HIV-1 Replication In Lymphocytes Research Articles

Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein.

ABSTRACTNullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.

Coumarins as Potential Inhibitors of DNA Polymerases and Reverse Transcriptases. Searching New Antiretroviral and Antitumoral Drugs.

Human Immunodeficiency Virus (HIV) is the viral agent of Acquired Immunodeficiency Syndrome (AIDS), and at present, there is no effective vaccine against HIV. Reverse Transcriptase (RT) is an essential enzyme for retroviral replication, such as HIV as well as for other RNA infectious viruses like Human T lymphocyte virus. Polymerases act in DNA metabolism, modulating different processes like mitosis, damage repair, transcription and replication. It has been widely documented that DNA Polymerases and Reverse Transcriptases serve as molecular targets for antiviral and antitumoral chemotherapy. Coumarins are oxygen heterocycles that are widely distributed throughout the plant kingdom. Natural coumarins have attraction due to their bioactive properties such as tumor promotion inhibitory effects, and anti-HIV activity. Coumarins and derivates exhibit potent inhibitory effects on HIV-1 replication in lymphocytes and compounds isolated from Calophyllum inophyllum or DCK derivates showed inhibitory activity against human RT. Furthermore, natural isocoumarins isolated from cultures of fungi or hydroxycoumarins were able to inhibit human DNA polymerase. In view of their importance as drugs and biologically active natural products, and their medicinally useful properties, extensive studies have been carried out on the synthesis of coumarin compounds in recent years. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), a class of antiretroviral chemotherapeutic agents, act by binding to an allosteric pocket showing, generally, low toxicity. This work tries to summarize the investigation about natural and synthetic coumarins with the ability to inhibit key enzymes that play a crucial role in DNA metabolism and their possible application as antiretroviral and antitumoral agents.

Peroxisome proliferator-activated receptor-γ activation suppresses HIV-1 replication in an animal model of encephalitis

Poor penetration of antiretroviral therapy across the blood-brain barrier poses an impediment on control of HIV-1 infection in brain macrophages. Peroxisome proliferator-activated receptor (PPAR)-gamma, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. As PPARgamma agonists are rapidly absorbed by oral administration and efficiently permeate the blood-brain barrier, we hypothesized that PPARgamma stimulation may suppress HIV-1 replication. We investigated the effect of PPARgamma ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages and in vivo using a murine model (immunodeficient mice reconstituted with human lymphocytes and intracerebrally inoculated with HIV-1 infected macrophages) of HIV-1 encephalitis. Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARgamma stimulation inhibited virus replication by modulating NF-kappaB activation in a receptor-dependent manner, leading to downregulation of HIV-1 long terminal repeat (LTR) promoter activity and suppression of HIV-1 replication. These effects were PPARgamma specific as PPARgamma silencing or addition of PPARgamma antagonist abolished effects of PPARgamma stimulation on HIV-1 LTR and virus replication. Using a murine model for HIV-1 encephalitis, we demonstrated that PPARgamma ligand suppressed HIV-1 replication in macrophages in brain tissue and reduced viremia by 50%. In vitro data delineated the novel mechanism by which PPARgamma activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARgamma agonists to reduce HIV-1 replication in lymphocytes and brain macrophages, thus offering a new therapeutic intervention in brain and systemic infection.

Therapies directed against the rev axis of HIV autoregulation

The Rev axis contains two positively acting elements, the Rev protein and its RNA target sequence, the Rev response element (RRE). Scattered throughout the HIV genome in the genes coding for virion structural proteins are CRSs (constitutive repressor sequences) that act in cis to constitutively downregulate the expression of the messenger RNAs (mRNAs) that contain them. Rev binds to the RRE as a multimer. Evidence indicates that the Rev protein, through its nuclear export signal (NES), binds exportin/CRM1 and Ran guanosine triphosphate (GTP) cooperatively when it is bound to the RRE-containing RNA. Numerous steps in the Rev pathway are appropriate targets for gene therapy. Most efforts have focused on blocking the Rev access to the RRE by transdominant Rev inhibitors that bind to the RRE without promoting expression. Major efforts have also focused on sequestering Rev protein with various RRE decoys. Although the role of eIF-5A in the Rev axis is not as well established as that of CRM-1, it is of interest that there is a reported inhibition of HIV-1 replication in lymphocytes by mutants of eIF-5A. Despite the promise afforded antiRev-directed therapies by encouraging in vitro results, considerable hurdles remain before the attainment of effective in vivo therapies is likely to be obtained. Even the elegant clinical studies, establishing that RevTDs introduced by gene therapy can have a protective effect, are far from demonstrating any clinical benefit.

VITAMIN A AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1(HIV-1) IN VITRO INFECTION IN CORD BLOOD LYMPHOCYTES AND MONOCYTES. • 1760

Vitamin A deficiency maybe a risk factor for perinatal transmission of HIV-1 (Lancet. 1994;343:1593). Vitamin A enhances T-cell function and leukocyte phagocytic activity in vitro. Levels of vitamin A are decreased in newborns and infected HIV-1 pregnant mothers. Our hypothesis is that vitamin A may effect HIV-1 infection in cord blood lymphocytes and monocyte derived macrophages. We studied the effect of different concentrations of vitamin A and HIV-1 infection in cord blood lymphocytes and monocytes from 4 healthy(HIV negative) term infants. Lymphocytes and monocytes were isolated and cultured separately, then infected with HIV-1 (BAL strain). Supernatant was collected in triplicate on successive days for reverse transcriptase(RT) and p24 antigen analysis by radioimmunoassay and ELISA(Coulter, Miami, FL) respectively. Vitamin A significantly inhibited p24 viral replication by 50% at concentrations greater than 34.9 μmol/L in cord lymphocytes(101.1± 30.1 vs 219.4 ± 39.2, pg/ml, M±SD, p<0.0001, n=2) and 75% in monocytes(117.6 ±63.4 vs 507.0 ± 8.5 pg/ml p24, M±SD, p<0.025, n=2). Monocyte RT activity was decreased by 60%(3632± 1206 vs 9096 ± 1670 cpm/5μl, M±SEM, p<0.025, n=2). At concentrations less than 7.0 μmol/L p24 viral replication in lymphocytes was increased twofold(457.5 ± 77.2 vs 219.4 ± 39.2 pg/ml, M±SD, p<0.0001, n=2). In macrophages, RT was stimulated nearly threefold at concentrations less than 34.9 μmol/L (26078 ± 4757 vs 9096 ± 1670 cpm/5μl, M±SEM, p<0.025, n=2), but p24 antigen levels were unchanged. There was no cytotoxicity at 139.6 μmol/L. In summary, vitamin A significantly suppresses HIV-1 expression in both cord blood lymphocytes and macrophages at concentrations greater than 34.9μmol/L in a concentration dependent fashion. However, at concentrations less than 7.0 μmol/L vitamin A significantly stimulates viral replication. Thus, these observations may indicate that at high concentrations vitamin A may have the potential to inhibit HIV-1 replication in lymphocytes and monocytes in vivo and prevent perinatal transmission.

The Synthetic (Tyr 5,12,Lys 7)-Polyphemusin II Peptide (T22) Binds to the CD4 Cell Surface Molecule

The [Tyr 5,12, Lys 7]-polyphemusin II peptide (T22) inhibits HIV-1 replication in lymphocytes. The mechanism of T22 inhibition of HIV-1 replication may involve T22 competition with HIV-1 for attachment sites on the plasma membrane of targeted cells. Here we find that the T22 peptide binds to the CD4 molecule in affinity columns. We also find that antiserum to CD4 inhibits cell attachment to T22. Further CD4+ transfected cells attach to T22 while their parental cells which do not express CD4 do not attach to T22. These data demonstrate that T22 binds to the CD4 molecule and supports the hypothesis that T22 inhibits HIV-1 replication by binding to the cell surface CD4 molecule and inhibiting uptake of the virus.

Lymphocytes and Promonocytes Attach to the Synthetic [Tyr 5,12, Lys 7]-Polyphemusin II Peptide (T22)

The [Tyr 5,12, Lys 7]-polyphemusin II peptide (T22) has been shown to inhibit HIV-1 replication in lymphocytes. The mechanism of T22 inhibition of HIV-1 replication is not known but may involve T22 competition with HIV-1 for attachment sites on the plasma membrane of targeted cells. Here we find that three human immunocyte cell lines (H9, Jurkat, and U-937) attach to T22. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), has been shown to activate intracellular protein kinase C and to stimulate lymphocyte attachment to various substrates through specific cell surface receptors. Here we find that TPA treatment enhances attachment of the immunocytes to T22 by three- to four-fold. These data demonstrate that T22 binds to immunocyte cell surfaces and support the hypothesis that T22 may inhibit HIV-1 replication by competing with the virus for a common cell surface receptor(s).

Inhibition of HIV-1 protease by short peptides derived from the terminal segments of the protease

The active HIV-1 protease is a homodimeric enzyme. A β-sheet consisting of N- and C-terminal segments provides the main driving force for dimerization of the inactive protomers. Several short peptides with sequences derived from the N- and C-termini of the protease were tested for inhibition of protease activity and for inhibition of HIV-1 replication in lymphocytes. Medium inhibitory activity was found with each of the peptides in the enzyme test and no inhibition of the lymphocytes was found up to 200 μg/ml. The enzyme tests indicate that HIV-1 protease is the target of the inhibitory action. Synergistic action could not be found with pairs of the peptides derived from the two different termini. Prolonged incubation with one of the peptides increased inhibition indicating a slow dissociation of the protease dimers. No cytotoxic effect of the inhibitors could be found below 200 μg/ml.

A phase I/II study of trichosanthin treatment of HIV disease

Trichosanthin, a ribosomal inhibitor protein, blocks HIV replication in lymphocytes and macrophages. This agent was used to treat 51 patients with advanced HIV disease in a dose-escalation study in which three injections were administered over a 9-21-day period in a dose range of 10-30 micrograms/kg per injection. The maximum tolerated dose was estimated to be 30 micrograms/kg. Reversible but severe fatigue and myalgias were the major dose-limiting side-effects; mild leucocytosis and elevations in serum transaminases were noted and were reversible. Non-dose-related reversible mental status changes were seen in six patients and were considered to be associated with the drug. This was usually manifest as dementia, but progressed to coma in two patients. This reversed, but the sequelae resulted in death in one patient. Decreases in serum p24 antigen levels were noted 1 month after the first infusion in 10 of 18 patients who entered the study with elevated levels; one converted to negative. Values usually remained low to the end of the study period (2 months). In those patients with CD4+ cell levels greater than 50 x 10(6) cells/l significant decreases in sedimentation rate and increases in CD4+ cell numbers were also noted. These changes were found at all dose levels but only in patients receiving three infusions.

Failure of dideoxynucleosides to inhibit human immunodeficiency virus replication in cultured human macrophages.

Primary human monocyte-derived macrophages (MDM) were shown to have diminished deoxynucleoside kinase activities compared to T lymphoblasts, and a reduced ability to phosphorylate dideoxynucleosides with anti-human immunodeficiency virus (HIV) activity. These drugs, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyadenosine (ddA), which are potent anti-HIV agents in CD4 lymphocytes, did not inhibit HIV replication in MDM, even at concentrations of 100 microM. This drug concentration of AZT is approximately 100-fold higher than the levels attained in the serum of treated patients and the levels required to inhibit HIV replication in lymphocytes. These observations may explain the failure of AZT therapy to clear viremia, consistent with the presence of a drug-resistant reservoir of infected cells in vivo. New therapeutic approaches to inhibit the replication of HIV in MDM may be needed.