Inhibit Human Immunodeficiency Virus-1 Replication Research Articles

HIV-1 Transcription Inhibitor 1E7-03 Decreases Nucleophosmin Phosphorylation

Transcription activation of latent human immunodeficiency virus-1 (HIV-1) occurs due to HIV-1 rebound, the interruption of combination antiretroviral therapy, or development of drug resistance. Thus, novel HIV-1 inhibitors, targeting HIV-1 transcription are needed. We previously developed an HIV-1 transcription inhibitor, 1E7-03, that binds to the noncatalytic RVxF-accommodating site of protein phosphatase 1 and inhibits HIV-1 replication in cultured cells and HIV-1–infected humanized mice by impeding protein phosphatase 1 interaction with HIV-1 Tat protein. However, host proteins and regulatory pathways targeted by 1E7-03 that contribute to its overall HIV-1 inhibitory activity remain to be identified. To address this issue, we performed label-free quantitative proteome and phosphoproteome analyses of noninfected and HIV-1–infected CEM T cells that were untreated or treated with 1E7-03. 1E7-03 significantly reprogramed the phosphorylation profile of proteins including PPARα/RXRα, TGF-β, and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue in PPARα/RXRα pathway was significantly reduced (>20-fold, p = 1.37 × 10−9), followed by the reduced phosphorylation of transforming growth factor-beta 2 at Ser-46 (TGF-β2, >12-fold, p = 1.37 × 10−3). Downregulation of NPM1’s Ser-125 phosphorylation was further confirmed using Western blot. Phosphorylation mimicking NPM1 S125D mutant activated Tat-induced HIV-1 transcription and exhibited enhanced NPM1–Tat interaction compared to NPM1 S125A mutant. Inhibition of Aurora A or Aurora B kinases that phosphorylate NPM1 on Ser-125 residue inhibited HIV-1, further supporting the role of NPM1 in HIV-1 infection. Taken together, 1E7-03 reprogrammed PPARα/RXRα and TGF-β pathways that contribute to the inhibition of HIV-1 transcription. Our findings suggest that NPM1 phosphorylation is a plausible target for HIV-1 transcription inhibition.

Pharmacological activation of the circadian component REV-ERB inhibits HIV-1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks a curative therapy or vaccine. Despite the reduction in AIDS-related deaths achieved by current antiretroviral therapies, drawbacks including drug resistance and the failure to eradicate infection highlight the need to identify new pathways to target the infection. Circadian rhythms are endogenous 24-h oscillations which regulate physiological processes including immune responses to infection, and there is an emerging role for the circadian components in regulating viral replication. The molecular clock consists of transcriptional/translational feedback loops that generate rhythms. In mammals, BMAL1 and CLOCK activate rhythmic transcription of genes including the nuclear receptor REV-ERBα, which represses BMAL1 and plays an essential role in sustaining a functional clock. We investigated whether REV-ERB activity regulates HIV-1 replication and found REV-ERB agonists inhibited HIV-1 promoter activity in cell lines, primary human CD4 T cells and macrophages, whilst antagonism or genetic disruption of REV-ERB increased promoter activity. The REV-ERB agonist SR9009 inhibited promoter activity of diverse HIV-subtypes and HIV-1 replication in primary T cells. This study shows a role for REV-ERB synthetic agonists to inhibit HIV-1 LTR promoter activity and viral replication, supporting a role for circadian clock components in regulating HIV-1 replication.

Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability

Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N4 position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discrete sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor remodelin could inhibit HIV-1 replication at concentrations that have no effect on cell viability, thus identifying ac4C addition as a potential target for antiviral drug development.

Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability

Epitranscriptomic RNA modifications, including methylation of A and C residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N4 position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discreet sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor Remodelin could inhibit HIV-1 replication at levels that have no effect on cell viability, thus identifying ac4C addition as a potential novel target for antiviral drug development.

Novel host restriction factors implicated in HIV-1 replication

Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection

Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4+ lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

Target silencing of components of the conserved oligomeric Golgi complex impairs HIV-1 replication

All viruses require host cell factors to replicate. A large number of host factors have been identified that participate at numerous points of the human immunodeficiency virus 1 (HIV-1) life cycle. Recent evidence supports a role for components of the trans-Golgi network (TGN) in mediating early steps in the HIV-1 life cycle. The conserved oligomeric Golgi (COG) complex is a heteroctamer complex that functions in coat protein complex I (COPI)-mediated intra-Golgi retrograde trafficking and plays an important role in the maintenance of Golgi structure and integrity as well as glycosylation enzyme homeostasis. The targeted silencing of components of lobe B of the COG complex, namely COG5, COG6, COG7 and COG8, inhibited HIV-1 replication. This inhibition of HIV-1 replication preceded late reverse transcription (RT) but did not affect viral fusion. Silencing of the COG interacting protein the t-SNARE syntaxin 5, showed a similar defect in late RT product formation, strengthening the role of the TGN in HIV replication.

HIV-1 Gag-Virus-Like Particles Inhibit HIV-1 Replication in Dendritic Cells and T Cells through IFN-α-Dependent Upregulation of APOBEC3G and 3F

Human immunodeficiency virus-1 (HIV-1) infection and the acquired immune deficiency syndrome (AIDS) pandemic remain global threats in the absence of a protective or a therapeutic vaccine. HIV-1 replication is reportedly inhibited by some cellular factors, including APOBEC3G (A3G) and APOBEC3F (A3F), which are well known inhibitors of HIV-1. Recently, HIV-1 Gag-virus-like particles (Gag-VLPs) have been shown to be safe and potent HIV-1 vaccine candidates that can elicit strong cellular and humoral immunity without need of any adjuvant. In this report, we stimulated human monocyte-derived dendritic cells (DCs) with Gag-VLPs and we demonstrated that Gag-VLP-treated DCs (VLP-DCs) produced interferon alpha (IFN-α), along with an increase in mRNA and protein expression of A3G and A3F. Gag-VLPs inhibited HIV-1 replication not only in DCs themselves, but also in cocultured T cells in an IFN-α-dependent manner. In addition, A3G/3F content in HIV virions released from VLP-DCs increased. Both the increase in A3G/3F expression and the inhibition of HIV-1 replication were reversed by anti-IFN-α or anti-IFNAR antibodies. Our findings in this study provide insight into the mechanism of Gag-VLP-induced inhibition of HIV-1 replication in DCs and T cells.

Heme arginate potentiates latent HIV-1 reactivation while inhibiting the acute infection

Human immunodeficiency virus-1 (HIV-1) successfully escapes from host immune surveillance, vaccines and antiretroviral agents. The available antiretroviral compounds can only control viremia, but it is impossible to eliminate the virus from the organism, namely because HIV-1 provirus persists in the reservoir cells from which the virus repeatedly disseminates into new cells. Current therapeutic approaches, however, do not specifically address the stage of virus reactivation.Heme has been demonstrated as very efficient in inhibiting HIV-1 reverse transcription, while its derivative hemin ameliorated HIV-1 infection via induction of heme oxygenase-1. Normosang (heme arginate; HA) is a human hemin-containing compound used to treat acute porphyria. In this work, we studied the effects of HA in HIV-1-acutely infected T-cell lines, and in cell lines harboring either a complete HIV-1 provirus (ACH-2 cells) or an HIV-1 “mini-virus” (Jurkat clones expressing EGFP under control of HIV LTR). We demonstrate that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA alone stimulated the reactivation of HIV-1 “mini-virus” and synergized with phorbol ester or TNF-α in the reactivation of HIV-1 provirus. The stimulatory effects of HA were inhibited by N-acetyl cysteine, suggesting an increased redox stress and activation of NF-κB. Further, HA induced expression of heme oxygenase-1 (HO-1) in ACH-2 cells, while HO-1 was found expressed in untreated Jurkat clones. Inhibitor of HO-1 activity, tin protoporphyrin IX, further increased HA-mediated reactivation of HIV-1 “mini-virus” in Jurkat clones, and this effect was also inhibited by N-acetyl cysteine. The stimulatory effects of HA on HIV-1 reactivation thus seem to involve HO-1 and generation of free radicals. Additionally, the effective concentrations of HA did neither affect normal T-cell activation with PMA nor induce activation of the unstimulated cells.In conclusion, HA appears to possess a combination of unique properties that could help to decrease the pool of latently infected reservoir cells, while simultaneously inhibiting HIV-1 replication in newly infected cells. Our results thus suggest a new direction to explore in treatment of HIV/AIDS disease.

Hormonally Active Vitamin D3 (1α,25-Dihydroxycholecalciferol) Triggers Autophagy in Human Macrophages That Inhibits HIV-1 Infection

Autophagy is a self-digestion pathway essential for maintaining cellular homeostasis and cell survival and for degrading intracellular pathogens. Human immunodeficiency virus-1 (HIV-1) may utilize autophagy for replication as the autophagy-related protein-7 (ATG-7), microtubule-associated protein 1 light chain 3, ATG-12, and ATG-16L2 are required for productive HIV-1 infection; however, the effects of autophagy induction on HIV-1 infection are unknown. HIV-1-infected individuals have lower levels of 1α,25-dihydroxycholecalciferol, the hormonally active form of vitamin D, than uninfected individuals. with the lowest concentrations found in persons with AIDS. Using human macrophages and RNA interference for ATG-5 and Beclin-1 and chemical inhibition of phosphatidylinositol 3-kinase, we have found that physiologically relevant concentrations of 1α,25-dihydroxycholecalciferol induce autophagy in human macrophages through a phosphatidylinositol 3-kinase-, ATG-5-, and Beclin-1-dependent mechanism that significantly inhibits HIV-1 replication in a dose-dependent manner. We also show that the inhibition of basal autophagy inhibits HIV-1 replication. Furthermore, although 1α,25-dihydroxycholecalciferol induces the secretion of human cathelicidin, at the concentrations produced in vitro, cathelicidin does not trigger autophagy. Our findings support an important role for autophagy during HIV-1 infection and provide new insights into novel approaches to prevent and treat HIV-1 infection and related opportunistic infections.

Inhibition of HIV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A.

Human immunodeficiency virus 1 (HIV-1) multiplication depends on a cellular protein, cyclophilin A (CyPA), that gets integrated into viral particles. Because CyPA is not required for cell viability, we attempted to block its synthesis in order to inhibit HIV-1 replication. For this purpose, we used antisense U7 small nuclear RNAs (snRNAs) that disturb CyPA pre-mRNA splicing and short interfering RNAs (siRNAs) that target CyPA mRNA for degradation. With dual-specificity U7 snRNAs targeting the 3' and 5' splice sites of CyPA exons 3 or 4, we obtained an efficient skipping of these exons and a strong reduction of CyPA protein. Furthermore, short interfering RNAs targeting two segments of the CyPA coding region strongly reduced CyPA mRNA and protein levels. Upon lentiviral vector-mediated transduction, prolonged antisense effects were obtained for both types of antisense RNAs in the human T-cell line CEM-SS. These transduced CEM-SS cells showed a delayed, and for the siRNAs also reduced, HIV-1 multiplication. Since the two types of antisense RNAs function by different mechanisms, combining the two approaches may result in a synergistic effect.

The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes.

Although human immunodeficiency virus-1 (HIV-1) infects quiescent and proliferating CD4+ lymphocytes, the virus replicates poorly in resting T cells. Factors that block viral replication in these cells might help to prolong the asymptomatic phase of HIV infection; however, the molecular mechanisms that control this process are not fully understood. Here we show that Murr1, a gene product known previously for its involvement in copper regulation, inhibits HIV-1 growth in unstimulated CD4+ T cells. This inhibition was mediated in part through its ability to inhibit basal and cytokine-stimulated nuclear factor (NF)-kappaB activity. Knockdown of Murr1 increased NF-kappaB activity and decreased IkappaB-alpha concentrations by facilitating phospho-IkappaB-alpha degradation by the proteasome. Murr1 was detected in CD4+ T cells, and RNA-mediated interference of Murr1 in primary resting CD4+ lymphocytes increased HIV-1 replication. Through its effects on the proteasome, Murr1 acts as a genetic restriction factor that inhibits HIV-1 replication in lymphocytes, which could contribute to the regulation of asymptomatic HIV infection and the progression of AIDS.

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense, decoy, and sense RNAs, and trans-dominant negative mutant Gag and Env proteins.

Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Psi(e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Psi(e) signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense decoy and sense RNAS and trans-dominant negative mutant gag and env proteins

Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Psi(e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Psi(e) signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.

Caspase Inhibitor Blocks Human Immunodeficiency Virus 1–Induced T-Cell Death Without Enhancement of HIV-1 Replication and Dimethyl Sulfoxide Increases HIV-1 Replication Without Influencing T-Cell Survival

To determine the relationship, if any, between reagents that modulate survival of T-cells and replication of human immunodeficiency virus 1 (HIV-1) and to determine the effects of the solvent dimethyl sulfoxide (DMSO) and drugs such as cyclosporin A and all-trans retinoic acid on HIV-1 replication. To first establish the direct effects of solvent alone (ie, DMSO) at various concentrations on HIV-1 replication, followed by the ability of various compounds such as the caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethylketone (z-VAD-fmk), cyclosporin A, and all-trans retinoic acid on HIV-1 replication. Next, to determine if HIV-1 induces T-cell apoptosis using TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assays and DNA fragmentation and poly-(ADP-ribose)-polymerase (PARP) cleavage, and then to examine how the various compounds influence T-cell survival after HIV-1 exposure. The human T-cell line, CEM cells, were exposed to HIV(IIIB) and viral replication monitored using reverse transcription assays at 3, 6, and 9 days following infection. Cells were pretreated with various compounds dissolved in DMSO over a wide range of concentrations, and DMSO itself was also examined. T-cell death and apoptosis were assessed using TUNEL staining to detect 3'-OH DNA strand breaks and agarose gel electrophoresis to detect DNA fragmentation (laddering). Furthermore, PARP cleavage implicated in the apoptotic process was also examined. At very low levels, such as 0.002%, DMSO itself appears to enhance HIV-1 replication at 6 and 9 days after infection. At low levels of cyclosporin A, such as 0.01 microgram/mL, HIV-1 replication was further enhanced above the solvent effect, but at 1 microgram/mL, cyclosporin A strongly inhibited HIV-1 replication. Retinoic acid between 0.01 and 1 microgram/mL did not influence HIV-1 replication. In addition, a discrepancy was noted in that HIV-1-infected T-cells were TUNEL positive, indicating DNA strand breaks; however, more complete DNA fragmentation was not detected nor was PARP cleavage identified. The induction of TUNEL positivity was blocked by the caspase inhibitor z-VAD-fmk but not by DMSO or cyclosporin A. Even though z-VAD-fmk blocked the appearance of TUNEL-positive T-cells, there was not a consistently observed increase in HIV-1 replication. Low levels of DMSO and cyclosporin A can enhance HIV-1 replication in CEM cells. At higher levels, cyclosporin A inhibits HIV-1 replication with no significant effects by all-trans retinoic acid. No evidence for classic apoptosis was detected in CEM cells after HIV-1 infection, although DNA strand breaks may be present as revealed by TUNEL positivity. There was no correlation between levels of HIV-1 replication and T-cell survival or death. The mechanism of T-cell death after HIV-1 infection requires further study, and investigators who add compounds dissolved in DMSO must include controls to carefully examine the direct effects of even trace levels of this solvent on HIV-1 replication.

Regulated Lentiviral Packaging Cell Line Devoid of Most Viralcis-Acting Sequences

Packaging cell lines derived from human immunodeficiency virus-1 (HIV-1) are promising tools forin vivosomatic cell gene therapy protocols due to the ability of lentiviruses to infect nondividing cells. We describe here the generation of a safe, stable HIV-1 packaging cell line capable of expressing all of the HIV-1 structural, enzymatic, and regulatory proteins but lacking majority of thecis-acting sequences. The use of an inducible expression system circumvented the cytotoxic and cytostatic effects associated with the expression of some of the HIV-1 viral proteins. Reverse transcriptase activity was detectable in the supernatant from the stable packaging line 1 day after induction, while vector titers peaked 5 days postinduction. Vector titers of up to 3.5 × 104infectious units/ml (IU/ml) were maintained through 8 months following the establishment of the cell line. Lineage-specific gene delivery can be achieved from this established cell line as viral stocks obtained specifically infect CD4+target cells. Moreover, this cell line provides a safe and easy to use system for screening of drugs that inhibit HIV-1 replication.

Infection of Human Marrow Stroma by Human Immunodeficiency Virus-1 (HIV-1) Is Both Required and Sufficient for HIV-1–Induced Hematopoietic Suppression In Vitro: Demonstration by Gene Modification of Primary Human Stroma

AbstractPatients with human immunodeficiency virus-1 (HIV-1) infection often present with bone marrow (BM) failure that may affect all hematopoietic lineages. It is presently unclear whether this failure reflects a direct viral impairment of the CD34+ hematopoietic progenitor cells or whether the virus affects the BM microenvironment. To study the effects of HIV-1 on the BM microenvironment, we examined the stromal cell monolayers in long-term BM culture (LTBMC), which are the in vitro equivalent of the hematopoietic microenvironment. We assessed the hematopoietic support function (HSF ) of human stromal layers by determining the cellular proliferation and colony-forming ability of hematopoietic progenitors from BM cells grown on the stromal layers. We show that the HSF is reduced by in vitro infection of the human stromal cell layer by a monocytotropic isolate of HIV-1 (JR-FL). There is no loss of HSF when the stromal cell layer is resistant to HIV-1 replication, either using murine stromal cell layers that are innately resistant to HIV-1 infection or using human stromal cells genetically modified to express a gene that inhibits HIV-1 replication (an RRE decoy). Decreased HSF was seen using either human or murine hematopoietic cells, if the stromal cells were human cells that were susceptible to HIV-1 infection. These in vitro studies implicate HIV-1 replication in the stroma as the essential component causing decreased hematopoietic cell production in HIV-1 infection.

Inhibition of Human Immunodeficiency Virus-1 (HIV-1) Replication After Transduction of Granulocyte Colony-Stimulating Factor–Mobilized CD34+ Cells From HIV-1–Infected Donors Using Retroviral Vectors Containing Anti–HIV-1 Genes

AbstractTransfer of “anti-HIV-1 genes” into hematopoietic stem cells of human immunodeficiency virus-1 (HIV-1)–infected individuals may be a potent therapeutic approach to render mature cells arising from transduced stem cells resistant to the destructive events associated with HIV-1 infection. To determine the feasibility of gene therapy for acquired immunodeficiency syndrome in individuals already infected with HIV-1, granulocyte colony-stimulating factor mobilized peripheral blood CD34+ cells were isolated from HIV-1–infected individuals and transduced with retroviral vectors containing three different anti–HIV-1-genes: the Rev binding domain of the Rev Responsive Element (RRE decoy) (L-RRE-neo), a double hammerhead ribozyme vector targeted to cleave the tat and rev transcripts (L-TR/TAT-neo), and the trans-dominant mutant of rev (M10) (L-M10-SN). As a control, a vector mediating only neomycin resistance (LN) was used. After 3 days of transduction on allogeneic stroma in the presence of stem cell factor, interleukin-6 (IL-6), and IL-3, the cultures were G418-selected, and then challenged with HIV-1JR-FL and a primary HIV-1 isolate. Compared with the control cultures, the L-RRE-neo–, L-TR/TAT-neo–, and L-M10-SN–transduced cultures displayed up to 1,000-fold inhibition of HIV-1 replication after challenge with HIV-1JR-FL and the primary HIV-1 isolate. Growth of the hematopoietic cells in long-term bone marrow culture was not perturbed by the presence of any of the anti–HIV-1 genes. This study shows that anti–HIV-1 genes can be introduced into CD34+ cells from individuals already infected with HIV-1, and strongly inhibit HIV-1 replication in primary monocytes derived from the CD34+ progenitors.

C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells.

Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C-C chemokines (RANTES, MIP-1 alpha, and MIP-1 beta) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

Discovery and characterization of an HIV-1 Tat antagonist.

Ro 5-3335, 7-chloro-5-(2-pyrryl)-3H-1,4-benzo-diazepin-2-(H)-one, has been shown to inhibit gene expression controlled by the human immunodeficiency virus-1 (HIV-1) LTR promoter. The inhibition was specific for the viral transcriptional transactivator Tat. The compound did not inhibit the basal activity of the HIV-1 LTR or the activity of promoters not responsive to Tat. Consistent with its mode of action, Ro 5-3335 inhibited HIV-1 replication (IC50 = 0.1-1 microM) by reducing viral RNA synthesis in acutely, as well as chronically, infected cells in vitro. The compound was active against HIV-1 and HIV-2, and AZT-resistant clinical isolates.