Bharat Ramratnam

Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy.

BACKGROUND In patients infected with human immunodeficiency virus type 1 (HIV-1), combination antiretroviral therapy can result in sustained suppression of plasma levels of the virus. However, replication-competent virus can still be recovered from latently infected resting memory CD4 lymphocytes; this finding raises serious doubts about whether antiviral treatment can eradicate HIV-1. METHODS We looked for evidence of residual HIV-1 replication in eight patients who began treatment soon after infection and in whom plasma levels of HIV-1 RNA were undetectable after two to three years of antiretroviral therapy. We examined whether there had been changes over time in HIV-1 proviral sequences in peripheral-blood mononuclear cells, which would indicate residual viral replication. We also performed in situ hybridization studies on tissues from one patient to identify cells actively expressing HIV-1 RNA. We estimated the rate of decrease of latent, replication-competent HIV-1 in resting CD4 lymphocytes on the basis of the decrease in the numbers of proviral sequences identified during primary infection and direct sequential measurements of the size of the latent reservoir. RESULTS Six of the eight patients had no significant variations in proviral sequences during treatment. However, in two patients there was sequence evolution but no evidence of drug-resistant viral genotypes. In one patient, extensive in situ studies provided additional evidence of persistent viral replication in lymphoid tissues. Using two independent approaches, we estimated that the half-life of the latent, replication-competent virus in resting CD4 lymphocytes was approximately six months. CONCLUSIONS These findings suggest that combination antiretroviral regimens suppress HIV-1 replication in some but not all patients. Given the half-life of latently infected CD4 lymphocytes of about six months, it may require many years of effective antiretroviral treatment to eliminate this reservoir of HIV-1.

The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy.

Replication-competent HIV-1 can be isolated from infected patients despite prolonged plasma virus suppression by anti-retroviral treatment. Recent studies have identified resting, memory CD4+ T lymphocytes as a long-lived latent reservoir of HIV-1 (refs. 4,5). Cross-sectional analyses indicate that the reservoir is rather small, between 103 and 107 cells per patient. In individuals whose plasma viremia levels are well suppressed by anti-retroviral therapy, peripheral blood mononuclear cells containing replication-competent HIV-1 were found to decay with a mean half-life of approximately 6 months, close to the decay characteristics of memory lymphocytes in humans and monkeys. In contrast, little decay was found in a less-selective patient population. We undertook this study to address this apparent discrepancy. Using a quantitative micro-culture assay, we demonstrate here that the latent reservoir decays with a mean half-life of 6.3 months in patients who consistently maintain plasma HIV-1 RNA levels of fewer than 50 copies/ml. Slower decay rates occur in individuals who experience intermittent episodes of plasma viremia. Our findings indicate that the persistence of the latent reservoir of HIV-1 despite prolonged treatment is due not only to its slow intrinsic decay characteristics but also to the inability of current drug regimens to completely block HIV-1 replication.

Human Immunodeficiency Virus Type 1 Escape from RNA Interference

ABSTRACT Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.

Rapid production and clearance of HIV-1 and hepatitis C virus assessed by large volume plasma apheresis

BACKGROUND In chronic HIV-1 infection, dynamic equilibrium exists between viral production and clearance. The half-life of free virions can be estimated by inhibiting virion production with antiretroviral agents and modelling the resulting decline in plasma HIV-1 RNA. To define HIV-1 and hepatitis C virus (HCV) dynamics, we used plasma apheresis to increase virion clearance temporarily while leaving virion production unaffected. METHODS Plasma virus loads were measured frequently before, during, and after apheresis in four HIV-1-infected patients, two of whom were also co-infected with HCV. Rates of virion clearance were derived by non-linear least-square fitting of plasma virus load to a model of viral dynamics. FINDINGS Virion clearance rate constants were 0.0063/min (9.1/day) to 0.025/min (36.0/day; half-life 28-110 min) for HIV-1 and 0.0038/min (5.5/day) to 0.0069/min (9.9/day; half-life 100-182 min) for HCV. These values provided estimates of daily particle production of 9.3 log10-10.2 log10 particles for HIV-1 and 11.6 log10-13.0 log10 particles for HCV. INTERPRETATION Our findings confirm that HIV-1 and HCV are produced and cleared extremely rapidly. New estimates for HIV-1 clearance are up to ten times higher than previous ones, whereas HCV clearance is similar to previous estimates.

A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals.

OBJECTIVES HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (ELISPOT) assay. METHODS Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase. RESULTS In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 10(6) PBMC. In HLA A*0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays. CONCLUSIONS Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies.

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- And tissue-specific microRNAs

Significance MicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology. Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

Genetic characterization of rebounding HIV-1 after cessation of highly active antiretroviral therapy

Despite prolonged treatment with highly active antiretroviral therapy (HAART), infectious HIV-1 continues to replicate and to reside latently in resting memory CD4(+) T lymphocytes, creating a major obstacle to HIV-1 eradication. It is therefore not surprising to observe a prompt viral rebound after discontinuation of HAART. The nature of the rebounding virus, however, remains undefined. We now report on the genetic characterization of rebounding viruses in eight patients in whom plasma viremia was undetectable throughout about 3 years of HAART. Taking advantage of the extensive length polymorphism in HIV-1 env, we found that in five patients who did not show HIV-1 replication during treatment, the rebound virus was identical to those isolated from the latent reservoir. In three other patients, two of whom had been free of plasma viremia but had showed some residual viral replication, the rebound virus was genetically different from the latent reservoir virus, corresponding instead to minor viral variants detected during the course of treatment in lymphoid tissues. We conclude that in cases with apparent complete HIV-1 suppression by HAART, viral rebound after cessation of therapy could have originated from the activation of virus from the latent reservoir. In patients with incomplete suppression by chemotherapy, however, the viral rebound is likely triggered by ongoing, low-level replication of HIV-1, perhaps occurring in lymphoid tissues.

Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription

OBJECTIVE Microvesicles have been shown to mediate intercellular communication. Previously, we have correlated entry of murine lung-derived microvesicles into murine bone marrow cells with expression of pulmonary epithelial cell-specific messenger RNA (mRNA) in these marrow cells. The present studies establish that entry of lung-derived microvesicles into marrow cells is a prerequisite for marrow expression of pulmonary epithelial cell-derived mRNA. MATERIALS AND METHODS Murine bone marrow cells cocultured with rat lung, but separated from them using a cell-impermeable membrane (0.4-microm pore size), were analyzed using species-specific primers (for rat or mouse). RESULTS These studies revealed that surfactant B and C mRNA produced by murine marrow cells were of both rat and mouse origin. Similar results were obtained using murine lung cocultured with rat bone marrow cells or when bone marrow cells were analyzed for the presence of species-specific albumin mRNA after coculture with rat or murine liver. These studies show that microvesicles both deliver mRNA to marrow cells and mediate marrow cell transcription of tissue-specific mRNA. The latter likely underlies the longer-term stable change in genetic phenotype that has been observed. We have also observed microRNA in lung-derived microvesicles, and studies with RNase-treated microvesicles indicate that microRNA negatively modulates pulmonary epithelial cell-specific mRNA levels in cocultured marrow cells. In addition, we have also observed tissue-specific expression of brain, heart, and liver mRNA in cocultured marrow cells, suggesting that microvesicle-mediated cellular phenotype change is a universal phenomena. CONCLUSION These studies suggest that cellular systems are more phenotypically labile than previously considered.

Acetylation-Dependent Signal Transduction for Type I Interferon Receptor

Cytokine-activated receptors initiate intracellular signaling by recruiting protein kinases that phosphorylate the receptors on tyrosine residues, thus enabling docking of SH2 domain-bearing activating factors. Here we report that in response to type 1 interferon (IFNalpha), IFNalpha receptors recruit cytoplasmic CREB-binding protein (CBP). By binding to IFNalphaR2 within the region where two adjacent proline boxes bear phospho-Ser364 and phospho-Ser384, CBP acetylates IFNalphaR2 on Lys399, which in turn serves as the docking site for interferon regulatory factor 9 (IRF9). IRF9 interacts with the acetyl-Lys399 motif by means of its IRF homology2 (IH2) domain, leading to formation of the ISGF3 complex that includes IRF9, STAT1, and STAT2. All three components are acetylated by CBP. Remarkably, acetylation within the DNA-binding domain (DBD) of both IRF9 and STAT2 is critical for the ISGF3 complex activation and its associated antiviral gene regulation. These results have significant implications concerning the central role of acetylation in cytokine receptor signal transduction.

Discontinuation of antiretroviral therapy commenced early during the course of human immunodeficiency virus type 1 infection, with or without adjunctive vaccination.

Sixteen subjects were treated with highly active antiretroviral therapy within 120 days of the onset of symptoms of newly acquired human immunodeficiency virus type 1 (HIV-1) infection. Eleven of the 16 participated in an adjunctive therapeutic vaccine trial. After a mean of 3.2 years of treatment, they elected to discontinue therapy. Virus rebound occurred in all subjects and was followed by a spontaneous, transient although significant reduction in log plasma HIV-1 RNA level, ranging from 0.3 to 3.1 log(10) copies/mL. Despite evidence of the induction of HIV-1-specific cell-mediated immune responses, plasma viremia was not persistently suppressed to <500 copies/mL in any subject. The magnitude and dynamics of virus rebound were similar in both vaccinated and unvaccinated subjects. Nevertheless, given the transient suppression of viremia observed in nearly all subjects after treatment has been discontinued, further investigations of adjunctive vaccination with optimized antiretroviral therapy in treating HIV-1 infection are warranted.