Clonal expansion of SIV-infected cells in macaques on antiretroviral therapy is similar to that of HIV-infected cells in humans.

Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes. We obtained approximately 8000 integration sites from blood samples taken from SIV-infected macaques prior to the initiation of ART, and from blood, spleen, and lymph node samples taken at necropsy. Seven clones were identified in the pre-ART samples; one persisted for a year on ART. An additional 100 clones were found only in on-ART samples; a number of these clones were found in more than one tissue. The timing and extent of clonal expansion of SIV-infected cells in macaques and HIV-infected cells in humans is quite similar. This suggests that SIV-infected macaques represent a useful model of the clonal expansion of HIV infected cells in humans that can be used to evaluate strategies intended to control or eradicate the viral reservoir.

Preclinical Development of a Hematopoietic Stem and Progenitor Cell Bioengineered Factor VIII Lentiviral Vector Gene Therapy for Hemophilia A.

Genetically modified, autologous hematopoietic stem and progenitor cells (HSPCs) represent a new class of genetic medicine. Following this therapeutic paradigm, we are developing a product candidate, designated CD68-ET3-LV CD34+, for the treatment of the severe bleeding disorder, hemophilia A. The product consists of autologous CD34+ cells transduced with a human immunodeficiency virus 1-based, monocyte lineage-restricted, self-inactivating lentiviral vector (LV), termed CD68-ET3-LV, encoding a bioengineered coagulation factor VIII (fVIII) transgene, termed ET3, designed for enhanced expression. This vector was shown capable of high-titer manufacture under clinical scale and Good Manufacturing Practice. Biochemical and immunogenicity testing of recombinant ET3, as well as safety and efficacy testing of CD68-ET3-LV HSPCs, were utilized to demonstrate overall safety and efficacy in murine models. In the first model, administration of CD68-ET3-LV-transduced stem-cell antigen-1+ cells to hemophilia A mice resulted in sustained plasma fVIII production and hemostatic correction without signs of toxicity. Patient-derived, autologous mobilized peripheral blood (mPB) CD34+ cells are the clinical target cells for ex vivo transduction using CD68-ET3-LV, and the resulting genetically modified cells represent the investigational drug candidate. In the second model, CD68-ET3-LV gene transfer into mPB CD34+ cells isolated from normal human donors was utilized to obtain in vitro and in vivo pharmacology, pharmacokinetic, and toxicology assessment. CD68-ET3-LV demonstrated reproducible and efficient gene transfer into mPB CD34+ cells, with vector copy numbers in the range of 1 copy per diploid genome equivalent without affecting clonogenic potential. Differentiation of human CD34+ cells into monocytes was associated with increased fVIII production, supporting the designed function of the CD68 promoter. To assess in vivo pharmacodynamics, CD68-ET3-LV CD34+ cell product was administered to immunodeficient mice. Treated mice displayed sustained plasma fVIII levels and no signs of product related toxicity. Collectively, the findings of the current study support the preclinical safety and efficacy of CD68-ET3-LV CD34+.

Bushen recipe and its disassembled prescriptions inhibit inflammation of liver injury associated with Concanavalin A through Toll­like receptor 3/9 signaling pathway.

The aim of the present study was to explore the effect of Bushen recipe and its disassembled prescriptions on liver injury and chronic hepatitis B. Liver injury was induced in normal and hepatitis B virus (HBV)­transgenic mice through injection of Concanavalin A, followed by treatment with Bushen recipe and its disassembled prescriptions including the Bushen­yang, the Bushen­yin and the QingHua groups as well as the GanYanLing group (positive control). Subsequently, their liver function indexes were investigated by a microplate method and liver sections were blindly evaluated using an optical microscope by a pathologist. Subsequently, the activation state of Toll­like receptor (TLR)3/9 signaling pathway in liver tissues was analyzed by western blotting. Additionally, the inflammatory factors produced following liver injury in peripheral blood were detected via ELISA. Following intervention with the Bushen recipe and its disassembled prescriptions, the liver function indexe alanine aminotransferase had declined, whereas cholinesterase increased. The pathological alterations of liver tissue in HBV transgenic mice were reversed by Bushen recipe and its disassembled prescriptions. In addition, the TLR3/9 signaling pathway in liver tissues of HBV transgenic mice was inhibited and inflammatory factors such as interleukin (IL)­6, IL­1, tumor necrosis factor­α and interferon­Î³ were reduced significantly. In conclusion, the present study demonstrated that Bushen recipe and its disassembled prescriptions repaired liver injury induced by Concanavalin A through inhibition of TLR3/9 signaling pathway.

SJP-L-5, a novel small-molecule compound, inhibits HIV-1 infection by blocking viral DNA nuclear entry.

BACKGROUND: Small-molecule compounds that inhibit human immunodeficiency virus type 1 (HIV-1) infection can be used not only as drug candidates, but also as reagents to dissect the life cycle of the virus. Thus, it is desirable to have an arsenal of such compounds that inhibit HIV-1 infection by various mechanisms. Until now, only a few small-molecule compounds that inhibit nuclear entry of viral DNA have been documented. RESULTS: We identified a novel, small-molecule compound, SJP-L-5, that inhibits HIV-1 infection. SJP-L-5 is a nitrogen-containing, biphenyl compound whose synthesis was based on the dibenzocyclooctadiene lignan gomisin M2, an anti-HIV bioactive compound isolated from Schisandra micrantha A. C. Smith. SJP-L-5 displayed relatively low cytotoxicity (50% cytoxicity concentrations were greater than 200 µg/ml) and high antiviral activity against a variety of HIV strains (50% effective concentrations (EC50)) of HIV-1 laboratory-adapted strains ranged from 0.16-0.97 µg/ml; EC50s of primary isolates ranged from 1.96-5.33 µg/ml). Analyses of the viral DNA synthesis indicated that SJP-L-5 specifically blocks the entry of the HIV-1 pre-integration complex (PIC) into the nucleus. Further results implicated that SJP-L-5 inhibits the disassembly of HIV-1 particulate capsid in the cytoplasm of the infected cells. CONCLUSIONS: SJP-L-5 is a novel small-molecule compound that inhibits HIV-1 nuclear entry by blocking the disassembly of the viral core.

Analysis of protein kinase C theta inhibitors for the control of HIV-1 replication in human CD4+ T cells reveals an effect on retrotranscription in addition to viral transcription.

HIV-1 infection cannot be cured due to reservoirs formed early after infection. Decreasing the massive CD4+ T cell activation that occurs at the beginning of the disease would delay reservoir seeding, providing a better prognosis for patients. CD4+ T cell activation is mediated by protein kinase C (PKC) theta (θ), which is involved in T-cell proliferation, as well as NF-κB, NF-AT, and AP-1 activation. We found that PKCθ activity increased viral replication, but also that HIV-1 induced higher activation of PKCθ in infected CD4+ T cells, creating a feedback loop. Therefore, specific inhibition of PKCθ activity could contribute to control HIV-1 replication. We tested the efficacy of seven PKCθ specific inhibitors to control HIV-1 replication in CD4+ T cells and selected two of the more potent and safer: CGX1079 and CGX0471. They reduced PKCθ phosphorylation at T538 and its translocation to the plasma membrane, which correlated with decreased HIV-1 retrotranscription through partial inhibition of SAMHD1 antiviral activity, rendering lower proviral integration. CGX1079 and CGX0471 also interfered with viral transcription, which would reduce the production of new virions, as well as the subsequent spread and infection of new targets that would increase the reservoir size. CGX1079 and CGX0471 did not completely abrogate T-cell functions such as proliferation and CD8-mediated release of IFN-γ in PBMCs from HIV-infected patients, thereby avoiding general immunosuppresion. Consequently, using PKCθ inhibitors as adjuvant of antiretroviral therapy in recently infected patients would decrease the pool of activated CD4+ T cells, thwarting proviral integration and reducing the reservoir size.

Wikstroelide M potently inhibits HIV replication by targeting reverse transcriptase and integrase nuclear translocation.

AIM: To evaluate the anti-HIV activity and mechanism of action of wikstroelide M, a daphnane diterpene from Daphne acutiloba Rehder (Thymelaeaceae). METHODS: The anti-HIV activities of wikstroelide M against different HIV strains were evaluated by cytopathic effect assay and p24 quantification assay with ELISA. The inhibitory effect of wikstroelide M on HIV reverse transcription was analyzed by real-time PCR and ELISA. The effect of wikstroelide M on HIV-1 integrase nuclear translocation was observed with a cell-based imaging assay. The effect of wikstroelide M on LEDGF/p75-IN interaction was assayed by molecular docking. RESULTS: Wikstroelide M potently inhibited different HIV-1 strains, including HIV-1IIIB, HIV-1A17, and HIV-19495, induced a cytopathic effect, with EC50 values ranging from 3.81 to 15.65 ng·mL⁻¹. Wikstroelide M also had high inhibitory activities against HIV-2ROD and HIV-2CBL-20-induced cytopathic effects with EC50 values of 18.88 and 31.90 ng·mL⁻¹. The inhibitory activities of wikstroelide M on the three HIV-1 strains were further confirmed by p24 quantification assay, with EC50 values ranging from 15.16 to 35.57 ng·mL⁻¹. Wikstroelide M also potently inhibited HIV-1IIIB induced cytolysis in MT-4 cells, with an EC50 value of 9.60 ng·mL⁻¹. The mechanistic assay showed that wikstroelide M targeted HIV-1 reverse transcriptase and nuclear translocation of integrase through disrupting the interaction between integrase and LEDGF/p75. CONCLUSION: Wikstroelide M may be a potent HIV-1 and HIV-2 inhibitor, the mechanisms of action may include inhibition of reverse trascriptase activity and inhibition of integrase nuclear translocation through disrupting the interaction between integrase and LEDGF/p75.

Wikstroelide M potently inhibits HIV replication by targeting reverse transcriptase and integrase nuclear translocation / 中国天然药物

AIM@#To evaluate the anti-HIV activity and mechanism of action of wikstroelide M, a daphnane diterpene from Daphne acutiloba Rehder (Thymelaeaceae).@*METHODS@#The anti-HIV activities of wikstroelide M against different HIV strains were evaluated by cytopathic effect assay and p24 quantification assay with ELISA. The inhibitory effect of wikstroelide M on HIV reverse transcription was analyzed by real-time PCR and ELISA. The effect of wikstroelide M on HIV-1 integrase nuclear translocation was observed with a cell-based imaging assay. The effect of wikstroelide M on LEDGF/p75-IN interaction was assayed by molecular docking.@*RESULTS@#Wikstroelide M potently inhibited different HIV-1 strains, including HIV-1IIIB, HIV-1A17, and HIV-19495, induced a cytopathic effect, with EC50 values ranging from 3.81 to 15.65 ng·mL⁻¹. Wikstroelide M also had high inhibitory activities against HIV-2ROD and HIV-2CBL-20-induced cytopathic effects with EC50 values of 18.88 and 31.90 ng·mL⁻¹. The inhibitory activities of wikstroelide M on the three HIV-1 strains were further confirmed by p24 quantification assay, with EC50 values ranging from 15.16 to 35.57 ng·mL⁻¹. Wikstroelide M also potently inhibited HIV-1IIIB induced cytolysis in MT-4 cells, with an EC50 value of 9.60 ng·mL⁻¹. The mechanistic assay showed that wikstroelide M targeted HIV-1 reverse transcriptase and nuclear translocation of integrase through disrupting the interaction between integrase and LEDGF/p75.@*CONCLUSION@#Wikstroelide M may be a potent HIV-1 and HIV-2 inhibitor, the mechanisms of action may include inhibition of reverse trascriptase activity and inhibition of integrase nuclear translocation through disrupting the interaction between integrase and LEDGF/p75.

A new chimeric protein represses HIV-1 LTR-mediated expression by DNA methylase.

Once the human immunodeficiency virus (HIV) genome is inserted into the host genome, the virus cannot be removed, which results in latency periods and makes it difficult to eradicate. The majority of strategies to eradicate HIV have been based on preventing virus latency, thereby enabling antiretroviral drugs to act against HIV replication. Another innovative strategy is permanently silencing the integrated virus to prevent the spread of infection. Epigenetic processes are natural mechanisms that can silence viral replication. We describe a new chimeric protein (IN3b) that consists of a HIV-1 integrase domain, which recognises the HIV long terminal repeat (LTR) and the catalytic domain of DNA methyltransferase DNMT3b. Our objective was to silence HIV replication by the specific delivery of the catalytic methyltransferase domain to the LTR promoter to induce its methylation. We found that our IN3b chimeric protein was expressed in the nucleus and decreased LTR-associated HIV genome expression and HIV replication. Therefore, the IN3b chimeric protein may be an effective tool against HIV replication and maybe used in a new line of research to induce or maintain HIV latency.

Role of antiviral therapy in the management of hepatocellular carcinoma.

Globally, hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection leads to liver fibrosis and cirrhosis, which in turn causes resultant hepatocellular carcinoma (HCC). Frequently, HCC recurs very soon even after a potentially curative treatment such as surgical interference or locoregional ablative therapies. Chronic HBV/HCV infection is often responsible for this recurrence, through secondary carcinogenesis. Antiviral therapy after a curative treatment of HCC plays an important role in preventing or delaying recurrence and improves survival in patients with HBV/HCV infection-related HCC. This article reviews the worldwide epidemiology of HBV/HCV infection, the association of viral infection with HCC, the mechanism of hepatitis virus-related hepatocarcinogenesis, and the paramount importance of antiviral therapy in the management of HCC.

Cimicifuga foetida L. inhibited human respiratory syncytial virus in HEp-2 and A549 cell lines.

Human respiratory syncytial virus (HRSV) causes serious pediatric infection of the lower respiratory tract without effective therapeutic modality. Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) has been proven to be effective at inhibiting HRSV-induced plaque formation, and Cimicifuga foetida is the major constituent of SMGGT. We tested the hypothesis that C. foetida effectively inhibited the cytopathic effects of HRSV by a plaque reduction assay in both human upper (HEp2) and lower (A549) respiratory tract cell lines. Its ability to stimulate anti-viral cytokines was evaluated by an enzyme-linked immunosorbent assay (ELISA). C. foetida dose-dependently inhibited HRSV-induced plaque formation (p < 0.0001) before and after viral inoculation, especially in A549 cells (p < 0.0001). C. foetida dose-dependently inhibited viral attachment (p < 0.0001) and could increase heparins effect on viral attachment. In addition, C. foetida time-dependently and dose-dependently (p < 0.0001) inhibited HRSV internalization. C. foetida could stimulate epithelial cells to secrete IFN-ß to counteract viral infection. However, C. foetida did not stimulate TNF-α secretion. Therefore, C. foetida could be useful in managing HRSV infection. This is the first evidence to support that C. foetida possesses antiviral activity.

Evaluation of a system to screen for stimulators of non-specific DNA nicking by HIV-1 integrase: application to a library of 50,000 compounds.

BACKGROUND: In addition to activities needed to catalyse integration, retroviral integrases exhibit non-specific endonuclease activity that is enhanced by certain small compounds, suggesting that integrase could be stimulated to damage viral DNA before integration occurs. METHODS: A non-radioactive, plate-based, solution phase, fluorescence assay was used to screen a library of 50,080 drug-like chemicals for stimulation of non-specific DNA nicking by HIV-1 integrase. RESULTS: A semi-automated workflow was established and primary hits were readily identified from a graphic output. Overall, 0.6% of the chemicals caused a large increase in fluorescence (the primary hit rate) without also having visible colour that could have artifactually caused this result. None of the potential stimulators from this moderate-size library, however, passed a secondary test that included an inactive integrase mutant that assessed whether the increased fluorescence depended on the endonuclease activity of integrase. CONCLUSIONS: This first attempt at identifying integrase stimulator compounds establishes the necessary logistics and workflow required. The results from this study should encourage larger scale high-throughput screening to advance the novel antiviral strategy of stimulating integrase to damage retroviral DNA.

High-throughput screening uncovers a compound that activates latent HIV-1 and acts cooperatively with a histone deacetylase (HDAC) inhibitor.

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

Proanthocyanidin-enriched extract from Myrothamnus flabellifolia Welw. exerts antiviral activity against herpes simplex virus type 1 by inhibition of viral adsorption and penetration.

AIM OF THE STUDY: Extracts from the aerial parts of the South African resurrection plant Myrothamnus flabellifolia Welw. have been used traditionally against infections of the upper respiratory tract and skin diseases. A polyphenol-enriched extract was investigated for potential antiviral effects against herpes simplex virus type 1 (HSV-1) and adenovirus, and the underlying mode of action was to be studied. MATERIALS AND METHODS: Antiviral effects of an acetone-water extract (MF) from Myrothamnus flabellifolia on HSV-1 and adenovirus type 3 were tested in infected Vero cells by plaque reduction assay, MTT test and immunofluorescence. The influence of the extract on the HSV-1 envelope glycoprotein D was shown by Western blot. Organotypic full thickness skin models consisting of multilayer skin equivalents were used for the investigation of MF effects on HSV-1 replication. RESULTS: MF exhibited strong antiviral activity against HSV-1. The HSV-1-specific inhibitory concentration (IC(50)) was determined as 0.4 µg/mL and the cytotoxic concentration (CC(50)) against Vero cells as 50 µg/mL. A selectivity index (SI) (ratio of CC(50) to IC(50)) of approximately 120 was calculated when MF was added to the virus inoculum for 1h at 37°C prior to infection. The replication of adenovirus 3 was not affected by MF. MF abolished virus entry into the host cell by blocking viral attachment to the cell surface. When added after attachment at a concentration of >6 µg/mL, the extract also inhibited penetration of HSV-1 into the host cell. Polyphenolic compounds from MF directly interacted with viral particles, leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein D (gD). Using organotypic full thickness tissue cultures, it was shown that treatment of HSV-1 infected cultures with the MF resulted in reduced viral spread. CONCLUSIONS: A polyphenol-enriched extract from Myrothamnus flabellifolia strongly acts against HSV-1 by blocking viral entry into the cells.

In vivo expansion of MDR1-transduced cells accompanied by a post-transplantation chemotherapy regimen with mitomycin C and methotrexate.

BACKGROUND: A recurrent breast cancer patient received high-dose chemotherapy, a transplant of multidrug resistance 1 (MDR1)-transduced cells and four different protocols of post-transplantation chemotherapy. We report the analysis of MDR1-transduced cells in this patient. METHODS: MDR1 transgene levels in the peripheral blood mononuclear cells of the patient were evaluated by polymerase chain reaction (PCR). Retroviral integration sites of the MDR1-transduced cells were identified by linear amplification-mediated (LAM)-PCR. RESULTS: Twelve days after transplantation, approximately 1% of the peripheral blood mononuclear cells were MDR1 transgene-positive. The transgene levels decreased quickly, and were at low levels until day 504. A remarkable increase in MDR1 transgene-positive cells was observed on day 532, during combination chemotherapy with mitomycin C and methotrexate. Using LAM-PCR, 31 MDR1-transduced clones were identified, and eight of these were long-life clones that survived for more than 500 days. Among the 31 clones, ten had a retroviral integration site near genes listed in the Retroviral Tagged Cancer Gene (RTCG) Database. Two long-life clones, N-30 and N-31, had retroviral integration sites within the MDS1-EVI1 locus. Another two long-life clones had integration sites close to PRDM16 or CUEDC1. CONCLUSIONS: These results suggest that MDR1-transduced cells were enriched in vivo by an MDR1 substrate, mitomycin C. The possible activation of EVI1 or other RTCGs by retroviral insertion may have affected the survival and persistence of a proportion of the transduced cells.

Transcription factor YY1 interacts with retroviral integrases and facilitates integration of moloney murine leukemia virus cDNA into the host chromosomes.

Retroviral integrases associate during the early viral life cycle with preintegration complexes that catalyze the integration of reverse-transcribed viral cDNA into the host chromosomes. Several cellular and viral proteins have been reported to be incorporated in the preintegration complex. This study demonstrates that transcription factor Yin Yang 1 binds to Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. The results of coimmunoprecipitation and in vitro pulldown assays revealed that Yin Yang 1 interacted with the catalytic core and C-terminal domains of Moloney murine leukemia virus and human immunodeficiency virus type 1 integrases, while the transcriptional repression and DNA-binding domains of the Yin Yang 1 molecule interacted with Moloney murine leukemia virus integrase. Immunoprecipitation of the cytoplasmic fraction of virus-infected cells followed by Southern blotting and chromatin immunoprecipitation demonstrated that Yin Yang 1 associated with Moloney murine leukemia virus cDNA in virus-infected cells. Yin Yang 1 enhanced the in vitro integrase activity of Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. Furthermore, knockdown of Yin Yang 1 in host cells by small interfering RNA reduced Moloney murine leukemia virus cDNA integration in vivo, although viral cDNA synthesis was increased, suggesting that Yin Yang 1 facilitates integration events in vivo. Taking these results together, Yin Yang 1 appears to be involved in integration events during the early viral life cycle, possibly as an enhancer of integration.

Prediction of the interaction of HIV-1 integrase and its dicaffeoylquinic acid inhibitor through molecular modeling approach.

An essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is integration of the double-stranded retroviral DNA into the genome of the host cell. HIV-1 integrase, the enzyme that inserts the vital DNA into the host chromosome, is an attractive and rational target for anti-AIDS drug design because it is essential for HIV replication and there are no known counterparts in the host cell. Inhibitors of this enzyme have a great potential to complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. Natural products have provided a source of new drug candidates for anti-AIDS therapy. Dicaffeoylquinic acids, isolated from traditional medicinal plants, are a novel class of integrase inhibitors. These compounds are potent inhibitors of HIV-1 replication in cultured cell lines and catalytic activities of integrase in vitro. They are therefore promising compounds for developing new anti-AIDS drugs. To understand how the inhibitors work and therefore design more potent and specific inhibitors, we have used molecular modeling techniques to investigate the binding modes of 3,4-dicaffeoylquinic acid. Our computational modeling study demonstrated that the inhibitor of this compound on HIV integrase is likely to proceed by two different but equivalent mechanisms with one bound to the active site region of the enzyme and another docked into the binding pocket located on the other side of the catalytic site. Our study will be of help to design new pharmaceuticals for the treatment of AIDS.

The mechanism of HIV-1 Tat-directed nucleic acid annealing supports its role in reverse transcription.

The main function of the HIV-1 trans-activator of transcription (Tat protein) is to promote the transcription of the proviral DNA by the host RNA polymerase which leads to the synthesis of large quantities of the full length viral RNA. Tat is also thought to be involved in the reverse transcription (RTion) reaction by a still unknown mechanism. The recently reported nucleic acid annealing activity of Tat might explain, at least in part, its role in RTion. To further investigate this possibility, we carried out a fluorescence study on the mechanism by which the full length Tat protein (Tat(1-86)) and the basic peptide (44-61) direct the annealing of complementary viral DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, essential for the early steps of RTion. Though both Tat(1-86) and the Tat(44-61) peptide were unable to melt the lower half of the cTAR stem, they strongly promoted cTAR/dTAR annealing through non-specific attraction between the peptide-bound oligonucleotides. Using cTAR and dTAR mutants, this Tat promoted-annealing was found to be nucleated through the thermally frayed 3'/5' termini, resulting in an intermediate with 12 intermolecular base pairs, which then converts into the final extended duplex. Moreover, we found that Tat(1-86) was as efficient as the nucleocapsid protein NCp7, a major nucleic acid chaperone of HIV-1, in promoting cTAR/dTAR annealing, and could act cooperatively with NCp7 during the annealing reaction. Taken together, our data are consistent with a role of Tat in the stimulation of the obligatory strand transfers during viral DNA synthesis by reverse transcriptase.

HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects.

Highly active antiretroviral therapy (HAART) results in potent and durable suppression of HIV-1 viremia. However, HIV-1 replication resumes if therapy is interrupted. Although it is generally believed that active replication has been halted in individuals on HAART, immune activation and inflammation continue at abnormal levels, suggesting continued, low-level viral replication. To assess whether active replication might be driving immune activation in HAART, we examined the impact of treatment intensification with the integrase inhibitor raltegravir on viral complementary DNA and immune activation parameters. In the presence of raltegravir, linear HIV-1 cDNA is prevented from integrating into chromatin and is subsequently converted to episomal cDNAs. Raltegravir intensification of a three-drug suppressive HAART regimen resulted in a specific and transient increase in episomal DNAs in a large percentage of HAART-suppressed subjects. Furthermore, in subjects with these episomal DNAs, immune activation was higher at baseline and was subsequently normalized after raltegravir intensification. These results suggest that, despite suppressive HAART, active replication persists in some infected individuals and drives immune activation. The ability of raltegravir intensification to perturb the reservoir that supports active replication has implications for therapeutic strategies aimed at achieving viral eradication.

Polysaccharides from Sargassum tenerrimum: structural features, chemical modification and anti-viral activity.

Herpes simplex viruses (HSVs) display affinity for cell-surface heparan sulfate proteoglycans with biological relevance in virus entry. Here, we exploit an approach to inhibiting HSV infection by using a sulfated fucoidan, and a guluronic acid-rich alginate derived from Sargassum tenerrimum, mimicking the active domain of the entry receptor. These macromolecules have apparent molecular masses of 30+/-5 and 26+/-5 kDa, respectively. They and their chemically sulfated derivatives showed activity against herpes simplex virus type 1 (HSV-1). Their inhibitory concentration 50% (IC(50)) values were in the range 0.5-15 microg/ml and they lacked cytotoxicity at concentrations up to 1000 microg/ml. The anti-HSV activity increased with increasing sulfate ester content. Our results suggest the feasibility of inhibiting HSV infection by blocking viral entry with polysaccharide having specific structure.

Ellagitannins from Tuberaria lignosa as entry inhibitors of HIV.

Screening of plants from the Iberian Peninsula for anti-human immunodeficiency virus (-HIV) activity revealed that aqueous extract of Tuberaria lignosa gave positive results. Following an activity-guided procedure, the crude extract was counterextracted, and the subsequent fractions obtained tested for their anti-HIV activity in vitro. The bioassay-guided fractionation of the extract afforded an ellagitannin enriched fraction (EEF) isolated for the first time from this species. This EEF exhibited antiviral activity against HIV in MT-2 infected cells, with an IC(50) value of 2.33mug/ml (selectivity index greater than 21). Inhibition of HIV infection by EEF appears to be mediated by CD4 down-regulation, the main receptor for HIV entry. CXCR4 and CCR5 receptors were not affected by EEF, explaining why EEF is able to inhibit R5 and X4 infections.