authorized users.

• Diversity
• Elite controllers
• Evolution
• HIV-1
• Reseeding
• Reservoir
• Viremic controllers

In most patients, current antiretroviral therapy (ART) regimens can rapidly reduce plasma viral load. However, even after years of effective treatment, a significant proportion of patients show residual plasma viremia below the clinical detection limit. Although residual viremia might be associated with increased chronic immune activation and morbidity, its origin and its potential role in the replenishment of the viral reservoir during suppressive ART is not completely understood. We performed an in-depth genetic analysis of the total and episomal cell-associated viral DNA (vDNA) repertoire in purified CD4⁺ T cell subsets of three HIV-infected individuals, and used phylogenetic analysis to explore its relationship with plasma viruses.

The predominant proviral reservoir was established in naïve or memory (central and transitional) CD4⁺ T cell subsets in patients harboring X4- or R5-tropic viruses, respectively. Regardless of the viral tropism, most plasma viruses detected under suppressive ART resembled the proviral reservoir identified in effector and transitional memory CD4⁺ T-cell subsets in blood, suggesting that residual viremia originates from these cells in either blood or lymphoid tissue. Most importantly, sequences in episomal vDNA in CD4⁺ T-cells were not well represented in residual viremia.

Viral tropism determines the differential distribution of viral reservoir among CD4⁺ T-cell subsets. In spite of viral tropism, the effector and transitional memory CD4⁺ T-cells subsets are the main source of residual viremia during suppressive ART, even though their contribution to the total proviral pool is small. However, the lack of concordance between residual viremia and viral variants driving de novo infection of CD4⁺ T cells on ART may reflect the predominance of defective plasma HIV RNA genomes. These findings highlight the need for monitoring the multiple viral RNA/DNA persistence markers, based on their differential contribution to viral persistence.

The online version of this article (doi:10.1186/s12977-016-0282-9) contains supplementary material, which is available to authorized users.

• CD4+ T cell subsets
• HIV-1
• Persistence
• Residual viremia
• Viral reservoir

• Emerging technologies
• HIV-1/AIDS
• Microfluidics
• Nanotechnology
• Point-of-care

• Anti-HIV Agents/pharmacology
• Anti-HIV Agents/therapeutic use
• Biosensing Techniques/methods
• Biosensing Techniques/trends
• HIV Infections/diagnosis
• HIV Infections/therapy
• HIV Infections/virology
• HIV-1/drug effects
• HIV-1/isolation & purification
• Humans
• Microfluidics/methods
• Microfluidics/trends
• Nanomedicine/methods
• Nanomedicine/trends
• Point-of-Care Systems/trends
• Viral Load/drug effects

• R21 AI110277 NIAID NIH HHS
• R01 AI120683 NIAID NIH HHS
• R21 AI113117 NIAID NIH HHS
• R01 DE024971 NIDCR NIH HHS
• R01 AI093282 NIAID NIH HHS
• U54 EB015408 NIBIB NIH HHS

The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4–2.6) between time point 1 and 2; and median of 31 days (IQR: 28–36) between time point 2 and 3. Patients were median of 6 years (IQR: 3–12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2–8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R² = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log₁₀, and 2.23 log₁₀ between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication-competent virus in ART suppressed patients.

Current HIV-1 research aims to find a cure for HIV-1, either by pursuing viral eradication or by attempting to attain an immune-mediated functional cure. For the purpose of interpreting the findings of these eradication strategies, a validated representative biomarker of the replication-competent latent HIV-1 reservoir is urgently needed. In this study we have evaluated several cell-associated HIV-1 persistence markers, and we have measured replication-competent reservoir using the viral outgrowth assay (VOA). The results show a correlation between the pool of HIV-1 DNA and the replication-competent reservoir. Our data show that the pool of HIV-1 DNA (total or integrated HIV-1 DNA) can predict the amount of replication-competent latent HIV-1 in patients receiving treatment. Hence, PCR based assays quantifying integrated and/or total HIV-1 DNA can play an important role in future studies aiming at HIV-1 eradication.

• Animals
• Anti-Retroviral Agents/adverse effects
• Anti-Retroviral Agents/therapeutic use
• CD4-Positive T-Lymphocytes/immunology
• CD4-Positive T-Lymphocytes/virology
• HIV Infections/drug therapy
• HIV Infections/immunology
• HIV-1/immunology
• Humans
• Proviruses/immunology
• Virus Integration/drug effects
• Virus Integration/immunology

• cns
• phylogenetics
• phylogeography
• hiv-1
• reservoir
• siv
• phyloanatomy

• Animals
• CD4-Positive T-Lymphocytes/virology
• Central Nervous System/virology
• Disease Reservoirs/virology
• HIV Infections/virology
• HIV-1/pathogenicity
• HIV-1/physiology
• Humans
• Macrophages/virology
• Phylogeny
• Simian Acquired Immunodeficiency Syndrome/virology
• Simian Immunodeficiency Virus/pathogenicity
• Simian Immunodeficiency Virus/physiology
• Virus Latency/physiology

• R01 NS063897 NINDS NIH HHS

Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure.

DOI:http://dx.doi.org/10.7554/eLife.09115.001

Many people with HIV infections are able to live relatively normal lives thanks to major advances in drug therapies. A cure, however, remains elusive. One reason for this is that the virus can hide in certain types of human cells, where it is protected from the immune system and the effects of “antiretroviral” drugs. This creates reservoirs of virus particles in the body that can quickly multiply and spread if treatment stops.

Some people who become infected with HIV are able to contain the virus without the help of drug treatments. These individuals – known as long-term non-progressors – do not become ill and only have low numbers of HIV particles in reservoirs. People who receive treatment early in the course of an HIV infection also have fewer viruses in reservoirs and are less likely to develop severe illness. Therefore, it might be possible to develop a “functional” cure that may not completely eliminate the virus from the body, but would prevent illness and allow the individuals to eventually stop taking antiretroviral drugs.

Now, Malatinkova, De Spiegelaere et al. studied samples from 84 patients with HIV-1 to find how much effect an early start to treatment has on the amount of the virus in reservoirs. People who started treatment soon after infection had lower levels of HIV-1 in their blood than people who started treatment later (even after 10 years of treatment). However, patients that started treatment early had higher levels of HIV-1 in the blood than the patients who were long-term non-progressors. All the patients had similar levels of HIV-1 in tissue samples taken from the rectum, regardless of when they started treatment.

The experiments suggest that HIV-1 reservoirs form very soon after infection. Malatinkova, De Spiegelaere et al. found that in addition to reducing reservoirs of HIV-1, an early start to drug treatment reduced the ability of the virus to make copies of its genetic code. People who started treatment earlier also had healthier immune cells. Together, the experiments support the benefits of starting drug treatments as soon as possible after a person is infected with HIV-1. It is important to further characterize thoroughly the viral reservoir in patients with limited HIV-1 reservoirs and to look for other immune factors involved in virus control, in the search for a functional cure of HIV.

DOI:http://dx.doi.org/10.7554/eLife.09115.002

• HIV-1 reservoir
• early long-term ART
• human
• infectious disease
• long-term non-progressors
• microbiology
• seroconversion
• viruses

While human immunodeficiency virus 1 (HIV-1) infection is controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference (RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by non-coding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing (TGS), as well as fine-tuning their expression through post-transcriptional gene silencing (PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells.

• Human immunodeficiency virus 1
• RNA interference
• Reservoirs
• Epigenetics
• Latency
• Transcriptional gene silencing
• Post-transcriptional gene silencing

• Anti-HIV drug
• Cell-free
• Cell-to-cell
• Circular HIV-1 DNA
• HIV-1
• Integration

• Anti-HIV Agents/pharmacology
• Cell Line
• HIV-1/drug effects
• HIV-1/physiology
• Humans
• Virus Internalization/drug effects
• Virus Release/drug effects
• Virus Replication/drug effects

• 2LTR circular HIV DNA
• HCV
• HIV
• IFN-α

• RNA editing
• alternative splicing
• quantitative genetics

• Alternative Splicing/genetics
• Animals
• Base Sequence
• High-Throughput Nucleotide Sequencing
• Humans
• Mice
• Phenotype
• Polymorphism, Single Nucleotide
• Protein Isoforms/genetics
• Proteomics
• Quantitative Trait Loci/genetics
• RNA Editing/genetics
• RNA, Messenger/genetics
• Sequence Analysis, RNA
• Transcriptome

• R01 AI080621 NIAID NIH HHS
• R01-AI080621 NIAID NIH HHS
• U54 AI057159 NIAID NIH HHS
• U54-AI057159 NIAID NIH HHS
• 104111 Wellcome Trust
• Wellcome Trust

• ADN
• Clinical use
• DNA
• HIV-1
• Integrated
• Intégré
• Non intégré
• PCR
• Quantification
• Unintegrated
• Usage clinique
• VIH-1

• HDAC inhibitors
• HIV latency
• HIV/AIDS
• antiviral therapy
• hematopoietic progenitor cells
• resting memory CD4+ T cells

• Antiretroviral Therapy, Highly Active
• Base Sequence
• Boston
• CD4-Positive T-Lymphocytes/cytology
• CD4-Positive T-Lymphocytes/virology
• Cluster Analysis
• Disease Reservoirs/virology
• Evolution, Molecular
• Flow Cytometry
• HIV Infections/drug therapy
• HIV Infections/virology
• HIV-1/genetics
• Humans
• Lymphoid Progenitor Cells/cytology
• Molecular Sequence Data
• Phylogeny
• Reverse Transcriptase Polymerase Chain Reaction
• Sequence Analysis, DNA
• Species Specificity
• Statistics, Nonparametric
• Virus Latency
• env Gene Products, Human Immunodeficiency Virus/genetics

• K08 AI100699 NIAID NIH HHS
• R21 AI106468 NIAID NIH HHS
• AI098480 NIAID NIH HHS
• AI106468 NIAID NIH HHS
• AI100699 NIAID NIH HHS
• P01 AI074415 NIAID NIH HHS
• AI089339 NIAID NIH HHS
• AI098487 NIAID NIH HHS
• AI074415 NIAID NIH HHS
• K23 AI098480 NIAID NIH HHS
• R01 AI089339 NIAID NIH HHS
• Howard Hughes Medical Institute
• R01 AI098487 NIAID NIH HHS

• Cryptic viremia
• Drug sanctuary
• HIV
• Mathematical biology

Analyzing the time-course of several viral infections using mathematical models based on experimental data can provide important quantitative insights regarding infection dynamics. Over the past decade, the importance and significance of mathematical modeling has been gaining recognition among virologists. In the near future, many animal models of human-specific infections and experimental data from high-throughput techniques will become available. This will provide us with the opportunity to develop new quantitative approaches, combining experimental and mathematical analyses. In this paper, we review the various quantitative analyses of viral infections and discuss their possible applications.

• animal experiment
• computer simulation
• mathematical modeling
• quantification
• virus infection

• hiv-1
• viral reservoirs
• hematopoietic stem cell transplantation
• hiv-1 persistence
• ccr5
• reduced-intensity allogeneic stem cell transplantation

• Anti-Retroviral Agents/administration & dosage
• Antiretroviral Therapy, Highly Active/methods
• Gene Deletion
• HIV Infections/drug therapy
• HIV Infections/virology
• HIV-1/isolation & purification
• HIV-1/pathogenicity
• Heterozygote
• Humans
• Male
• Receptors, CCR5/genetics
• Receptors, CCR5/metabolism
• Receptors, HIV/genetics
• Receptors, HIV/metabolism
• Stem Cell Transplantation
• Transplantation, Homologous
• Viral Load

• K08 AI081545 NIAID NIH HHS
• U19 AI096109 NIAID NIH HHS
• 1K23AI098480-01A1 NIAID NIH HHS
• R01 CA183560 NCI NIH HHS
• K23 AI098480 NIAID NIH HHS
• R01 CA183559 NCI NIH HHS
• P30 AI060354 NIAID NIH HHS
• UM1 AI068636 NIAID NIH HHS

Ultra-sensitive laboratory assays based on the Polymerase Chain Reaction (PCR) are playing an increasingly important role in HIV cure-related research. This article reviews the different assays available and how they have evolved. There is a great need for their standardization and for the establishment of reference reagents and testing algorithms to evaluate potential HIV cure-related treatments.

• 2-LTR
• Eradication
• HIV
• Nucleic acid tests
• PCR
• Proviral DNA
• Standardization

• HIV
• cryptic viremia
• mathematical biology

The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.

The release of the transcription elongation factor P-TEFb from the 7SK RNP complex and its binding to the HIV Tat transactivator protein enables the efficient transcription of HIV proviruses. In resting memory T-cells, which carry the bulk of the latent HIV viral pool, limiting the cellular levels of P-TEFb ensures that the provirus remains silenced unless the host cell is activated. Here we demonstrate that T-cell receptor (TCR) activation induces phosphorylation of Ser175, a residue which is located at the interface between CycT1, CDK9 and Tat. Phosphorylation of Ser175 occurs on free or 7SK-dissociated P-TEFb and genetic experiments indicate that this modification enhances P-TEFb interaction with Tat resulting in Tat-dependent reactivation of HIV proviral transcription. Modification of Ser175 appears critical for controlling the competitive binding of Tat and the bromodomain protein BRD4 to P-TEFb. Activation of P-TEFb in resting T-cells thus involves both the initial assembly of the 7SK snRNP complex and the subsequent mobilization of P-TEFb by cellular signaling and Tat. Therefore, pSer175 provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb that can be used to monitor the extent of T-cell activation during therapeutic interventions aimed at virus eradication.

• Biomarkers/metabolism
• CD4-Positive T-Lymphocytes/metabolism
• CD4-Positive T-Lymphocytes/virology
• Cell Cycle Proteins
• Cyclin T/genetics
• Cyclin T/metabolism
• Cyclin-Dependent Kinase 9/genetics
• Cyclin-Dependent Kinase 9/metabolism
• Female
• HIV/genetics
• HIV/metabolism
• HIV Infections/genetics
• HIV Infections/metabolism
• Humans
• Male
• Mutation
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Phosphorylation/genetics
• Positive Transcriptional Elongation Factor B/genetics
• Positive Transcriptional Elongation Factor B/metabolism
• Protein Binding/genetics
• Proviruses/genetics
• Proviruses/metabolism
• Transcription Factors/genetics
• Transcription Factors/metabolism
• Transcription, Genetic
• tat Gene Products, Human Immunodeficiency Virus/genetics
• tat Gene Products, Human Immunodeficiency Virus/metabolism

• DP1 DA028869 NIDA NIH HHS
• P30-AI036219 NIAID NIH HHS
• DP1-DA028869 NIDA NIH HHS
• P30 AI036219 NIAID NIH HHS
• R01-AI06709 NIAID NIH HHS
• R01 AI067093 NIAID NIH HHS
• U19 AI096113 NIAID NIH HHS

• hiv latency
• markers of hiv reservoirs
• quantification

• Biomarkers/analysis
• Carrier State/immunology
• Carrier State/virology
• Disease Reservoirs/virology
• HIV Infections/immunology
• HIV Infections/virology
• Humans
• Viral Load

• P01 AI080193 NIAID NIH HHS
• R01 AI047745 NIAID NIH HHS
• AI047745 NIAID NIH HHS
• R21 AI047745 NIAID NIH HHS
• AI306214 NIAID NIH HHS
• R56 AI047745 NIAID NIH HHS
• U19 AI096113 NIAID NIH HHS
• UM1 AI069432 NIAID NIH HHS
• AI080193 NIAID NIH HHS
• U01 AI069432 NIAID NIH HHS
• AI74621 NIAID NIH HHS
• AI69432 NIAID NIH HHS
• P01 AI074621 NIAID NIH HHS
• AI096113 NIAID NIH HHS

In most HIV-infected individuals adherent to modern antiretroviral therapy (ART), plasma viremia stays undetectable by clinical assays and therefore, additional virological markers for monitoring and predicting therapy responses and for measuring the degree of HIV persistence in patients on ART should be identified. For the above purposes, quantitation of cell-associated HIV biomarkers could provide a useful alternative to measurements of viral RNA in plasma. This review concentrates on cell-associated (CA) HIV RNA with the emphasis on its use as a virological biomarker. We discuss the significance of CA HIV RNA as a prognostic marker of disease progression in untreated patients and as an indicator of residual virus replication and the size of the dynamic viral reservoir in ART-treated patients. Potential value of this biomarker for monitoring the response to ART and to novel HIV eradication therapies is highlighted.

• Anti-HIV Agents/therapeutic use
• HIV Infections/drug therapy
• HIV Infections/virology
• HIV-1/drug effects
• HIV-1/physiology
• Humans
• Virus Latency/drug effects
• Virus Replication/drug effects

• Adult
• Anti-HIV Agents/pharmacology
• Anti-HIV Agents/therapeutic use
• Biomarkers/metabolism
• CD4 Lymphocyte Count
• CD4-Positive T-Lymphocytes/drug effects
• Clinical Trials as Topic
• DNA, Viral/drug effects
• Disease Reservoirs/virology
• Female
• HIV Infections/drug therapy
• HIV-1/drug effects
• HIV-1/genetics
• Humans
• Interferon-alpha/pharmacology
• Interferon-alpha/therapeutic use
• Male
• Middle Aged
• Polymerase Chain Reaction
• Viral Load/drug effects
• Viremia/virology
• Virus Integration/drug effects
• Virus Replication/drug effects

• P30 CA010815 NCI NIH HHS
• U01AI065279 NIAID NIH HHS
• 5K08AI073102 NIAID NIH HHS
• P30 CA10815 NCI NIH HHS
• P30 AI027763 NIAID NIH HHS
• P30 AI 045008 NIAID NIH HHS
• T32 AI007632 NIAID NIH HHS
• K08 AI073102 NIAID NIH HHS
• P30 AI045008 NIAID NIH HHS
• U01 AI065279 NIAID NIH HHS
• R21 AI087461 NIAID NIH HHS
• K02AI078766 NIAID NIH HHS
• K02 AI078766 NIAID NIH HHS

• Antiretroviral Therapy, Highly Active
• CD4-Positive T-Lymphocytes/immunology
• CD8-Positive T-Lymphocytes/immunology
• HIV/genetics
• HIV/immunology
• HIV Infections/immunology
• HIV Infections/prevention & control
• Humans
• Immunity, Cellular/physiology
• Immunotherapy/methods
• Mutation/genetics
• Therapies, Investigational/methods

• G108/626 Medical Research Council