Inhibition Of Virus Production Research Articles

Liposomal encapsulation enhances antiviral efficacy of SPC3 against human immunodeficiency virus type-1 infection in human lymphocytes

Because encapsulation of antiviral drugs in liposomes resulted generally in improved activity against retroviral replication in vivo, the antiviral effects of free-SPC3 and liposome-associated SPC3 were compared in cultured human lymphocytes infected with HIV-1. SPC3 was entrapped in various liposomal formulations, either different in size (mean diameter of 100 and 250 nm), SPC3 concentration or cholesterol content. Liposome-associated SPC3 were tested for both inhibition of cell–cell fusion and infection with HIV-1 clones. SPC3 inhibited HIV-1-induced fusion at a micromolar concentration range. When associated with liposomes, SPC3 was found to be about 10-fold more potent than free SPC3 in inhibiting syncytium formation. Continuous treatment with free SPC3 also inhibited virus production in a dose-dependent manner, with inhibition of HIV infection of C8166 T-cells or human peripheral blood lymphocytes (PBLs) at micromolar concentrations. Liposomal entrapment was found to increase the antiviral efficacy of SPC3 by more than 10- and 5-fold in C8166 and PBLs, respectively. These data suggest that the liposome approach may be used to improve SPC3 antiviral efficacy.

Advances in HIV molecular biology

Advances in HIV molecular biology

Effect of fatty acids on arenavirus replication: inhibition of virus production by lauric acid

To study the functional involvement of cellular membrane properties on arenavirus infection, saturated fatty acids of variable chain length (C10-C18) were evaluated for their inhibitory activity against the multiplication of Junin virus (JUNV). The most active inhibitor was lauric acid (C12), which reduced virus yields of several attenuated and pathogenic strains of JUNV in a dose dependent manner, without affecting cell viability. Fatty acids with shorter or longer chain length had a reduced or negligible anti-JUNV activity. Lauric acid did not inactivate virion infectivity neither interacted with the cell to induce a state refractory to virus infection. From mechanistic studies, it can be concluded that lauric acid inhibited a late maturation stage in the replicative cycle of JUNV. Viral protein synthesis was not affected by the compound, but the expression of glycoproteins in the plasma membrane was diminished. A direct correlation between the inhibition of JUNV production and the stimulation of triacylglycerol cell content was demonstrated, and both lauric-acid induced effects were dependent on the continued presence of the fatty acid. Thus, the decreased insertion of viral glycoproteins into the plasma membrane, apparently due to the increased incorporation of triacylglycerols, seems to cause an inhibition of JUNV maturation and release.

The rapid first phase decline in hepatitis C viremia is lower in previous nonresponders compared to treatment naive patients

Despite advances in antiviral therapy for hepatitis C Virus (HCV), a large proportion of patients fails to respond to treatment. Initiation of interferon therapy is associated with a rapid first phase decline in viremia that occurs over 24 to 48 hours and seems to reflect inhibition of viral production or release from infected cells. We compared the blocking effectiveness of consensus interferon aifacon-1 (CIFN) in previous nonresponders (NR) to that observed in naive patients. Methods: Twenty virological NR with genotype 1 infection and no cirrhosis were evaluated. Ten received a single 15 mcg dose of IFN and ten ware given a 30 mug dose. Blood samples were collected at time O, 4, 8, 24, 48, 72, 96, and 168 hours after interferon administration. Data from previously untreated patients with genotype 1 infestion who received 9 mcg (n = 12) or 15 mg (n = 13) of CIFN provided a basis for comparison (Neumann at al. Hepatology 2000;32:372A). HCV RNA levels were measured by quantitative PCR (NGI). Viral dynamics were evaluated after CIFN dosing using models described by Neumann at al. (Science 1998;282:103). Results: The effectiveness of CIFN in inhibiting HCV production or release is shown in the table. 15 mcg of CIFN was significantly less effective in blocking HCV production in NR compared to either 15 mcg (p=O.O01) or 9 mcg (p=O.03) in halves. The 30 mcg dose provided less blocking effectiveness in NR than 15 mcg in naive patients (p=O.03), and a similar effect to 9 mcg in naives. NR who had greater than a 50% decrease in HCV RNA level from baseline at the end of previous treatment had a larger reduction in viral load at 24 hours compared to those who had not achieved that level of response with prior therapy (p = 0.05). Conclusions: The blocking effectiveness of CIFN was significantly lower in previous NR compared to naive patients at similar doses, suggesting that response to further treatment in NR is limited by intrinsic HCV resistance. However, the resistance was partially overcome with higher CIFN doses.

Stopping the flu

Drugs that block cellular signaling cascades could be effective new treatments for flu. Most anti-influenza compounds target the virus itself, which leads to the selection of drug-resistant variants. Researchers in StephanLudwig's laboratory (Wurzburg University, Germany) took a different approach by targeting host cell proteins. They found that influenza A infection activates the Raf/MEK/ERK signaling cascade. Inhibiting Raf signaling caused nuclear retention of viral ribonucleoprotein complexes and inhibited virus production. In their paper, published in the March issue of Nature Cell Biology, the authors suggest that ‘MEK inhibitors may serve as potent non-toxic anti-influenza agents’. However, targeting virally infected cells will be necessary to avoid non-specific side effects. JW

Evaluation of (1R,2R)-1-(5'-methylfur-3'-yl)propane-1,2,3-triol, a sphydrofuran derivative isolated from a Streptomyces species, as an anti-herpesvirus drug.

(1R,2R)-1-(5'-Methylfur-3'-yl)propane-1,2,3-triol (MFPT), a stable anhydro derivative of sphydrofuran, was obtained from the culture broth of STREPTOMYCES: sp. strain FV60 as an inhibitor of herpes simplex virus type 1 (HSV-1). The compound showed antiherpetic activity with a 50% inhibitory concentration of 1.2 IM in an in vitro assay system. Although the binding of virus to host cells was not inhibited, the penetration of virus into cells was moderately blocked by MFPT. Some of the viruses, once they had penetrated cells, failed to form plaques in the presence of MFPT. When added to the late stages of HSV-1 replication, MFPT also inhibited virus production. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of isotope-labelled HSV-specific proteins revealed that a protein or proteins with reduced molecular weight (about 120 kDa) was clearly detected in cells treated with MFPT. Western blot analysis with antibodies against three HSV-specific glycoproteins (gB, gC and gD) showed a significant difference in gC synthesis between untreated and MFPT-treated cells. Release of progeny viruses was suppressed by MFPT. Syncytium formation by HSV-1 strain HF was inhibited and small plaques with rounded cells were formed in MFPT-treated cell cultures. When wild-type HSV-1 was serially propagated under the selective pressure of MFPT, resistant virus emerged. MFPT-resistant progeny were accompanied by the formation of plaques with rounded cells. These results, taken together, suggest that MFPT might act by limiting the maturation of HSV-specific glycoproteins, particularly of HSV-1 gC.

Inhibition of human immunodeficiency virus type 1 by packageable, multigenic antisense RNA.

Viral-based vectors can provide an efficient delivery mechanism for stable expression of antisense RNA. To enhance and propagate the antiviral effect of antisense RNA, two novel human immunodeficiency virus type 1 (HIV-1)-based vector DNAs, designated as pMAG7 and pMAG19, were constructed which contained HIV-1 cis-acting packaging elements and produced multigenic HIV-1 antisense RNA that could target the entire pol, env, vif, vpu, vpr, rev, and tat and portions of gag and nef. The two DNAs were identical except that pMAG19 had additional gag coding sequences. Cotransfection of pMAG DNA and infectious, cloned HIV-1 DNA in 293 cells inhibited virus production (81%-98% reduction in reverse transcriptase activity) of various T cell-tropic and macrophage-tropic clade B isolates, such as NL4-3, YU-2, and JR-CSF. In addition, virion-associated pMAG antisense RNA was detected in residual virus particles produced by pNL4-3 in the presence of pMAG7 DNA, and the antisense sequences were stably transferred by infection of 174 x CEM cells. The results suggest that pMAG DNA may confer broad protection against HIV-1 by reducing initial virus burden due to antisense RNA and subsequent virus spread by propagation of antisense sequences along with wild-type virus.

Effect of ABC transporters on HIV-1 infection: inhibition of virus production by the MDR1 transporter.

The MDR1 multidrug transporter P-gp (P-glycoprotein) is an efflux pump that extrudes diverse hydrophobic drugs and peptides from cells. Since the entry of HIV-1 into cells involves an initial interaction of the viral gp41 hydrophobic peptide with the plasma membrane, a potential effect of P-gp on HIV-1 infectivity was explored. Virus production was greatly decreased when P-gp was overexpressed at the surface of a continuous CD4(+) human T-leukemic cell line (12D7) infected with HIV-1(NL4-3), a T-tropic molecular clone of HIV-1. P-gp overexpression did not significantly alter the surface expression or distribution of either the HIV-1 receptor CD4 or the coreceptor CXCR4. Reduction of HIV-1 infectivity in P-gp-expressing cells occurred both during the fusion of viral and plasma membranes and at subsequent step(s) in the HIV-1 life cycle.

The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains.

Infection of target cells by HIV-1 requires initial binding interactions between the viral envelope glycoprotein gp120, the cell surface protein CD4, and one of the members of the seven-transmembrane G protein-coupled chemokine receptor family. Most primary isolates (R5 strains) use chemokine receptor CCR5, but some primary syncytium-inducing, as well as T cell line-adapted, strains (X4 strains) use the CXCR4 receptor. Signaling from both CCR5 and CXCR4 is mediated by pertussis toxin (PTX)-sensitive G(i) proteins and is not required for HIV-1 entry. Here, we show that the PTX holotoxin as well as its binding subunit, B-oligomer, which lacks G(i)-inhibitory activity, blocked entry of R5 but not X4 strains into primary T lymphocytes. Interestingly, B-oligomer inhibited virus production by peripheral blood mononuclear cell cultures infected with either R5 or X4 strains, indicating that it can affect HIV-1 replication at both entry and post-entry levels. T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1beta or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1beta or HIV-1 to such cells were not impaired. The inhibitory effect of B-oligomer on signaling from CCR5 and on entry of R5 HIV-1 strains was reversed by protein kinase C (PKC) inhibitors, indicating that B-oligomer activity is mediated by signaling events that involve PKC. B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1. These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1.

Improved titers of retroviral vectors from the human FLYRD18 packaging cell line in serum- and protein-free medium.

The influence of serum on the production of retroviral vectors by the HT1080 human fibrosarcoma-derived packaging cell line FLYRD18 was investigated. A fourfold increase in virus titer was observed under serum-free conditions, as compared with medium supplemented with 10% fetal calf serum. A similar improvement was also seen for bulk transduction efficiency. Serum had a negative and dose-dependent effect on titer without affecting cell growth, virus stability, or infectivity. In contrast to virus from NIH 3T3-derived packaging cells [Hanenberg, H., et al. (1996). Nature Med. 2, 876-882], the FLYRD18-derived virus did not adhere to fibronectin or serum proteins adsorbed at the surface of culture flasks. Electron microscopy supports the conclusion that the effect of serum is at the level of virus production by the cells. Addition of soybean trypsin inhibitor had an inhibitory effect on virus production, while pretreatment of serum with trypsin was found to enhance the retroviral titer. These results suggest that protease inhibitors present in serum may be responsible for the inhibition of virus production. The exact mechanism remains, however, to be determined. As compared with medium supplemented with 10% serum, the combination of increased virus titer and absence of exogenous protein under serum-free conditions resulted in a 300-fold increase in the virus:total protein ratio in the supernatants harvested from the FLYRD18 packaging line. This improvement enhances prospects for further concentration and purification of the virus.

Anti-HIV activity of amphotericin B-cholesteryl sulfate colloidal dispersion in vitro

We examined whether the anti-HIV-1 activity of the polyene antibiotic Amphotericin B (AMB) is retained following incorporation into sterically stabilized ‘Stealth’ liposomes (L-AMB) with prolonged circulation in vivo, or cholesteryl sulfate colloidal dispersions (CD-AMB). The effects of the different preparations on acute infection of H9 cells with HIV-1 IIIB, spreading of the virus from chronically infected H9/HTLV-IIIB cells to SupT1 cells, and HIV-1-induced syncytium formation were evaluated. Infection was monitored by p24 levels in culture supernatants. L-AMB did not affect HIV-1 infection. When present only during initial infection, AMB (3–20 μg/ml) reduced p24 levels by 70–80% after 7 and 10 days post-infection, while CD-AMB inhibited p24 production by ∼30–40% at day 7 and 50–60% at day 10. The inhibitory effect of CD-AMB and AMB was enhanced by continuous treatment of acutely infected cells. The reduction of p24 production during continuous treatment was not due to cytotoxicity. During spreading of infection from infected to uninfected cells, AMB almost completely inhibited virus production while CD-AMB reduced both p24 production and the cytopathic effect in a dose-dependent manner. HIV-1 induced syncytium formation was slightly inhibited by AMB but not by CD-AMB. Because CD-AMB is considerably less cytotoxic than AMB, its ability to inhibit HIV infection in vivo needs to be evaluated further.

Repression of the human immunodeficiency virus type 1 promoter by the human KRAB domain results in inhibition of virus production

The Krüppel-associated box (KRAB) domain has been described as a eukaryotic repressor of transcription. We show that fusion of KRAB to DNA-binding-domains provides a novel approach to inhibit expression of a replication-competent human immunodeficiency virus (HIV) genome. The KRAB domain from the human zinc finger protein KOX1 was combined with the DNA binding domain of the Escherichia coli tetracycline repressor (TetR). Constitutive expression of the TetR–KRAB protein in HeLa cells inhibited virus production from an HIV genome encoding TetR target sequences by 80%. The same inhibition was observed with HIV-promoter-driven reporter plasmids. The specificity of inhibition was shown with informative KRAB mutants, plasmids lacking the respective target sequences, and by reversal of the TetR–KRAB-mediated inhibition with tetracycline. Virus production was suppressed by binding of TetR–KRAB at a distance of 6 kbp to the promoter. We therefore conclude that any site of the genuine HIV genome could serve as target of a chimeric KRAB repressor protein. Specific targeting of the KRAB domain by artificially selected binding domains may be generally applicable to control transcription in mammalian cells.

Alpha interferon inhibits human herpesvirus 8 (HHV-8) reactivation in primary effusion lymphoma cells and reduces HHV-8 load in cultured peripheral blood mononuclear cells.

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-alpha), we analyzed the effects of IFN-alpha or anti-IFN-alpha antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-alpha inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-alpha inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-alpha was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-alpha Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-alpha Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-alpha reduced the HHV-8 load in PBMC. Thus, IFN-alpha appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-alpha may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.

Liposome-mediated delivery of antiviral agents to human immunodeficiency virus-infected cells.

Intracellular delivery of novel macromolecular drugs against human immunodeficiency virus type-1 (HIV-1), including antisense oligodeoxynucleotides, ribozymes and therapeutic genes, may be achieved by encapsulation in or association with certain types of liposomes. Liposomes may also protect these drugs against nucleases. Low-molecular-weight, charged antiviral drugs may also be delivered more efficiently via liposomes. Liposomes were targeted to HIV-1-infected cells via covalently coupled soluble CD4. An HIV-1 protease inhibitor encapsulated in conventional negatively charged multilamellar liposomes was about 10-fold more effective and had a lower EC90 than the free drug in inhibiting HIV-1 production in human monocyte-derived macrophages. The drug encapsulated in sterically stabilized liposomes was as effective as the free drug. The EC50 of the reverse transcriptase inhibitor 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was reduced by an order of magnitude when delivered to HIV-1-infected macrophages in pH-sensitive liposomes. A 15-mer antisense oligodeoxynucleotide against the Rev response element was ineffective in free form against HIV-1 replication in macrophages, while delivery of the oligonucleotide in pH-sensitive liposomes inhibited virus replication. The oligodeoxynucleotide encapsulated in sterically stabilized pH-sensitive liposomes with prolonged circulation in vivo, which were recently developed in the laboratories of the authors, was also highly effective. A ribozyme complementary to HIV-1 5'-LTR delivered in pH-sensitive liposomes inhibited virus production by 90%, while the free ribozyme caused only a slight inhibition. Cationic liposome-mediated co-transfection of the HIV-regulated diphtheria toxin A fragment gene and a proviral HIV clone into HeLa cells completely inhibited virus production, while the frame-shifted mutant gene was ineffective. Co-transfection of the proviral genome and a gene encoding a Rev-binding aptamer into HeLa cells via transferrin-associated cationic liposomes inhibited virus production. These studies indicate that liposomes can be used to facilitate the intracellular delivery of certain anti-HIV agents and to enhance their therapeutic effects. These properties may be particularly advantageous in the development of novel macromolecular drugs, which may be necessary because of the emergence of virus strains resistant to the currently available drugs.

Peptides that block hepatitis B virus assembly: analysis by cryomicroscopy, mutagenesis and transfection.

Peptides selected to bind to hepatitis B virus (HBV) core protein block interaction with the long viral surface antigen (L-HBsAg) in vitro. High resolution electron cryomicroscopy showed that one such peptide binds at the tips of the spikes of the core protein shell. The peptides contain two basic residues; changing either of two acidic residues at the spike tip to an alanine greatly reduced the binding affinity. Transfection of hepatoma cells with a replication-competent HBV plasmid gave significantly reduced production of virus in the presence of peptide, in a dose-dependent manner. These experiments show that the interaction of L-HBsAg with core particles is critical for HBV assembly, and give proof of principle for its disruption in vivo by small molecules.

Delivery of an anti-HIV-1 ribozyme into HIV-infected cells via cationic liposomes

Cationic liposome-mediated intracellular delivery of a fluorescein-labeled chimeric DNA–RNA ribozyme targeted to the HIV-1 5′ LTR was investigated, using THP-1, THP-1/HIV-1 IIIB or HeLa/LAV cells. Different fluorescence patterns were observed when the cells were exposed to Lipofectamine, Lipofectin or DMRIE:DOPE (1:1) complexed to the ribozyme. With Lipofectamine intense cell-associated fluorescence was found. Incubation with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE an intense but sporadic fluorescence was observed. Differentiated THP-1/HIV-1 IIIB cells were more susceptible to killing by liposome–ribozyme complexes than THP-1 cells. Under non-cytotoxic conditions (a 4-h treatment) complexes of 5, 10 or 15 μM Lipofectin or DOTAP:DOPE (1:1) and ribozyme, at lipid:ribozyme ratios of 8:1 or 4:1, did not affect p24 production in THP-1/HIV-1 IIIB cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP-1/HIV-1 IIIB cells to 5 μM Lipofectin or DOTAP:DOPE (1:1) complexed with either the functional or a modified control ribozyme reduced virus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, the free ribozyme at a relatively high concentration inhibited virus production by 30%, while the control ribozyme was ineffective, indicating a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 μM after a 24-h treatment. A 4-h treatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 μM was not toxic to the cells, but also did not inhibit p24 production. In contrast, treatment of HeLa CD4 + cells immediately after infection with HIV-1 IIIB at the same lipid concentrations and lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ribozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vivo applications.

Isolation and characterization of the fungal metabolite 3-O-methylviridicatin as an inhibitor of tumour necrosis factor alpha-induced human immunodeficiency virus replication.

The cytokine tumour necrosis factor alpha (TNF-alpha) has been shown to play a role in human immunodeficiency virus (HIV) replication by activating transcription of the provirus in both T cells and macrophages. Therefore, agents that block TNF-alpha-induced HIV expression could have therapeutic value in the treatment of AIDS. We have sought to identify antiviral agents that block TNF-alpha induction of HIV LTR-directed transcription, using a cell-based, virus-free assay system in automated high-throughput screening. HeLa cells were transfected with an HIV LTR-luciferase reporter plasmid and a stable line was isolated in which TNF-alpha increased luciferase production by two- to threefold. This cell line was used to screen approximately 15,000 fungal extracts. An inhibitory activity specific for TNF-alpha-induced HIV LTR transcription was observed in culture OS-F67406. The active component was isolated and identified as a known metabolite, 3-O-methylviridicatin, by NMR and mass spectrometry. No biological activity has been associated with this compound previously. This compound blocks TNF-alpha activation of the HIV LTR in the HeLa-based system, with an IC50 of 5 microM, and inhibited virus production in the OM-10.1 cell line, a model of chronic infection responsive to induction by TNF-alpha, with an IC50 of 2.5 microM.

Current concepts in mucosal immunity. IV. How epithelial transport of IgA antibodies relates to host defense.

The humoral arm of the mucosal immune system is principally composed of locally synthesized polymeric IgA, whose Fc portion is adapted for binding to the polymeric immunoglobulin receptor that is expressed on the basolateral surface of mucosal epithelial cells, including enterocytes. This receptor mediates the endocytosis and transcytosis of polymeric IgA, which allows IgA to function in host defense at three anatomic levels in relation to mucosal epithelium: IgA antibodies in the lamina propria can bind antigens and excrete them through the epithelium into the lumen; antiviral IgA antibodies in transit through epithelial cells can inhibit virus production by an intracellular action; and IgA antibodies secreted into the lumen can prevent antigens and microbes from adhering to and penetrating the epithelium. The ways in which IgA antibodies function in mucous membranes provide challenging investigative opportunities for cell physiologists and cell biologists.

Receptor Ligand-Facilitated Cationic Liposome Delivery of Anti-HIV-1 Rev-Binding Aptamer and Ribozyme DNAs

We examined whether HIV-1 gene expression could be inhibited by the anti-HIV Rev-binding aptamer [RBE(apt)], and whether the antiviral effect of the aptamer could be enhanced by a ribozyme directed against the HIV-1 env gene. Since cationic liposomes are relatively safe and non-immunogenic for in vivo gene delivery, we tested the effectiveness of the aptamer and ribozyme DNAs in HeLa cells, using Lipofectin reagent in a transient transfection assay. To increase the transfection efficiency, lipofectin was mixed with transferrin before subsequent addition of DNA. Co-transfection of HeLa cells with the RBE(apt) and the proviral HIV clone, HXBΔBgl, resulted in inhibition of virus production. Specific inhibition of viral p24 production following co-transfection of the RBE(apt) and HIV proviral DNAs was observed. These data provide strong support for the use of in vitro evolved ligands as potential anti-HIV agents. The addition of the anti-env ribozyme to the aptamer construct did not further enhance the antiviral activity, suggesting either that we had reached the limits of inhibition in this assay, or that the ribozyme was not able to access its target site with Rev bound to the RBE aptamer. The observed inhibition of p24 production could not be attributed to the non-specific toxicity of the transfection procedure, because no difference in viability was observed between the RBE(apt)- and the vector control-treated cells. All of the aptamer-ribozyme constructs as well as the RBE(apt) were similarly effective.

IL-13 acts on macrophages to block the completion of reverse transcription, inhibit virus production, and reduce virus infectivity.

An understanding of the immune suppression of HIV-1 replication in macrophages continues to be a major goal of AIDS research due to the central role this cell type has in AIDS pathogenesis. We have previously discussed the potential clinical benefits of the anti-inflammatory cytokine interleukin-13 (IL-13), which, unlike IL-4 or IL-10, had limited effects on T cell functions. In this report are extend our observations on the effects of IL-13 on HIV-1 replication in monocyte-derived macrophages (MDM) and show redundancy with IL-4, IL-13 or IL-4 have similar effects on HIV-1 replication in MDM when added at different times after infection, with the ability to decrease infection virus release when added for up to 7 days after infection. Removal of IL-13 from MDM revealed a reduction of infection by 16- to 81-fold based on the absence of viral re-emergence from lower multiplicity of infection (m.o.i.). The reduction of HIV-1 infectivity in MDM caused by IL-13 was further characterized by studies on the formation of viral DNA over a range of m.o.i. IL-13 increased the formation of LTR DNA at the lowest m.o.i. of 0.007 while concurrently inhibiting the formation of gag DNA, a later reverse transcription product, at the highest m.o.i. tested, 0.62. Overall, our data indicate that IL-13 can act on macrophages before and after HIV-1 infection by blocking the completion of reverse transcription, decreasing virus production, and reducing the infectivity of the progeny virions.