Manohar R. Furtado

PERSISTENCE OF HIV-1 TRANSCRIPTION IN PERIPHERAL-BLOOD MONONUCLEAR CELLS IN PATIENTS RECEIVING POTENT ANTIRETROVIRAL THERAPY

BACKGROUND AND METHODSnAlthough potent antiretroviral therapy can control infection with human immunodeficiency virus type 1 (HIV-1), a long-lived reservoir of infectious virus persists in CD4+ T cells. We investigated this viral reservoir by measuring the levels of cell-associated viral DNA and messenger RNA (mRNA) that are essential for HIV-1 replication. Approximately every 6 months, we obtained samples of peripheral-blood mononuclear cells from five men with long-standing HIV-1 infection who had had undetectable levels of plasma HIV-1 RNA for 20 months or more during treatment with potent antiretroviral drugs.nnnRESULTSnBefore treatment, plasma levels of HIV-1 RNA correlated with the levels of cell-associated unintegrated HIV-1 DNA and unspliced viral mRNA. After treatment, plasma levels of HIV-1 RNA fell by more than 2.7 log to undetectable levels. The decrease in cell-associated integrated and unintegrated HIV-1 DNA and mRNA occurred in two phases. The first phase occurred during the initial 500 days of treatment and was characterized by substantial decreases in the levels of DNA and mRNA, but not to undetectable levels. The concentrations of cell-associated unintegrated viral DNA, integrated proviral DNA, and unspliced viral mRNA decreased by 1.25 to 1.46 log. The second phase occurred during the subsequent 300 days or more of treatment and was characterized by a plateau in the levels of HIV-1 DNA and unspliced mRNA. After an initial rapid decline, the ratio of unspliced to multiply spliced viral mRNA (a measure of active viral transcription) stabilized and remained greater than zero at each measurement.nnnCONCLUSIONSnDespite treatment with potent antiretroviral drugs and the suppression of plasma HIV-1 RNA to undetectable levels for 20 months or more, HIV-1 transcription persists in peripheral-blood mononuclear cells. Unless the quasi-steady state levels of HIV DNA and mRNA eventually disappear with longer periods of therapy, these findings suggest that HIV-1 infection cannot be eradicated with current treatments.

Adaptive Evolution of Human Immunodeficiency Virus-Type 1 During the Natural Course of Infection

The rate of progression to disease varies considerably among individuals infected with human immunodeficiency virus-type 1 (HIV-1). Analyses of semiannual blood samples obtained from six infected men showed that a rapid rate of CD4 T cell loss was associated with relative evolutionary stasis of the HIV-1 quasispecies virus population. More moderate rates of CD4 T cell loss correlated with genetic evolution within three of four subjects. Consistent with selection by the immune constraints of these subjects, amino acid changes were apparent within the appropriate epitopes of human leukocyte antigen class I-restricted cytotoxic T lymphocytes. Thus, the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations under natural selection are compatible with adaptive evolution.

Analysis of alternatively spliced human immunodeficiency virus type-1 mRNA species, one of which encodes a novel tat-env fusion protein.

A polymerase chain reaction-based analysis was used to define the structures of the mRNAs that encode human immunodeficiency virus type-1 (HIV-1) regulatory and structural proteins in infected H9 cells. Twenty alternatively spliced mRNAs encoding the vif, vpr, env, nef, tat, and rev proteins were characterized. An evaluation of the coding potentials of these transcripts recognized both leaky scanning and reinitiation at downstream initiation codons as mechanisms that may operate during translation of many of the polycistronic messages. Two new splice acceptor sites, one at nt 6018 defining a new mRNA coding for the env and vpu proteins and another at nt 8671 defining a novel tat-env fusion transcript, were characterized. The latter transcript expressed a novel protein p17tev that was immunoprecipitated by both polyclonal tat antibodies and monoclonals directed towards the C-terminal region of gp41. The p17tev protein was able to transactivate transcription from the HIV-1 LTR in transient transfection assays. The use of multiple alternative splice donor and acceptor sites and the generation of novel proteins may confer evolutionary advantages on the viral mutants encoding them and influence the course of clinical disease.

DETECTION OF HIV-RNA-POSITIVE MONOCYTES IN PERIPHERAL BLOOD OF HIV-POSITIVE PATIENTS BY SIMULTANEOUS FLOW CYTOMETRIC ANALYSIS OF INTRACELLULAR HIV RNA AND CELLULAR IMMUNOPHENOTYPE

Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV-1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor-alpha stimulated and unstimulated ACH-2 cells demonstrated the ability to detect < 5% HIV RNA positive cells with a sensitivity of < 30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV-seropositive patients on no, single, combination, or triple drug therapy and 8 HIV-seronegative patients were examined. The majority of HIV-positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV-infected cell populations in the peripheral blood of HIV-positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease.

Up-Regulation of HOXC6, HOXD1, and HOXD8 Homeobox Gene Expression in Human Neuroblastoma Cells following Chemical Induction of Differentiation

An early event in the pathogenesis of neuroblastoma (NB), a tumor derived from embryonal neural crest tissue, appears to be the arrested differentiation of neuroblasts. However, NB cells can be induced to differentiate in vitro with numerous chemicals including retinoic acid (RA) and dibutyryl cyclic AMP (db-cAMP). One family of transcription factors, encoded by the homeobox (HOX) genes, plays a crucial role in Drosophila, Xenopus, and mammalian embryonic differentiation and development. We have previously identified six HOX genes (HOXC6, HOXC8, HOXD1, HOXD4, HOXD8, and HOXD9), by a sensitive PCR-based approach, in a cDNA library prepared from the human LA-N-5 NB cell line induced to differentiate with RA. In this report, we studied the regulation of these six HOX genes in a series of NB cell lines chemically induced to differentiate. Untreated NB cells express low or undetectable levels of HOX mRNA, and HOXC8 remains undetectable in the induced cells. However, a significant induction of HOXC6, HOXD1, and HOXD8 expression is seen in the RA-treated NB cell lines, albeit with different patterns and degree of up-regulation. db-cAMP treatment also induced HOXC6 and HOXD8 expression in two of the three NB cell lines analyzed. Low levels of HOXD4 and HOXD9 induction were observed in two and one RA-treated NB cell line, respectively. Up-regulation of HOXC6, HOXD1, and HOXD8 expression in human NB cells, chemically induced to differentiate, appears to be associated with maturation toward a differentiated neuronal phenotype.

Persistent HIV-1-specific cellular responses despite prolonged therapeutic viral suppression

Design Antiretroviral therapy (ART) currently represents the best way to avert the lethal consequences of chronic persistent HIV-1 infection. It leads to significant reductions of plasma viremia, often to undetectable levels, but it can also be linked with the reduction and disappearance of detectable HIV-specific CD8 T-cell responses. Results Here we describe a group of patients in whom ongoing replication of HIV, particularly transcription of Nef mRNA species, was detected despite prolonged and clinically successful antiretroviral treatment. Modest, but significant, numbers of HIV-specific CD8 T cells and CD4 T-cell responses were found in these subjects, with the strongest responses directed towards Nef epitopes. Detailed phenotypic analysis of the HIV-specific CD8 cells demonstrated low perforin levels and persistent expression of CD27, a phenotype associated with incomplete differentiation of cytotoxic T lymphocytes (CTL). Conclusion This immature CTL phenotype has been described previously in association with chronic HIV disease, but its continued persistence is surprising in the setting of prolonged viral suppression on therapy and the presence of HIV-specific CD4 cell activity.

Down-modulation of TCR/CD3 surface complexes after HIV-1 infection is associated with differential expression of the viral regulatory genes.

We have investigated the mechanism(s) involved in progressive abrogation of CD3‐γ gene expression after HIV‐1 or HIV‐2 infection. A comparison of intracellular virus expression with T cell receptor surface density, revealed both high and low levels of viral p24 antigen in the TCR/CD3hi, TCR/CD3lo, and TCR/CD3– cells. Furthermore, in non‐productively infected cells expressing the multiply spliced, virally encoded tat, rev, and nef regulatory gene transcripts, the same progressive loss of surface TCR/CD3 complexes was observed. We treated HIV‐1‐infected cells with antisense (AS) phosphorothioate oligodeoxynucleotides (P‐OdN) targeted to the viral regulatory genes. All of the HIV‐1 sequence‐specific AS‐P‐OdN′s inhibited intracellular p24 antigen expression in a time‐ and dose‐dependent manner; although, blocking p24 expression alone was not sufficient to modulate TCR/CD3 surface density. Only Tat‐AS and Nef‐AS were able to delay TCR/CD3 down‐modulation on receptor‐positive cells or drive receptor up‐regulation on receptor‐negative cells. In contrast, Rev‐AS accelerated TCR/CD3 loss on receptor‐positive cells. RT‐PCR revealed that Tat‐AS and Nef‐AS reduce the level of tat, nef, and rev transcripts, while Rev‐AS increases the level of tat and nef transcripts in infected cells. Thus, when intracellular conditions favor expression of tat and/or nef in the absence of rev, CD3‐γ gene transcripts and TCR/CD3 surface density are down‐modulated.