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Sample records for inhibits hiv replication

  1. A nucleolar localizing Rev binding element inhibits HIV replication

    PubMed Central

    Michienzi, Alessandro; De Angelis, Fernanda G; Bozzoni, Irene; Rossi, John J

    2006-01-01

    The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment. PMID:16712721

  2. Hili inhibits HIV replication in activated T cells.

    PubMed

    Peterlin, B Matija; Liu, Pingyang; Wang, Xiaoyun; Cary, Daniele; Shao, Wei; Leoz, Marie; Hong, Tian; Pan, Tao; Fujinaga, Koh

    2017-03-22

    Piwil proteins restrict the replication of mobile genetic elements in the germline. They are also expressed in many transformed cell lines. In this report, we discovered that the human piwil 2 (hili) can also inhibit HIV replication, especially in activated CD4+ T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express hili, it was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of hili increased levels of viral proteins and new viral particles. Further studies revealed that hili binds to tRNA. Some of them represent rare tRNA species, whose codons are over-represented in the viral genome. Targeting tRNA(Arg)(UCU) with an antisense oligonucleotide replicated effects of hili and also inhibited HIV replication. Finally, hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements.IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germline. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small piRNAs. However, in some species and in human somatic cells, piwil proteins bind primarily to tRNA. In this report, we demonstrate that human piwil proteins, especially hili, not only bind to select tRNA species that include rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of hili in CD4+ T cells. Since hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements.

  3. GADD45 proteins inhibit HIV-1 replication through specific suppression of HIV-1 transcription.

    PubMed

    Liang, Zhibin; Liu, Ruikang; Zhang, Hui; Zhang, Suzhen; Hu, Xiaomei; Tan, Juan; Liang, Chen; Qiao, Wentao

    2016-06-01

    GADD45 proteins are a group of stress-induced proteins and participate in various cellular pathways including cell cycle regulation, cell survival and death, DNA repair and demethylation. It was recently shown that HIV-1 infection induces the expression of GADD45 proteins. However, the effect of GADD45 on HIV-1 replication has not been studied. Here, we report that overexpression of GADD45 proteins reduces HIV-1 production through suppressing transcription from the HIV-1 LTR promoter. This inhibitory effect is specific to HIV-1, since GADD45 proteins neither inhibit the LTR promoters from other retroviruses nor reduce the production of these viruses. Knockdown of endogenous GADD45 modestly activates HIV-1 in the J-Lat A72 latency cell line, which suggests GADD45 proteins might play a role in maintaining HIV-1 latency.

  4. Methamphetamine inhibits HIV-1 replication in CD4+ T cells by modulating anti-HIV-1 miRNA expression.

    PubMed

    Mantri, Chinmay K; Mantri, Jyoti V; Pandhare, Jui; Dash, Chandravanu

    2014-01-01

    Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4(+) T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4(+) T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti-HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4(+) T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis.

  5. Methamphetamine Inhibits HIV-1 Replication in CD4+ T Cells by Modulating Anti–HIV-1 miRNA Expression

    PubMed Central

    Mantri, Chinmay K.; Mantri, Jyoti V.; Pandhare, Jui; Dash, Chandravanu

    2015-01-01

    Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4+ T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4+ T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti–HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4+ T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis. PMID:24434277

  6. Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors.

    PubMed

    Eekels, Julia J M; Geerts, Dirk; Jeeninga, Rienk E; Berkhout, Ben

    2011-01-01

    In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected.

  7. Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Zarling, Joyce M.; Moran, Patricia A.; Haffar, Omar; Sias, Joan; Richman, Douglas D.; Spina, Celsa A.; Myers, Dorothea E.; Kuebelbeck, Virginia; Ledbetter, Jeffrey A.; Uckun, Fatih M.

    1990-09-01

    FUNCTIONAL impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells1,2. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA3,4. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000 (ref. 5), which inhibits replication of certain plant RNA viruses6-8, and of herpes simplex virus, poliovirus and influenza virus9-11. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CDS, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.

  8. Soybean-derived Bowman-Birk Inhibitor (BBI) Inhibits HIV Replication in Macrophages

    PubMed Central

    Ma, Tong-Cui; Zhou, Run-Hong; Wang, Xu; Li, Jie-Liang; Sang, Ming; Zhou, Li; Zhuang, Ke; Hou, Wei; Guo, De-Yin; Ho, Wen-Zhe

    2016-01-01

    The Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, is known to have anti-inflammatory effect in both in vitro and in vivo systems. Macrophages play a key role in inflammation and immune activation, which is implicated in HIV disease progression. Here, we investigated the effect of BBI on HIV infection of peripheral blood monocyte-derived macrophages. We demonstrated that BBI could potently inhibit HIV replication in macrophages without cytotoxicity. Investigation of the mechanism(s) of BBI action on HIV showed that BBI induced the expression of IFN-β and multiple IFN stimulated genes (ISGs), including Myxovirus resistance protein 2 (Mx2), 2′,5′-oligoadenylate synthetase (OAS-1), Virus inhibitory protein (viperin), ISG15 and ISG56. BBI treatment of macrophages also increased the expression of several known HIV restriction factors, including APOBEC3F, APOBEC3G and tetherin. Furthermore, BBI enhanced the phosphorylation of IRF3, a key regulator of IFN-β. The inhibition of IFN-β pathway by the neutralization antibody to type I IFN receptor (Anti-IFNAR) abolished BBI-mediated induction of the anti-HIV factors and inhibition of HIV in macrophages. These findings that BBI could activate IFN-β-mediated signaling pathway, initialize the intracellular innate immunity in macrophages and potently inhibit HIV at multiple steps of viral replication cycle indicate the necessity to further investigate BBI as an alternative and cost-effective anti-HIV natural product. PMID:27734899

  9. Inhibition of HIV-1 replication by newly developed adamantane-containing polyanionic agents.

    PubMed

    Burstein, M E; Serbin, A V; Khakhulina, T V; Alymova, I V; Stotskaya, L L; Bogdan, O P; Manukchina, E E; Jdanov, V V; Sharova, N K; Bukrinskaya, A G

    1999-04-01

    Newly developed antiviral compounds consisting of an adamantane derivative chemically linked to a water-soluble polyanionic matrix were shown to inhibit HIV-1 infection in lymphoblastoid cells, HeLa CD4+ beta-galactosidase (MAGI) cells and macrophages. The effect of the compounds was recorded by measuring viral reverse transcriptase activity and p24 by ELISA in culture supernatant and by immunoblotting of cell lysates. In this paper we describe the data obtained with one of the most promising compounds, Amant. Amant was not toxic for the host cells at concentrations as high as 1 mg/ml. The inhibition of HIV-1 replication in MT-4 and MAGI cells was observed when Amant was added either before infection or with the virus (0 h of infection), and was expressed even when the compound added at 0 h was removed 1.5 h after infection. Its inhibitory concentration (IC50) against HIV-1 and HIV-2 replication was 2-6 and 93 microg/ml, respectively. The anti-HIV-1 effect of the compound was gradually decreased when it was added 1 and 2 h post infection, and no inhibition was observed when the compound was added 4 h after infection, suggesting that the compound as a membranotropic drug blocks an early step of replication. It completely prevented the transport of Gag proteins into the nuclei. Pretreatment of the virus with Amant did not reduce its infectious activity. The classical adamantane derivatives amantadine and rimantadine hydrochloride did not inhibit HIV replication.

  10. Natural Plant Alkaloid (Emetine) Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity.

    PubMed

    Chaves Valadão, Ana Luiza; Abreu, Celina Monteiro; Dias, Juliana Zanatta; Arantes, Pablo; Verli, Hugo; Tanuri, Amilcar; de Aguiar, Renato Santana

    2015-06-22

    Ipecac alkaloids are secondary metabolites produced in the medicinal plant Psychotria ipecacuanha. Emetine is the main alkaloid of ipecac and one of the active compounds in syrup of Ipecac with emetic property. Here we evaluated emetine's potential as an antiviral agent against Human Immunodeficiency Virus. We performed in vitro Reverse Transcriptase (RT) Assay and Natural Endogenous Reverse Transcriptase Activity Assay (NERT) to evaluate HIV RT inhibition. Emetine molecular docking on HIV-1 RT was also analyzed. Phenotypic assays were performed in non-lymphocytic and in Peripheral Blood Mononuclear Cells (PBMC) with HIV-1 wild-type and HIV-harboring RT-resistant mutation to Nucleoside Reverse Transcriptase Inhibitors (M184V). Our results showed that HIV-1 RT was blocked in the presence of emetine in both models: in vitro reactions with isolated HIV-1 RT and intravirion, measured by NERT. Emetine revealed a strong potential of inhibiting HIV-1 replication in both cellular models, reaching 80% of reduction in HIV-1 infection, with low cytotoxic effect. Emetine also blocked HIV-1 infection of RT M184V mutant. These results suggest that emetine is able to penetrate in intact HIV particles, and bind and block reverse transcription reaction, suggesting that it can be used as anti-HIV microbicide. Taken together, our findings provide additional pharmacological information on the potential therapeutic effects of emetine.

  11. Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity

    PubMed Central

    Martinez, Zachary S.; Castro, Edison; Seong, Chang-Soo; Cerón, Maira R.

    2016-01-01

    Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2 showed strong antiviral activity on the replication of HIV-1 in human CD4+ T cells. However, these compounds did not inhibit infection by single-round infection vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped viruses, indicating no effect on the early steps of the viral life cycle. In contrast, analysis of single-round infection VSV-G-pseudotyped HIV-1 produced in the presence of compound 1 or 2 showed a complete lack of infectivity in human CD4+ T cells, suggesting that the late stages of the HIV-1 life cycle were affected. Quantification of virion-associated viral RNA and p24 indicates that RNA packaging and viral production were unremarkable in these viruses. However, Gag and Gag-Pol processing was affected, as evidenced by immunoblot analysis with an anti-p24 antibody and the measurement of virion-associated reverse transcriptase activity, ratifying the effect of the fullerene derivatives on virion maturation of the HIV-1 life cycle. Surprisingly, fullerenes 1 and 2 did not inhibit HIV-1 protease in an in vitro assay at the doses that potently blocked viral infectivity, suggesting a protease-independent mechanism of action. Highlighting the potential therapeutic relevance of fullerene derivatives, these compounds block infection by HIV-1 resistant to protease and maturation inhibitors. PMID:27431232

  12. Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

    PubMed Central

    Levy, David N.; Li, Yonghua; Kumar, Rajnish; Burke, Sean A.; Dawson, Rodney; Hioe, Catarina E.; Borkowsky, William; Rom, William N.; Hoshino, Yoshihiko

    2014-01-01

    While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages. PMID:25272020

  13. Dicaffeoyltartaric acid analogues inhibit human immunodeficiency virus type 1 (HIV-1) integrase and HIV-1 replication at nontoxic concentrations.

    PubMed

    Reinke, Ryan A; King, Peter J; Victoria, Joseph G; McDougall, Brenda R; Ma, Guoxiang; Mao, Yingqun; Reinecke, Manfred G; Robinson, W Edward

    2002-08-15

    The human immunodeficiency virus type 1 (HIV-1) is a major health problem worldwide. In this study, 17 analogues of L-chicoric acid, a potent inhibitor of HIV integrase, were studied. Of these analogues, five submicromolar inhibitors of integrase were discovered and 13 compounds with activity against integrase at less than 10 microM were identified. Six demonstrated greater than 10-fold selectivity for HIV replication over cellular toxicity. Ten analogues inhibited HIV replication at nontoxic concentrations. Alteration of the linkages between the two bis-catechol rings, including the use of amides, mixed amide esters, cholate, and alkyl bridges, was explored. Amides were as active as esters but were more toxic in tissue culture. Alkyl and cholate bridges were significantly less potent against HIV-1 integrase in vitro and were inactive against HIV-1 replication. Two amino acid derivates and one digalloylderivative of L-chicoric acid (L-CA) showed improved selectivity over L-CA against integration in cell culture. These data suggest that in addition to the bis-catechols and free carboxylic acid groups reported previously, polar linkages are important constituents for optimal activity against HIV-1 integrase and that new derivatives can be developed with increased specificity for integration over HIV entry in vivo.

  14. Inhibition of HIV replication in vitro by clinical immunosuppressants and chemotherapeutic agents

    PubMed Central

    2013-01-01

    Background Recent studies have suggested that a functional cure for HIV-1 infection, purportedly resultant from allogeneic bone marrow transplantation, may be possible. Additionally, the first such patient was treated with whole-body irradiation, immunosuppressants, and the chemotherapeutic, cytarabine. However, the precise role of the coinciding medical interventions in diminishing detectable HIV reservoirs remains unstudied. Findings In this article, we demonstrate that the immunosuppressants, mycophenolic acid and cyclosporine, and the chemotherapeutic, cytarabine, are potent antiretroviral agents at clinically relevant dosages. These drugs strongly inhibit HIV-1 replication in a GFP indicator T cell line and peripheral blood mononuclear cells (PBMC). Conclusions Our study suggests that certain clinical immunosuppressants and chemotherapeutic agents may act combinatorially to inhibit HIV infection. Additionally, chemotherapy-mediated cytotoxicity may also affect the stability of viral reservoirs. Thus, further study is needed to examine potential therapeutic value of these interventions in patients. PMID:23672887

  15. Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41.

    PubMed

    Rivero-Buceta, Eva; Doyagüez, Elisa G; Colomer, Ignacio; Quesada, Ernesto; Mathys, Leen; Noppen, Sam; Liekens, Sandra; Camarasa, María-José; Pérez-Pérez, María-Jesús; Balzarini, Jan; San-Félix, Ana

    2015-12-01

    Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity.

  16. Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    PubMed Central

    Gupta, Sachin; Termini, James M.; Issac, Biju; Guirado, Elizabeth; Stone, Geoffrey W.

    2016-01-01

    Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG), including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1). Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-β and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5) expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration. PMID:26849062

  17. CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape.

    PubMed

    Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T

    2016-03-01

    Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications.

  18. Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway.

    PubMed

    Sancho, Rocío; Márquez, Nieves; Gómez-Gonzalo, Marta; Calzado, Marco A; Bettoni, Giorgio; Coiras, Maria Teresa; Alcamí, José; López-Cabrera, Manuel; Appendino, Giovanni; Muñoz, Eduardo

    2004-09-03

    Coumarins and structurally related compounds have been recently shown to present anti-human immunodeficiency virus, type 1 (HIV-1) activity. Among them, the dietary furanocoumarin imperatorin is present in citrus fruits, in culinary herbs, and in some medicinal plants. In this study we report that imperatorin inhibits either vesicular stomatitis virus-pseudotyped or gp160-enveloped recombinant HIV-1 infection in several T cell lines and in HeLa cells. These recombinant viruses express luciferase as a marker of viral replication. Imperatorin did not inhibit the reverse transcription nor the integration steps in the viral cell cycle. Using several 5' long terminal repeat-HIV-1 constructs where critical response elements were either deleted or mutated, we found that the transcription factor Sp1 is critical for the inhibitory activity of imperatorin induced by both phorbol 12-myristate 13-acetate and HIV-1 Tat. Moreover in transient transfections imperatorin specifically inhibited phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein. Since Sp1 is also implicated in cell cycle progression we further studied the effect of imperatorin on cyclin D1 gene transcription and protein expression and in HeLa cell cycle progression. We found that imperatorin strongly inhibited cyclin D1 expression and arrested the cells at the G(1) phase of the cell cycle. These results highlight the potential of Sp1 transcription factor as a target for natural anti-HIV-1 compounds such as furanocoumarins that might have a potential therapeutic role in the management of AIDS.

  19. Inhibition of hepatitis B virus and human immunodeficiency virus (HIV-1) replication by Warscewiczia coccinea (Vahl) Kl. (Rubiaceae) ethanol extract.

    PubMed

    Quintero, A; Fabbro, R; Maillo, M; Barrios, M; Milano, M B; Fernández, A; Williams, B; Michelangeli, F; Rangel, H R; Pujol, F H

    2011-09-01

    The primary objective of this study was to search for natural products capable of inhibiting hepatitis B virus (HBV) replication. The research design, methods and procedures included testing hydro-alcoholic extracts (n = 66) of 31 species from the Venezuelan Amazonian rain forest on the cell line HepG2 2.2.15, which constitutively produces HBV. The main outcomes and results were as follows: the species Euterpe precatoria, Jacaranda copaia, Jacaranda obtusifolia, Senna silvestris, Warscewiczia coccinea and Vochysia glaberrima exerted some degree of inhibition on HBV replication. The leaves of W. coccinea showed a significant antiviral activity: 80% inhibition with 100 µg mL⁻¹ of extract. This extract also exerted inhibition on covalently closed circular deoxyribonucleic acid (cccDNA) production and on HIV-1 replication in MT4 cells (more than 90% inhibition with 50 µg mL⁻¹ of extract). Initial fractionation using organic solvents of increasing polarity and water showed that the ethanol fraction was responsible for most of the antiviral inhibitory activities of both the viruses. It was concluded that Warscewiczia coccinea extract showed inhibition of HBV and HIV-1 replication. Bioassay-guided purification of this fraction may allow the isolation of an antiviral compound with inhibitory activity against both viruses.

  20. Development of benzimidazole derivatives to inhibit HIV-1 replication through protecting APOBEC3G protein.

    PubMed

    Pan, Ting; He, Xin; Chen, Bing; Chen, Hui; Geng, Guannan; Luo, Haihua; Zhang, Hui; Bai, Chuan

    2015-05-05

    Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hypermutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor) counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which provided the best potency, with IC50 values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1 replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we suggest that these two benzimidazole derivatives can be further developed as a new category of anti-HIV-1 leads.

  1. Cold Atmospheric Plasma Inhibits HIV-1 Replication in Macrophages by Targeting Both the Virus and the Cells

    PubMed Central

    Volotskova, Olga; Dubrovsky, Larisa; Keidar, Michael; Bukrinsky, Michael

    2016-01-01

    Cold atmospheric plasma (CAP) is a specific type of partially ionized gas that is less than 104°F at the point of application. It was recently shown that CAP can be used for decontamination and sterilization, as well as anti-cancer treatment. Here, we investigated the effects of CAP on HIV-1 replication in monocyte-derived macrophages (MDM). We demonstrate that pre-treatment of MDM with CAP reduced levels of CD4 and CCR5, inhibiting virus-cell fusion, viral reverse transcription and integration. In addition, CAP pre-treatment affected cellular factors required for post-entry events, as replication of VSV-G-pseudotyped HIV-1, which by-passes HIV receptor-mediated fusion at the plasma membrane during entry, was also inhibited. Interestingly, virus particles produced by CAP-treated cells had reduced infectivity, suggesting that the inhibitory effect of CAP extended to the second cycle of infection. These results demonstrate that anti-HIV activity of CAP involves the effects on target cells and the virus, and suggest that CAP may be considered for potential application as an anti-HIV treatment. PMID:27783659

  2. Initial HIV-1 antigen-specific CD8+ T cells in acute HIV-1 infection inhibit transmitted/founder virus replication.

    PubMed

    Freel, Stephanie A; Picking, Ralph A; Ferrari, Guido; Ding, Haitao; Ochsenbauer, Christina; Kappes, John C; Kirchherr, Jennifer L; Soderberg, Kelly A; Weinhold, Kent J; Cunningham, Coleen K; Denny, Thomas N; Crump, John A; Cohen, Myron S; McMichael, Andrew J; Haynes, Barton F; Tomaras, Georgia D

    2012-06-01

    CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8(+) T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8(+) responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8(+) T cells during AHI. Autologous and heterologous CD8(+) T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8(+) T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8(+) antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8(+)-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8(+) T cell-mediated inhibition of virus replication. CD8(+) T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies.

  3. Anti-HIV-1 potency of the CRISPR/Cas9 system insufficient to fully inhibit viral replication.

    PubMed

    Ueda, Shuhei; Ebina, Hirotaka; Kanemura, Yuka; Misawa, Naoko; Koyanagi, Yoshio

    2016-07-01

    The range of genome-editing tools has recently been expanded. In particular, an RNA-guided genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) system, has many applications for human diseases. In this study, guide RNA (gRNA) to target gag, pol and a long terminal repeat of HIV-1 was designed and used to generate gRNA-expressing lentiviral vectors. An HIV-1-specific gRNA and Cas9 were stably dually transduced into a highly HIV-1-susceptible human T-cell line and the inhibitory ability of the anti-HIV-1 CRISPR/Cas9 lentiviral vector assessed. Although clear inhibition of the early phase of HIV-1 infection was observed, as evaluated by a VSV-G-pseudotyped HIV-1 reporter system, the anti-HIV-1 potency in multiple rounds of wild type (WT) viral replication was insufficient, either because of generation of resistant viruses or overcoming of the activity of the WT virus. Thus, there are potential difficulties that must be addressed when considering anti-HIV-1 treatment with the CRISPR/Cas9 system alone.

  4. Venom Components of Iranian Scorpion Hemiscorpius lepturus Inhibit the Growth and Replication of Human Immunodeficiency Virus 1 (HIV-1)

    PubMed Central

    Zabihollahi, Rezvan; Bagheri, Kamran Pooshang; Keshavarz, Zohreh; Motevalli, Fatemeh; Bahramali, Golnaz; Siadat, Seyed Davar; Momen, Seyed Bahman; Shahbazzadeh, Delavar; Aghasadeghi, Mohammad Reza

    2016-01-01

    Background: During the recent years, significant progress has been achieved on development of novel anti-viral drugs. Natural products are assumed as the potential sources of novel anti-viral drugs; therefore, there are some previous studies reporting the anti-viral compounds from venomous animals. Based on the significant value for tracing of non-toxic anti-viral agents from natural resources, this study was aimed to investigate the anti-viral activity of some HPLC purified fractions derived from the venom of Iranian scorpion, Hemiscorpius lepturus, against human immunodeficiency virus 1 (HIV-1) and herpes simplex virus 1 (HSV-1). Methods: H. Lepturus crude venom was subjected to reverse phase HPLC analysis to determine its active components precisely where four dominant fractions obtained at retention time of 156-160 minutes. The phospholipase A2 and hemolytic activities of the purified fractions were first evaluated. Then the anti-viral activity was measured using single cycle HIV (NL4-3) replication and HSV (KOS) plaque reduction assays. Results: The H. lepturus crude venom inhibited HIV replication by 73% at the concentration of 200 µg/ml, while it did not show significant anti-HSV activity. It also inhibited the cell-free viral particles in a virucidal assay, while it showed no toxicity for the target cells in a proliferation assay. The four HPLC fractions purified from H. lepturus inhibited HIV with IC50 of 20 µg/ml. Conclusion: H. lepturus venom contains components with considerable anti-HIV activity insofar as it has virucidal activity that offers a novel therapeutic approach against HIV infection. Our results suggest a promising pilot for anti-HIV drug discovery with H. lepturus scorpion venom. PMID:27594443

  5. CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape.

    PubMed

    Wang, Zhen; Pan, Qinghua; Gendron, Patrick; Zhu, Weijun; Guo, Fei; Cen, Shan; Wainberg, Mark A; Liang, Chen

    2016-04-19

    Cas9 cleaves specific DNA sequences with the assistance of a programmable single guide RNA (sgRNA). Repairing this broken DNA by the cell's error-prone non-homologous end joining (NHEJ) machinery leads to insertions and deletions (indels) that often impair DNA function. Using HIV-1, we have now demonstrated that many of these indels are indeed lethal for the virus, but that others lead to the emergence of replication competent viruses that are resistant to Cas9/sgRNA. This unexpected contribution of Cas9 to the development of viral resistance is facilitated by some indels that are not deleterious for viral replication, but that are refractory to recognition by the same sgRNA as a result of changing the target DNA sequences. This observation illustrates two opposite outcomes of Cas9/sgRNA action, i.e., inactivation of HIV-1 and acceleration of viral escape, thereby potentially limiting the use of Cas9/sgRNA in HIV-1 therapy.

  6. Celastrol Inhibits Tat-mediated Human Immunodeficiency Virus (HIV) Transcription and Replication

    PubMed Central

    Narayan, Vivek; Kodihalli, Ravindra C.; Chiaro, Chris; Cary, Daniele; Aggarwal, Bharat B.; Henderson, Andrew J.; Prabhu, K. Sandeep

    2011-01-01

    Current drugs used for anti-retroviral therapy against HIV have a narrow spectrum of activity, and more often have associated toxicities and severe side effects in addition to developing resistance. Thus, there is a need to develop new therapeutic strategies against HIV/AIDS to complement the already existing ones. Surprisingly, Tat, an early virus encoded protein required for the efficient transcription of the HIV genome, has not been developed as a target for small molecular therapeutics. We have previously described the ability of an endogenous Michael acceptor electrophile (MAE), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), to inhibit Tat-dependent transcription, by targeting its cysteine (Cys) rich domain. In an effort to identify other MAEs possessing inhibitory activity against HIV-1 Tat, we tested a collection of plant-derived compounds with electrophilic properties, including curcumin, rosmarinic acid, and gambogic acid, for their ability to inhibit Tat. Celastrol (Cel), a triterpenoid MAE isolated from T. wilfordii, exhibited the highest inhibitory activity against Tat. Using biochemical techniques, we demonstrate that Cel by covalently modifying the cysteine thiols inhibits Tat transactivation function. Using circular dichroism (CD) spectroscopy, we show that alklylation of Tat brought about a change in the secondary structure of Tat, which inhibited the transcription elongation of the HIV proviral genome by effecting mechanisms other than Tat-TAR interaction. Our results demonstrate the underlying mechanism of anti-retroviral activity of the plant-derived MAEs, and suggest that Cel could serve as a lead compound to develop novel anti-viral therapeutics. PMID:21763500

  7. HIV-1 replication.

    PubMed

    Freed, E O

    2001-11-01

    In general terms, the replication cycle of lentiviruses, including HIV-1, closely resembles that of other retroviruses. There are, however, a number of unique aspects of HIV replication; for example, the HIVs and SIVs target receptors and coreceptors distinct from those used by other retroviruses. Lentiviruses encode a number of regulatory and accessory proteins not encoded by the genomes of the prototypical "simple" retroviruses. Of particular interest from the gene therapy perspective, lentiviruses possess the ability to productively infect some types of non-dividing cells. This chapter, while reiterating certain points discussed in Chapter 1, will attempt to focus on issues unique to HIV-1 replication. The HIV-1 genome encodes the major structural and non-structural proteins common to all replication-competent retroviruses (Fig. 1, and Chapter 1). From the 5'- to 3'-ends of the genome are found the gag (for group-specific antigen), pol (for polymerase), and env (for envelope glycoprotein) genes. The gag gene encodes a polyprotein precursor whose name, Pr55Gag, is based on its molecular weight. Pr55Gag is cleaved by the viral protease (PR) to the mature Gag proteins matrix (also known as MA or p17), capsid (CA or p24), nucleocapsid (NC or p7), and p6. Two spacer peptides, p2 and p1, are also generated upon Pr55Gag processing. The pol-encoded enzymes are initially synthesized as part of a large polyprotein precursor, Pr160GagPol, whose synthesis results from a rare frameshifting event during Pr55Gag translation. The individual pol-encoded enzymes, PR, reverse transcriptase (RT), and integrase (IN), are cleaved from Pr160GagPol by the viral PR. The envelope (Env) glycoproteins are also synthesized as a polyprotein precursor (Fig. 1). Unlike the Gag and Pol precursors, which are cleaved by the viral PR, the Env precursor, known as gp160, is processed by a cellular protease during Env trafficking to the cell surface, gp160 processing results in the generation of the

  8. Persistent HIV-1 replication during antiretroviral therapy

    PubMed Central

    Martinez-Picado, Javier; Deeks, Steven G.

    2016-01-01

    Purpose of review The present review will highlight some of the recent findings regarding the capacity of HIV-1 to replicate during antiretroviral therapy (ART). Recent findings Although ART is highly effective at inhibiting HIV replication, it is not curative. Several mechanisms contribute to HIV persistence during ART, including HIV latency, immune dysfunction, and perhaps persistent low-level spread of the virus to uninfected cells (replication). The success in curing HIV will depend on efficiently targeting these three aspects. The degree to which HIV replicates during ART remains controversial. Most studies have failed to find any evidence of HIV evolution in blood, even with samples collected over many years, although a recent very intensive study of three individuals suggested that the virus population does shift, at least during the first few months of therapy. Stronger but still not definitive evidence for replication comes from a series of studies in which standard regimens were intensified with an integration inhibitor, resulting in changes in episomal DNA (blood) and cell-associated RNA (tissue). Limited drug penetration within tissues and the presence of immune sanctuaries have been argued as potential mechanisms allowing HIV to spread during ART. Mathematical models suggest that HIV replication and evolution is possible even without the selection of fully drug-resistant variants. As persistent HIV replication could have clinical consequences and might limit the efficacy of curative interventions, determining if HIV replicates during ART and why, should remain a key focus of the HIV research community. Summary Residual viral replication likely persists in lymphoid tissues, at least in a subset of individuals. Abnormal levels of immune activation might contribute to sustain virus replication. PMID:27078619

  9. Stem cell gene therapy for HIV: strategies to inhibit viral entry and replication.

    PubMed

    DiGiusto, David L

    2015-03-01

    Since the demonstration of a cure of an HIV+ patient with an allogeneic stem cell transplant using naturally HIV-resistant cells, significant interest in creating similar autologous products has fueled the development of a variety of "cell engineering" approaches to stem cell therapy for HIV. Among the more well-studied strategies is the inhibition of viral entry through disruption of expression of viral co-receptors or through competitive inhibitors of viral fusion with the cell membrane. Preclinical evaluation of these approaches often starts in vitro but ultimately is tested in animal models prior to clinical implementation. In this review, we trace the development of several key approaches (meganucleases, short hairpin RNA (shRNA), and fusion inhibitors) to modification of hematopoietic stem cells designed to impart resistance to HIV to their T-cell and monocytic progeny. The basic evolution of technologies through in vitro and in vivo testing is discussed as well as the pros and cons of each approach and how the addition of postentry inhibitors may enhance the overall antiviral efficacy of these approaches.

  10. Optimization of a Class of Tryptophan Dendrimers That Inhibit HIV Replication Leads to a Selective, Specific, and Low-Nanomolar Inhibitor of Clinical Isolates of Enterovirus A71

    PubMed Central

    Rivero-Buceta, Eva; Sun, Liang; Martínez-Gualda, Belén; Doyagüez, Elisa G.; Donckers, Kim; Quesada, Ernesto; Camarasa, María-José; Delang, Leen; Leyssen, Pieter

    2016-01-01

    Tryptophan dendrimers that inhibit HIV replication by binding to the HIV envelope glycoproteins gp120 and gp41 have unexpectedly also proven to be potent, specific, and selective inhibitors of the replication of the unrelated enterovirus A71. Dendrimer 12, a consensus compound that was synthesized on the basis of the structure-activity relationship analysis of this series, is 3-fold more potent against the BrCr lab strain and, surprisingly, inhibits a large panel of clinical isolates in the low-nanomolar/high-picomolar range. PMID:27246775

  11. Lipid-based delivery of combinations of antisense oligodeoxynucleotides for the in vitro inhibition of HIV-1 replication.

    PubMed

    Lavigne, C; Yelle, J; Sauvé, G; Thierry, A G

    2001-01-01

    We evaluated a new approach to AIDS therapy by using combinations of oligodeoxynucleotides (ODNs), delivered with a lipid-based carrier system, that target different HIV viral genome sites. We identified some of the factors that seem to influence the effectiveness of a combination strategy in cell cultures including ODN concentrations, type of infection (acute vs chronic), backbone modification of the ODN, and the number of sequences. When delivered by the DLS carrier system, some advantages of using a combination of ODNs over treatment with only one ODN could be observed in acute infection assays but not in the chronic infection model. These results suggest that in the acute infection model, the 3 different antisense ODNs in the "cocktail" might block an early step of virus replication by combined inhibitory effects. Various combinations of phosphorothioate-modified (PS) and unmodified oligonucleotides delivered by the DLS system were compared for their antiviral activity in a long-term acute assay using HIV-1 (IIIB strain)-infected MOLT-3 cells. The most effective combination had 3 phosphorothioate antisense ODNs: Srev, SDIS, and SPac (>99% inhibition at 100 pM). However, the additive effect determined when using ODN combinations was rather low, revealing the high level of nonsequence specificity in HIV-1 cell culture models. Data illustrated the high sequence nonspecific activity of ODNs, especially when comparing activity of antisense ODNs with activity of random control sequence ODNs. The latter exhibited an inhibitory effect similar to that of antisense ODNs under our experimental conditions. Nevertheless, we demonstrated that it is possible to achieve high anti-HIV activity by using, in combination, picomolar range concentrations of antisense oligonucleotides complexed to a lipid-based carrier system such as the DLS system, without increasing cell toxicity.

  12. Hyperthermia Stimulates HIV-1 Replication

    PubMed Central

    Roesch, Ferdinand; Meziane, Oussama; Kula, Anna; Nisole, Sébastien; Porrot, Françoise; Anderson, Ian; Mammano, Fabrizio; Fassati, Ariberto; Marcello, Alessandro; Benkirane, Monsef; Schwartz, Olivier

    2012-01-01

    HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42–45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38–40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity. PMID:22807676

  13. Consistent inhibition of HIV-1 replication in CD4+ T cells by acyclovir without detection of human herpesviruses.

    PubMed

    McMahon, Moira A; Parsons, Teresa L; Shen, Lin; Siliciano, Janet D; Siliciano, Robert F

    2011-05-01

    Acyclovir, a nucleoside analog, is thought to be specific for the human herpesviruses because it requires a virally encoded enzyme to phosphorylate it to acyclovir monophosphate. Recently, acyclovir triphosphate was shown to be a direct inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Here, we showed that acyclovir is an inhibitor of HIV-1 replication in CD4(+) T cells from cord blood that have undetectable levels of the eight human herpesviruses. Additionally, acyclovir phosphates were detected by reverse-phase-high performance liquid chromatography (RP-HPLC) and quantified in a primer extension assay from cord blood. The data support acyclovir as an inhibitor of HIV-1 replication in herpesvirus-negative cells.

  14. Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

    PubMed Central

    Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

    2010-01-01

    Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

  15. Oligoclonal CD8 lymphocytes from persons with asymptomatic human immunodeficiency virus (HIV) type 1 infection inhibit HIV-1 replication.

    PubMed

    Toso, J F; Chen, C H; Mohr, J R; Piglia, L; Oei, C; Ferrari, G; Greenberg, M L; Weinhold, K J

    1995-10-01

    CD8 lymphocytes from asymptomatic human immunodeficiency virus (HIV) type 1-infected patients can suppress virus production from infected CD4 cells. Suppressive activity is separate and distinct from cytotoxic T lymphocyte (CTL) reactivities and is likely mediated by a soluble factor(s). The majority of HIV-1 suppression studies have been done in the context of bulk CD8 cell cultures. In this study, viral suppression was characterized by clonal populations of CD8 cells derived from HIV-1-infected patients. Most of the suppressive clones were devoid of detectable CTL reactivity against env-, gag-, pol-, and nef-expressing targets. Among the suppressive clones derived from an individual patient, a marked heterogeneity was evident with respect to phenotypic markers, cytokine production, and T cell receptor V beta expression. These results suggest that noncytolytic virus suppression is oligoclonal in nature. Clones provide tools for future studies aimed at understanding the mechanism of suppression and identifying the suppressive factor.

  16. Efficient Inhibition of HIV Replication in the Gastrointestinal and Female Reproductive Tracts of Humanized BLT Mice by EFdA

    PubMed Central

    Shanmugasundaram, Uma; Kovarova, Martina; Ho, Phong T.; Schramm, Nathaniel; Wahl, Angela; Parniak, Michael A.; Garcia, J. Victor

    2016-01-01

    Background The nucleoside reverse transcriptase inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) in preclinical development exhibits improved safety and antiviral activity profiles with minimal drug resistance compared to approved NRTIs. However, the systemic antiviral efficacy of EFdA has not been fully evaluated. In this study, we utilized bone marrow/liver/thymus (BLT) humanized mice to investigate the systemic effect of EFdA treatment on HIV replication and CD4+ T cell depletion in the peripheral blood (PB) and tissues. In particular, we performed a comprehensive analysis of the female reproductive tract (FRT) and gastrointestinal (GI) tract, major sites of transmission, viral replication, and CD4+ T cell depletion and where some current antiretroviral drugs have a sub-optimal effect. Results EFdA treatment resulted in reduction of HIV-RNA in PB to undetectable levels in the majority of treated mice by 3 weeks post-treatment. HIV-RNA levels in cervicovaginal lavage of EFdA-treated BLT mice also declined to undetectable levels demonstrating strong penetration of EFdA into the FRT. Our results also demonstrate a strong systemic suppression of HIV replication in all tissues analyzed. In particular, we observed more than a 2-log difference in HIV-RNA levels in the GI tract and FRT of EFdA-treated BLT mice compared to untreated HIV-infected control mice. In addition, HIV-RNA was also significantly lower in the lymph nodes, liver, lung, spleen of EFdA-treated BLT mice compared to untreated HIV-infected control mice. Furthermore, EFdA treatment prevented the depletion of CD4+ T cells in the PB, mucosal tissues and lymphoid tissues. Conclusion Our findings indicate that EFdA is highly effective in controlling viral replication and preserving CD4+ T cells in particular with high efficiency in the GI and FRT tract. Thus, EFdA represents a strong potential candidate for further development as a part of antiretroviral therapy regimens. PMID:27438728

  17. HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

    PubMed Central

    Chaudhary, Priyanka; Khan, Sohrab Zafar; Rawat, Pratima; Augustine, Tracy; Raynes, Deborah A.; Guerriero, Vince; Mitra, Debashis

    2016-01-01

    HIV-1 efficiently hijacks host cellular machinery and exploits a plethora of host–viral interactions for its successful survival. Identifying host factors that affect susceptibility or resistance to HIV-1 may offer a promising therapeutic strategy against HIV-1. Previously, we have reported that heat shock proteins, HSP40 and HSP70 reciprocally regulate HIV-1 gene-expression and replication. In the present study, we have identified HSP70 binding protein 1 (HspBP1) as a host-intrinsic inhibitor of HIV-1. HspBP1 level was found to be significantly down modulated during HIV-1 infection and virus production inversely co-related with HspBP1 expression. Our results further demonstrate that HspBP1 inhibits HIV-1 long terminal repeat (LTR) promoter activity. Gel shift and chromatin immunoprecipitation assays revealed that HspBP1 was recruited on HIV-1 LTR at NF-κB enhancer region (κB sites). The binding of HspBP1 to κB sites obliterates the binding of NF-κB hetero-dimer (p50/p65) to the same region, leading to repression in NF-κB mediated activation of LTR-driven gene-expression. HspBP1 also plays an inhibitory role in the reactivation of latently infected cells, corroborating its repressive effect on NF-κB pathway. Thus, our results clearly show that HspBP1 acts as an endogenous negative regulator of HIV-1 gene-expression and replication by suppressing NF-κB-mediated activation of viral transcription. PMID:26538602

  18. C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir

    PubMed Central

    Aoki, Manabu; Hayashi, Hironori; Yedidi, Ravikiran S.; Martyr, Cuthbert D.; Takamatsu, Yuki; Aoki-Ogata, Hiromi; Nakamura, Teruya; Nakata, Hirotomo; Das, Debananda; Yamagata, Yuriko; Ghosh, Arun K.

    2015-01-01

    ABSTRACT We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1WT), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRVRP51); the three compounds remained active against HIV-1DRVRP51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR. IMPORTANCE Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRVR) has recently been observed in vivo and in

  19. Comparative Analysis of the Capacity of Elite Suppressor CD4+ and CD8+ T Cells To Inhibit HIV-1 Replication in Monocyte-Derived Macrophages

    PubMed Central

    Walker-Sperling, Victoria E. K.; Buckheit, Robert W.

    2014-01-01

    ABSTRACT Elite controllers or suppressors (ESs) are HIV-1-infected individuals who are able to maintain viral loads below the limit of detection of clinical assays without antiretroviral therapy. The mechanisms of virologic control are not fully understood, but ESs have been shown to have a more effective CD8+ T cell response to infected CD4+ T cells than chronic progressors (CPs). While macrophages are another cell type productively infected by HIV-1, few studies have examined the ability of primary effector T cells to suppress HIV-1 replication in these target cells. Here, we compared the ability of unstimulated primary CD4+ and CD8+ effector T cells to suppress viral replication in monocyte-derived macrophages (MDMs) in ESs and CPs. While CD4+ effector T cells were capable of inhibiting viral replication in MDMs, the magnitude of this response was not significantly different between ESs and CPs. In contrast, the CD8+ T cells from ESs were significantly more effective than those from CPs at inhibiting viral replication in MDMs. The CD4+ T cell response was partially mediated by soluble factors, while the CD8+ T cell response required cell-to-cell interaction. Our results suggest that the individual contributions of various effector cells should be considered in rational vaccine design and in ongoing eradication efforts. IMPORTANCE Elite suppressors are individuals capable of maintaining low-level viremia in HIV-1 infection without antiretroviral drugs. Their T cell responses have been implicated in eliminating infected CD4+ T cells, and as such, elite suppressors may represent a model of a functional cure of HIV-1 infection. Here, we sought to determine whether the suppressive T cell responses against infected CD4+ T cells also apply to infected macrophages by comparing the responses of elite suppressors and HIV-1-positive individuals on highly active antiretroviral therapy (HAART). Our results show that the CD8+ cells but not CD4+ T cells from elite suppressors

  20. Extracts from Acacia catechu suppress HIV-1 replication by inhibiting the activities of the viral protease and Tat

    PubMed Central

    2013-01-01

    Background Acacia catechu (Mimosa family) stem bark extracts have been used traditionally as a dietary supplement as well as a folk medicine given its reported anti-inflammatory, immunomodulatory, hepatoprotective, antioxidant, anti-microbial and anti-tumor activities. The present study was undertaken to evaluate the anti-HIV-1 activity of the extracts from stem bark of A. catechu. Methods The aqueous and 50% ethanolic extracts of A. catechu stem bark were prepared and 50% ethanolic extract was further fractioned by successively partitioning with petroleum ether, chloroform and n-butanol. All the extracts and fractions were evaluated for cytotoxicity and anti-HIV-1 activity using different in vitro assays. The active n-butanol fraction was evaluated for its inhibition against HIV-1 reverse transcriptase, integrase, protease, pro-viral genome integration and viral Tat protein mediated transactivation. The effect of n-butanol fraction on the induction of pro-inflammatory cytokines secretion in Vk2/E6E7 cells and transepithelial resistance in Caco-2 and HEC-1A cells was investigated. Results The aqueous and 50% ethanolic extracts of A. catechu showed IC50 values of 1.8 ± 0.18 μg/ml and 3.6 ± 0.31 μg/ml, respectively in cell-free virus based assay using TZM-bl cells and HIV-1NL4.3 (X-4 tropic). In the above assay, n-butanol fraction exhibited anti-HIV-1 activity with an IC50 of 1.7 ± 0.12 μg/ml. The n-butanol fraction showed a dose-dependent inhibition against HIV-1NL4.3 infection of the peripheral blood lymphocytes and against HIV-1BaL(R-5-tropic) as well as two different primary viral isolates of HIV-1 infection of TZM-bl cells. The n-butanol fraction demonstrates a potent inhibitory activity against the viral protease (IC50 = 12.9 μg/ml), but not reverse transcriptase or integrase. Further, in Alu-PCR no effect on viral integration was observed. The n-butanol fraction interfered with the Tat-mediated Long Terminal Repeat transactivation in

  1. A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread.

    PubMed Central

    Dropulić, B; Hĕrmánková, M; Pitha, P M

    1996-01-01

    Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8855316

  2. HIV-1 Vpr Inhibits Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by Inducing MicroRNA miR-942-5p and Activating NF-κB Signaling

    PubMed Central

    Yan, Qin; Shen, Chenyou; Qin, Jie; Li, Wan; Hu, Minmin; Lu, Hongmei; Qin, Di; Zhu, Jianzhong; Gao, Shou-Jiang

    2016-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) infection is required for the development of several AIDS-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The high incidence of AIDS-KS has been ascribed to the interaction of KSHV and HIV-1. We have previously shown that HIV-1-secreted proteins Tat and Nef regulate the KSHV life cycle and synergize with KSHV oncogenes to promote angiogenesis and tumorigenesis. Here, we examined the regulation of KSHV latency by HIV-1 viral protein R (Vpr). We found that soluble Vpr inhibits the expression of KSHV lytic transcripts and proteins, as well as viral particle production by activating NF-κB signaling following internalization into PEL cells. By analyzing the expression profiles of microRNAs combined with target search by bioinformatics and luciferase reporter analyses, we identified a Vpr-upregulated cellular microRNA (miRNA), miR-942-5p, that directly targeted IκBα. Suppression of miR-942-5p relieved the expression of IκBα and reduced Vpr inhibition of KSHV lytic replication, while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV lytic replication. Our findings collectively illustrate that, by activating NF-κB signaling through upregulating a cellular miRNA to target IκBα, internalized HIV-1 Vpr inhibits KSHV lytic replication. These results have demonstrated an essential role of Vpr in the life cycle of KSHV. IMPORTANCE Coinfection by HIV-1 promotes the aggressive growth of Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). In this study, we have shown that soluble HIV-1 Vpr inhibits KSHV lytic replication by activating NF-κB signaling following internalization into PEL cells. Mechanistic studies revealed that a cellular microRNA upregulated by Vpr, miR-942-5p, directly targeted IκBα. Suppression of miR-942-5p relieved IκBα expression and reduced Vpr inhibition

  3. 3-Phenylcoumarins as inhibitors of HIV-1 replication.

    PubMed

    Olmedo, Dionisio; Sancho, Rocío; Bedoya, Luis M; López-Pérez, José L; Del Olmo, Esther; Muñoz, Eduardo; Alcamí, José; Gupta, Mahabir P; San Feliciano, Arturo

    2012-08-02

    We have synthesized fourteen 3-phenylcoumarin derivatives and evaluated their anti-HIV activity. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase gene as reporter. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the HIV-LTR and Tat protein. Six compounds displayed NF-κB inhibition, four resulted Tat antagonists and three of them showed both activities. Three compounds inhibited HIV replication with IC₅₀ values < 25 µM. The antiviral effect of the 4-hydroxycoumarin derivative 19 correlates with its specific inhibition of Tat functions, while compound 8, 3-(2-chlorophenyl)coumarin, seems to act through a mechanism unrelated to the molecular targets considered in this research.

  4. Possible applications for replicating HIV 1 vectors

    PubMed Central

    Das, Atze T; Jeeninga, Rienk E; Berkhout, Ben

    2010-01-01

    Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication competent virus has been minimized. Nevertheless, there may be certain applications for replication competent HIV based vector systems, and we will review our activities in this particular field. This includes the generation of a conditionally replicating HIV 1 variant as a safe live attenuated virus vaccine, the construction of mini HIV variants as cancer selective viruses for virotherapy against leukemia, and the use of a conditionally live anti HIV gene therapy vector. Although safety concerns will undoubtedly remain for the use of replication competent HIV based vector systems, some of the results in cell culture systems are very promising and warrant further testing in appropriate animal models. PMID:20582153

  5. Possible applications for replicating HIV 1 vectors.

    PubMed

    Das, Atze T; Jeeninga, Rienk E; Berkhout, Ben

    2010-05-01

    Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication competent virus has been minimized. Nevertheless, there may be certain applications for replication competent HIV based vector systems, and we will review our activities in this particular field. This includes the generation of a conditionally replicating HIV 1 variant as a safe live attenuated virus vaccine, the construction of mini HIV variants as cancer selective viruses for virotherapy against leukemia, and the use of a conditionally live anti HIV gene therapy vector. Although safety concerns will undoubtedly remain for the use of replication competent HIV based vector systems, some of the results in cell culture systems are very promising and warrant further testing in appropriate animal models.

  6. In Vitro Drug Combination Studies of Letermovir (AIC246, MK-8228) with Approved Anti-Human Cytomegalovirus (HCMV) and Anti-HIV Compounds in Inhibition of HCMV and HIV Replication

    PubMed Central

    Wildum, Steffen; Zimmermann, Holger

    2015-01-01

    Despite modern prevention and treatment strategies, human cytomegalovirus (HCMV) remains a common opportunistic pathogen associated with serious morbidity and mortality in immunocompromised individuals, such as transplant recipients and AIDS patients. All drugs currently licensed for the treatment of HCMV infection target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Letermovir (AIC246, MK-8228) is a new anti-HCMV agent in clinical development that acts via a novel mode of action and has demonstrated anti-HCMV activity in vitro and in vivo. For the future, drug combination therapies, including letermovir, might be indicated under special medical conditions, such as the emergence of multidrug-resistant virus strains in transplant recipients or in HCMV-HIV-coinfected patients. Accordingly, knowledge of the compatibility of letermovir with other HCMV or HIV antivirals is of medical importance. Here, we evaluated the inhibition of HCMV replication by letermovir in combination with all currently approved HCMV antivirals using cell culture checkerboard assays. In addition, the effects of letermovir on the antiviral activities of selected HIV drugs, and vice versa, were analyzed. Using two different mathematical techniques to analyze the experimental data, (i) additive effects were observed for the combination of letermovir with anti-HCMV drugs and (ii) no interaction was found between letermovir and anti-HIV drugs. Since none of the tested drug combinations significantly antagonized letermovir efficacy (or vice versa), our findings suggest that letermovir may offer the potential for combination therapy with the tested HCMV and HIV drugs. PMID:25779572

  7. The Cellular TAR RNA Binding Protein, TRBP, Promotes HIV-1 Replication Primarily by Inhibiting the Activation of Double-Stranded RNA-Dependent Kinase PKR▿

    PubMed Central

    Sanghvi, Viraj R.; Steel, Laura F.

    2011-01-01

    The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5′-cap or by activating the dsRNA-dependent kinase, PKR. Several mechanisms by which TRBP can facilitate translation of viral transcripts have been proposed, including the binding and unwinding of TAR and the suppression of PKR activation. Further, TRBP has been identified as a cofactor of Dicer in the processing of microRNAs (miRNAs), and sequestration of TRBP by TAR in infected cells has been proposed as a viral countermeasure to potential host cell RNA interference-based antiviral activities. Here, we have addressed the relative importance of these various roles for TRBP in HIV-1 replication. Using Jurkat T cells, primary human CD4+ T cells, and additional cultured cell lines, we show that depletion of TRBP has no effect on viral replication when PKR activation is otherwise blocked. Moreover, the presence of TAR-containing mRNAs does not affect the efficacy of cellular miRNA silencing pathways. These results establish that TRBP, when expressed at physiological levels, promotes HIV-1 replication mainly by suppressing the PKR-mediated antiviral response, while its contribution to HIV-1 replication through PKR-independent pathways is minimal. PMID:21937648

  8. Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides.

    PubMed Central

    Goodchild, J; Agrawal, S; Civeira, M P; Sarin, P S; Sun, D; Zamecnik, P C

    1988-01-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs. PMID:3041414

  9. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

    NASA Astrophysics Data System (ADS)

    Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

    1988-08-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

  10. APOBEC4 Enhances the Replication of HIV-1

    PubMed Central

    Hofmann, Henning; Hanschmann, Kay-Martin; Mühlebach, Michael D.; Schumann, Gerald G.; König, Renate; Cichutek, Klaus; Häussinger, Dieter; Münk, Carsten

    2016-01-01

    APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral LTR. A4 did not show detectable cytidine deamination activity in vitro and weakly interacted with single-stranded DNA. The presence of A4 in virus producer cells enhanced HIV-1 replication by transiently transfected A4 or stably expressed A4 in HIV-susceptible cells. APOBEC4 was capable of similarly enhancing transcription from a broad spectrum of promoters, regardless of whether they were viral or mammalian. We hypothesize that A4 may have a natural role in modulating host promoters or endogenous LTR promoters. PMID:27249646

  11. Minichromosome replication in vitro: inhibition of re-replication by replicatively assembled nucleosomes.

    PubMed

    Krude, T; Knippers, R

    1994-08-19

    Single-stranded circular DNA, containing the SV40 origin sequence, was used as a template for complementary DNA strand synthesis in cytosolic extracts from HeLa cells. In the presence of the replication-dependent chromatin assembly factor CAF-1, defined numbers of nucleosomes were assembled during complementary DNA strand synthesis. These minichromosomes were then induced to semiconservatively replicate by the addition of the SV40 initiator protein T antigen (re-replication). The results indicate that re-replication of minichromosomes appears to be inhibited by two independent mechanisms. One acts at the initiation of minichromosome re-replication, and the other affects replicative chain elongation. To directly demonstrate the inhibitory effect of replicatively assembled nucleosomes, two types of minichromosomes were prepared: (i) post-replicative minichromosomes were assembled in a reaction coupled to replication as above; (ii) pre-replicative minichromosomes were assembled independently of replication on double-stranded DNA. Both types of minichromosomes were used as templates for DNA replication under identical conditions. Replicative fork movement was found to be impeded only on post-replicative minichromosome templates. In contrast, pre-replicative minichromosomes allowed one unconstrained replication cycle, but re-replication was inhibited due to a block in fork movement. Thus, replicatively assembled chromatin may have a profound influence on the re-replication of DNA.

  12. Ring Expanded Nucleoside Analogues Inhibit RNA Helicase and Intracellular Human Immunodeficiency virus type 1 Replication

    PubMed Central

    Yedavalli, Venkat S.R.K; Zhang, Ning; Cai, Hongyi; Zhang, Peng; Starost, Matthew F.; Hosmane, Ramachandra S.; Jeang, Kuan-Teh

    2008-01-01

    A series of ring expanded nucleoside (REN) analogues were synthesized and screened for inhibition of cellular RNA helicase activity and human immunodeficiency virus type 1 (HIV-1) replication. We identified two compounds 1 and 2 that inhibited the ATP dependent activity of human RNA helicase DDX3. Compounds 1 and 2 also suppressed HIV-1 replication in T cells and monocyte-derived macrophages. Neither compound at therapeutic doses was significantly toxic in ex vivo cell culture or in vivo in mice. Our findings provide proof-of-concept that a cellular factor, an RNA helicase, could be targeted for inhibiting HIV-1 replication. PMID:18680273

  13. EASY-HIT: HIV Full-Replication Technology for Broad Discovery of Multiple Classes of HIV Inhibitors▿ †

    PubMed Central

    Kremb, Stephan; Helfer, Markus; Heller, Werner; Hoffmann, Dieter; Wolff, Horst; Kleinschmidt, Andrea; Cepok, Sabine; Hemmer, Bernhard; Durner, Jörg; Brack-Werner, Ruth

    2010-01-01

    HIV replication assays are important tools for HIV drug discovery efforts. Here, we present a full HIV replication system (EASY-HIT) for the identification and analysis of HIV inhibitors. This technology is based on adherently growing HIV-susceptible cells, with a stable fluorescent reporter gene activated by HIV Tat and Rev. A fluorescence-based assay was designed that measures HIV infection by two parameters relating to the early and the late phases of HIV replication, respectively. Validation of the assay with a panel of nine reference inhibitors yielded effective inhibitory concentrations consistent with published data and allowed discrimination between inhibitors of early and late phases of HIV replication. Finer resolution of the effects of reference drugs on different steps of HIV replication was achieved in secondary time-of-addition assays. The EASY-HIT assay yielded high Z′ scores (>0.9) and signal stabilities, confirming its robustness. Screening of the LOPAC1280 library identified 10 compounds (0.8%), of which eight were known to inhibit HIV, validating the suitability of this assay for screening applications. Studies evaluating anti-HIV activities of natural products with the EASY-HIT technology led to the identification of three novel inhibitory compounds that apparently act at different steps of HIV-1 replication. Furthermore, we demonstrate successful evaluation of plant extracts for HIV-inhibitory activities, suggesting application of this technology for the surveillance of biological extracts with anti-HIV activities. We conclude that the EASY-HIT technology is a versatile tool for the discovery and characterization of HIV inhibitors. PMID:20876377

  14. EASY-HIT: HIV full-replication technology for broad discovery of multiple classes of HIV inhibitors.

    PubMed

    Kremb, Stephan; Helfer, Markus; Heller, Werner; Hoffmann, Dieter; Wolff, Horst; Kleinschmidt, Andrea; Cepok, Sabine; Hemmer, Bernhard; Durner, Jörg; Brack-Werner, Ruth

    2010-12-01

    HIV replication assays are important tools for HIV drug discovery efforts. Here, we present a full HIV replication system (EASY-HIT) for the identification and analysis of HIV inhibitors. This technology is based on adherently growing HIV-susceptible cells, with a stable fluorescent reporter gene activated by HIV Tat and Rev. A fluorescence-based assay was designed that measures HIV infection by two parameters relating to the early and the late phases of HIV replication, respectively. Validation of the assay with a panel of nine reference inhibitors yielded effective inhibitory concentrations consistent with published data and allowed discrimination between inhibitors of early and late phases of HIV replication. Finer resolution of the effects of reference drugs on different steps of HIV replication was achieved in secondary time-of-addition assays. The EASY-HIT assay yielded high Z' scores (>0.9) and signal stabilities, confirming its robustness. Screening of the LOPAC(1280) library identified 10 compounds (0.8%), of which eight were known to inhibit HIV, validating the suitability of this assay for screening applications. Studies evaluating anti-HIV activities of natural products with the EASY-HIT technology led to the identification of three novel inhibitory compounds that apparently act at different steps of HIV-1 replication. Furthermore, we demonstrate successful evaluation of plant extracts for HIV-inhibitory activities, suggesting application of this technology for the surveillance of biological extracts with anti-HIV activities. We conclude that the EASY-HIT technology is a versatile tool for the discovery and characterization of HIV inhibitors.

  15. Suppression of HIV-1 replication by microRNA effectors

    PubMed Central

    Chable-Bessia, Christine; Meziane, Oussama; Latreille, Daniel; Triboulet, Robinson; Zamborlini, Alessia; Wagschal, Alexandre; Jacquet, Jean-Marc; Reynes, Jacques; Levy, Yves; Saib, Ali; Bennasser, Yamina; Benkirane, Monsef

    2009-01-01

    The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART. PMID:19272132

  16. Adenovirus Vectors Block Human Immunodeficiency Virus–1 Replication in Human Alveolar Macrophages by Inhibition of the Long Terminal Repeat

    PubMed Central

    Kaner, Robert J.; Santiago, Francisco; Rahaghi, Franck; Michaels, Elizabeth; Moore, John P.; Crystal, Ronald G.

    2010-01-01

    Heterologous viruses may transactivate or suppress human immunodeficiency virus (HIV)–1 replication. An adenovirus type 5 gene transfer vector (Ad5) HIV-1 vaccine was recently evaluated in a clinical trial, without efficacy. In this context, it is relevant to ask what effect Ad vectors have on HIV-1 replication, particularly in cells that are part of the innate immune system. Infection of HIV-1–infected human alveolar macrophages (AMs) obtained from HIV-1+ individuals with an Ad vector containing no transgene (AdNull) resulted in dose-responsive inhibition of endogenous HIV-1 replication. HIV-1 replication in normal AMs infected with HIV-1 in vitro was inhibited by AdNull with a similar dose response. Ad reduced AM HIV-1 replication up to 14 days after HIV-1 infection. Fully HIV-1–infected AMs were treated with 3′-azido-3′-deoxythymidine, after which Ad infection still inhibited HIV-1 replication, suggesting a postentry step was affected. Substantial HIV-1 DNA was still produced after Ad infection, as quantified by TaqMan real-time PCR, suggesting that the replication block occurred after reverse transcription. AdNull blocked HIV-1 long terminal repeat (LTR) transcription, as assessed by an vesicular stomatitis virus G protein pseudotyped HIV-1 LTR luciferase construct. The formation of HIV-1 DNA integrated into the host chromosome was not inhibited by Ad, as quantified by a two-step TaqMan real-time PCR assay, implying a postintegration block to HIV-1 replication. These data indicate that Ad vectors are inhibitory to HIV-1 replication in human AMs based, in part, on their ability to inhibit LTR-driven transcription. PMID:19805482

  17. Iron chelators ICL670 and 311 inhibit HIV-1 transcription

    SciTech Connect

    Debebe, Zufan; Ammosova, Tatyana; Jerebtsova, Marina; Kurantsin-Mills, Joseph; Niu, Xiaomei; Charles, Sharroya; Richardson, Des R.; Ray, Patricio E.; Gordeuk, Victor R.; Nekhai, Sergei

    2007-10-25

    HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics.

  18. Inhibitory effect of aqueous dandelion extract on HIV-1 replication and reverse transcriptase activity

    PubMed Central

    2011-01-01

    Background Acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is an immunosuppressive disease that results in life-threatening opportunistic infections. The general problems in current therapy include the constant emergence of drug-resistant HIV strains, adverse side effects and the unavailability of treatments in developing countries. Natural products from herbs with the abilities to inhibit HIV-1 life cycle at different stages, have served as excellent sources of new anti-HIV-1 drugs. In this study, we aimed to investigate the anti-HIV-1 activity of aqueous dandelion extract. Methods The pseudotyped HIV-1 virus has been utilized to explore the anti-HIV-1 activity of dandelion, the level of HIV-1 replication was assessed by the percentage of GFP-positive cells. The inhibitory effect of the dandelion extract on reverse transcriptase activity was assessed by the reverse transcriptase assay kit. Results Compared to control values obtained from cells infected without treatment, the level of HIV-1 replication and reverse transcriptase activity were decreased in a dose-dependent manner. The data suggest that dandelion extract has a potent inhibitory activity against HIV-1 replication and reverse transcriptase activity. The identification of HIV-1 antiviral compounds from Taraxacum officinale should be pursued. Conclusions The dandelion extract showed strong activity against HIV-1 RT and inhibited both the HIV-1 vector and the hybrid-MoMuLV/MoMuSV retrovirus replication. These findings provide additional support for the potential therapeutic efficacy of Taraxacum officinale. Extracts from this plant may be regarded as another starting point for the development of an antiretroviral therapy with fewer side effects. PMID:22078030

  19. Effect of antiretroviral HIV therapy on hepatitis B virus replication and pathogenicity.

    PubMed

    Gürtler, Lutz G

    2014-01-01

    Coinfections with hepatitis B virus (HBV) and HIV are very frequent. Although HBV is a DNA virus, it replicates via reverse transcription like HIV. Structural similarities between the enzymatic pocket of the HBV DNA polymerase and HIV-1 reverse transcriptase are the basis that certain drugs inhibit both enzymes and thus the replication of both viruses. HBV components increase the pathogenic action of HIV and vice versa directly by certain proteins like HBsAg in the case of HBV and HIV-encoded Tat and Vpr and by disturbing the cytokine balance in affected cells. Antiretroviral therapy is highly beneficial for HIV/HBV-coinfected patients, but carries the risk of drug-induced resistance development and hepatotoxicity. Even with restoration of the immune capacity, signs of hepatic inflammation may develop even after 10 years of treatment.

  20. SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

    PubMed Central

    Amraoui, Sonia; di Nunzio, Francesca; Kieffer, Camille; Porrot, Françoise; Opp, Silvana; Diaz-Griffero, Felipe; Casartelli, Nicoletta

    2016-01-01

    ABSTRACT In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection. IMPORTANCE Cells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain

  1. Quantifying the Antiviral Effect of IFN on HIV-1 Replication in Cell Culture

    NASA Astrophysics Data System (ADS)

    Ikeda, Hiroki; Godinho-Santos, Ana; Rato, Sylvie; Vanwalscappel, Bénédicte; Clavel, François; Aihara, Kazuyuki; Iwami, Shingo; Mammano, Fabrizio

    2015-06-01

    Type-I interferons (IFNs) induce the expression of hundreds of cellular genes, some of which have direct antiviral activities. Although IFNs restrict different steps of HIV replication cycle, their dominant antiviral effect remains unclear. We first quantified the inhibition of HIV replication by IFN in tissue culture, using viruses with different tropism and growth kinetics. By combining experimental and mathematical analyses, we determined quantitative estimates for key parameters of HIV replication and inhibition, and demonstrate that IFN mainly inhibits de novo infection (33% and 47% for a X4- and a R5-strain, respectively), rather than virus production (15% and 6% for the X4 and R5 strains, respectively). This finding is in agreement with patient-derived data analyses.

  2. Mycobacterium xenopi multiplies within human macrophages and enhances HIV replication in vitro.

    PubMed

    Swords, W E; Guenthner, P C; Birkness, K A; Lal, R B; Dezzutti, C S; Quinn, F D

    2006-02-01

    Mycobacterium xenopi can cause opportunistic infections, particularly in persons infected with human immunodeficiency virus type 1 (HIV-1). The primary focus of this effort was to determine if M. xenopi isolates could survive and grow in human peripheral blood macrophage (MPhi), and if these isolates could promote the replication of HIV-1 in vitro. M. xenopi bacilli survived and replicated 10-fold within 48 h in human MPhi while avirulent Mycobacterium smegmatis, did not grow within the MPhi. M. xenopi bacilli when cultured with peripheral blood mononuclear cells enhanced HIV-1 replication 30- and 50-fold with the macrophage-tropic HIV-1(Ba-L) and 50- and 75-fold with T-cell-tropic strain HIV-1(LAI) by 6 days post-infection when compared to M. smegmatis. The enhanced HIV replication was associated with increased production of TNF-alpha. Partial inhibition of HIV-1 induction was observed using a neutralizing anti-TNF-alpha monoclonal antibody, pentoxifylline, and matrix metalloproteinase (MMP) inhibitor I. Similar mechanisms of pathogenesis among mycobacterial species may help elucidate better treatment approaches in HIV co-infected persons.

  3. Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication.

    PubMed

    Flores, O; Lee, G; Kessler, J; Miller, M; Schlief, W; Tomassini, J; Hazuda, D

    1999-06-22

    HIV-1 gene expression and viral replication require the viral transactivator protein Tat. The RNA polymerase II transcriptional elongation factor P-TEFb (cyclin-dependent kinase 9/cyclin T) is a cellular protein kinase that has recently been shown to be a key component of the Tat-transactivation process. For this report, we studied the requirement for P-TEFb in HIV-1 infection, and we now show that P-TEFb is both essential and limiting for HIV-1 replication. Attenuation of P-TEFb kinase activity either by expression of a dominant-negative cyclin-dependent kinase 9 transgene or through the use of small-molecule inhibitors suppresses HIV-1 gene expression and HIV-1 replication. Inhibition of HIV-1 replication is affected in a manner consistent with a direct and specific effect on P-TEFb and the known functional role of P-TEFb in Tat-activated transcription. Tat-activated expression of HIV-1 genes seems uniquely dependent on P-TEFb, as inhibition of P-TEFb activity and HIV-1 replication can be achieved without compromising cell viability or RNA polymerase II-dependent cellular gene transcription. Selective inhibition of the P-TEFb kinase may therefore provide a novel approach for developing chemotherapeutic agents against HIV-1.

  4. HIV Replication at Low Copy Number and its Correlation with the HIV Reservoir: A Clinical Perspective.

    PubMed

    Sarmati, Loredana; D'Ettorre, Gabriella; Parisi, Saverio Giuseppe; Andreoni, Massimo

    2015-01-01

    The efficacy of combination therapy (antiretroviral therapy--ARV) is demonstrated by the high rates of viral suppression achieved in most treated HIV patients. Whereas contemporary treatments may continuously suppress HIV replication, they do not eliminate the latent reservoir, which can reactivate HIV infection if ARV is discontinued. The persistence of HIV proviral DNA and infectious viruses in CD4+ T cells and others cells has long been considered a major obstacle in eradicating the HIV virus in treated patients. Moreover, recent studies have demonstrated the persistence of HIV replication at low copies in most patients on suppressive ARV. The source of this 'residual viraemia' and whether it declines over years of therapy remain unknown. Similarly, little is known regarding the biological relationships between the HIV reservoir and viral replication at low copies. The question of whether this 'residual viraemia' represents active replication or the release of non-productive virus from the reservoir has not been adequately resolved. From a clinical perspective, both the quantification of the HIV reservoir and the detection of low levels of replication in full-responder patients on prolonged ARV may provide important information regarding the effectiveness of treatment and the eradication of HIV. To date, the monitoring of these two parameters has been conducted only for research purposes; the routine use of standardised tests procedure is lacking. This review aims to assess the current data regarding the correlation between HIV replication at low copies and the HIV reservoir and to provide useful information for clinicians.

  5. Neoflavonoids as Inhibitors of HIV-1 Replication by Targeting the Tat and NF-κB Pathways.

    PubMed

    Olmedo, Dionisio A; López-Pérez, José Luis; Del Olmo, Esther; Bedoya, Luis M; Sancho, Rocío; Alcamí, José; Muñoz, Eduardo; Feliciano, Arturo San; Gupta, Mahabir P

    2017-02-19

    Twenty-eight neoflavonoids have been prepared and evaluated in vitro against HIV-1. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase reporter gene. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the HIV-LTR and Tat protein. Seven 4-phenylchromen-2-one derivatives showed HIV transcriptional inhibitory activity but only the phenylchrome-2-one 10 inhibited NF-κB and displayed anti-Tat activity simultaneously. Compounds 10, 14, and 25, inhibited HIV replication in both targets at concentrations <25 μM. The assays of these synthetic 4-phenylchromen-2-ones may aid in the investigation of some aspects of the anti-HIV activity of such compounds and could serve as a scaffold for designing better anti-HIV compounds, which may lead to a potential anti-HIV therapeutic drug.

  6. DYRK1A Controls HIV-1 Replication at a Transcriptional Level in an NFAT Dependent Manner

    PubMed Central

    Booiman, Thijs; Loukachov, Vladimir V.; van Dort, Karel A.; van ’t Wout, Angélique B.; Kootstra, Neeltje A.

    2015-01-01

    Background Transcription of the HIV-1 provirus is regulated by both viral and host proteins and is very important in the context of viral latency. In latently infected cells, viral gene expression is inhibited as a result of the sequestration of host transcription factors and epigenetic modifications. Results In our present study we analyzed the effect of host factor dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) on HIV-1 replication. We show that DYRK1A controls HIV-1 replication by regulating provirus transcription. Downregulation or inhibition of DYRK1A increased LTR-driven transcription and viral replication in cell lines and primary PBMC. Furthermore, inhibition of DYRK1A resulted in reactivation of latent HIV-1 provirus to a similar extent as two commonly used broad-spectrum HDAC inhibitors. We observed that DYRK1A regulates HIV-1 transcription via the Nuclear Factor of Activated T-cells (NFAT) by promoting its translocation from the nucleus to the cytoplasm. Therefore, inhibition of DYRK1A results in increased nuclear levels of NFAT and increased NFAT binding to the viral LTR and thus increasing viral transcription. Conclusions Our data indicate that host factor DYRK1A plays a role in the regulation of viral transcription and latency. Therefore, DYRK1A might be an attractive candidate for therapeutic strategies targeting the viral reservoir. PMID:26641855

  7. Antiviral Activity of Diterpene Esters on Chikungunya Virus and HIV Replication.

    PubMed

    Nothias-Scaglia, Louis-Félix; Pannecouque, Christophe; Renucci, Franck; Delang, Leen; Neyts, Johan; Roussi, Fanny; Costa, Jean; Leyssen, Pieter; Litaudon, Marc; Paolini, Julien

    2015-06-26

    Recently, new daphnane, tigliane, and jatrophane diterpenoids have been isolated from various Euphorbiaceae species, of which some have been shown to be potent inhibitors of chikungunya virus (CHIKV) replication. To further explore this type of compound, the antiviral activity of a series of 29 commercially available natural diterpenoids was evaluated. Phorbol-12,13-didecanoate (11) proved to be the most potent inhibitor, with an EC50 value of 6.0 ± 0.9 nM and a selectivity index (SI) of 686, which is in line with the previously reported anti-CHIKV potency for the structurally related 12-O-tetradecanoylphorbol-13-acetate (13). Most of the other compounds exhibited low to moderate activity, including an ingenane-type diterpene ester, compound 28, with an EC50 value of 1.2 ± 0.1 μM and SI = 6.4. Diterpene compounds are known also to inhibit HIV replication, so the antiviral activities of compounds 1-29 were evaluated also against HIV-1 and HIV-2. Tigliane- (4β-hydroxyphorbol analogues 10, 11, 13, 15, 16, and 18) and ingenane-type (27 and 28) diterpene esters were shown to inhibit HIV replication in vitro at the nanomolar level. A Pearson analysis performed with the anti-CHIKV and anti-HIV data sets demonstrated a linear relationship, which supported the hypothesis made that PKC may be an important target in CHIKV replication.

  8. Productive replication and evolution of HIV-1 in ferret cells.

    PubMed

    Fadel, Hind J; Saenz, Dyana T; Guevara, Rebekah; von Messling, Veronika; Peretz, Mary; Poeschla, Eric M

    2012-02-01

    A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1

  9. Persistent HIV-1 replication maintains the tissue reservoir during therapy

    PubMed Central

    Bedford, Trevor; Kim, Eun-Young; Archer, John; Pond, Sergei L. Kosakovsky; Chung, Yoon-Seok; Penugonda, Sudhir; Chipman, Jeffrey; Fletcher, Courtney V.; Schacker, Timothy W.; Malim, Michael H.; Rambaut, Andrew; Haase, Ashley T.; McLean, Angela R.; Wolinsky, Steven M.

    2015-01-01

    Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site of viral production, storage of viral particles in immune complexes, and viral persistence. Whilst combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. A spatial dynamic model of persistent viral replication and spread explains why the development of drug resistance is not a foregone conclusion under conditions where drug concentrations are insufficient to completely block virus replication. These data provide fresh insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and refill the viral reservoir despite potent antiretroviral therapy. PMID:26814962

  10. DNA Copy Number Control Through Inhibition of Replication Fork Progression

    PubMed Central

    Nordman, Jared T.; Kozhevnikova, Elena N.; Verrijzer, C. Peter; Pindyurin, Alexey V.; Andreyeva, Evgeniya N.; Shloma, Victor V.; Zhimulev, Igor F.; Orr-Weaver, Terry L.

    2014-01-01

    Summary Proper control of DNA replication is essential to ensure faithful transmission of genetic material and to prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass-spec identification of SUUR associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through inhibition of replication fork progression. PMID:25437540

  11. Comparison of antiviral activity of lambda-interferons against HIV replication in macrophages.

    PubMed

    Wang, Yizhong; Li, Jieliang; Wang, Xu; Zhou, Yu; Zhang, Ting; Ho, Wenzhe

    2015-03-01

    Lambda-interferons (IFN-λs) have been demonstrated as having the ability to inhibit HIV replication in macrophages. However, specific differences in signaling transduction and anti-HIV activity in macrophages between different IFN-λs are unclear. Here, we showed that although all 3 members of (IFN-λ1, λ2, and λ3) IFN-λ family induced the expression of a number of genes of janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway in monocyte-derived macrophages, IFN-λ1 or IFN-λ3 induced higher levels of antiviral IFN-stimulated genes (ISGs) expression than did IFN-λ2. In addition, IFN-λ1 or IFN-λ3 induced higher levels of several pattern recognition receptors (PPRs) than did IFN-λ2. Incubation of IFN-λs with HIV-infected macrophages showed that IFN-λ1 or IFN-λ3 is more potent in anti-HIV activity than IFN-λ2. We also showed that IFN-λ treatment before HIV infection was more potent in HIV inhibition than that after HIV infection. Further investigations showed that the inductions of ISGs and PPRs expression by IFN-λs were largely compromised by HIV infection. These findings provide further experimental evidence that IFN-λs have therapeutic potential in treatment of HIV infection.

  12. Heroin inhibits HIV-restriction miRNAs and enhances HIV infection of macrophages

    PubMed Central

    Wang, Xu; Ma, Tong-Cui; Li, Jie-Liang; Zhou, Yu; Geller, Ellen B.; Adler, Martin W.; Peng, Jin-Song; Zhou, Wang; Zhou, Dun-Jin; Ho, Wen-Zhe

    2015-01-01

    Although opioids have been extensively studied for their impact on the immune system, limited information is available about the specific actions of opioids on intracellular antiviral innate immunity against HIV infection. Thus, we investigated whether heroin, one of the most abused drugs, inhibits the expression of intracellular HIV restriction microRNA (miRNA) and facilitates HIV replication in macrophages. Heroin treatment of macrophages enhanced HIV replication, which was associated with the downregulation of several HIV restriction miRNAs. These heroin-mediated actions on the miRNAs and HIV could be antagonized by naltrexone, an opioid receptor antagonist. Furthermore, the in vitro negative impact of heroin on HIV-associated miRNAs was confirmed by the in vivo observation that heroin addicts had significantly lower levels of macrophage-derived HIV restriction miRNAs than those in the control subjects. These in vitro and in vivo findings indicate that heroin use compromises intracellular anti-HIV innate immunity, providing a favorable microenvironment for HIV survival in the target cells. PMID:26583016

  13. Factors secreted by human T lymphotropic virus type I (HTLV-I)-infected cells can enhance or inhibit replication of HIV-1 in HTLV-I-uninfected cells: implications for in vivo coinfection with HTLV-I and HIV-1.

    PubMed

    Moriuchi, H; Moriuchi, M; Fauci, A S

    1998-05-18

    It remains controversial whether human T lymphotropic virus type I (HTLV-I) coinfection leads to more rapid progression of human immunodeficiency virus (HIV) disease in dually infected individuals. To investigate whether HTLV-I infection of certain cells can modulate HIV-1 infection of surrounding cells, primary CD4(+) T cells were treated with cell-free supernatants from HTLV-I-infected MT-2 cell cultures. The primary CD4+ T cells became resistant to macrophage (M)-tropic HIV-1 but highly susceptible to T cell (T)-tropic HIV-1. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta in the MT-2 cell supernatants were identified as the major suppressive factors for M-tropic HIV-1 as well as the enhancers of T-tropic HIV-1 infection, whereas soluble Tax protein increased susceptibility to both M- and T-tropic HIV-1. The effect of Tax or CC chemokines on T-tropic HIV-1 was mediated, at least in part, by increasing HIV Env-mediated fusogenicity. Our data suggest that the net effect of HTLV-I coinfection in HIV-infected individuals favors the transition from M- to T-tropic HIV phenotype, which is generally indicative of progressive HIV disease.

  14. Macrophages sustain HIV replication in vivo independently of T cells

    PubMed Central

    Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Sharpe, Garrett; Haase, Ashley T.; Eron, Joseph J.; Garcia, J. Victor

    2016-01-01

    Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell–only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection. PMID:26950420

  15. Macrophages sustain HIV replication in vivo independently of T cells.

    PubMed

    Honeycutt, Jenna B; Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Madden, Victoria; Sharpe, Garrett; Haase, Ashley T; Eron, Joseph J; Garcia, J Victor

    2016-04-01

    Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

  16. A post-entry role for CD63 in early HIV-1 replication

    SciTech Connect

    Li Guangyu; Dziuba, Natallia; Friedrich, Brian; Murray, James L.; Ferguson, Monique R.

    2011-04-10

    Macrophages and CD4{sup +} lymphocytes are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in early HIV-1 replication events in both macrophages and a CD4{sup +} cell line. Further analysis revealed that viral attachment and cell-cell fusion were unaffected by CD63 silencing. However, CD63-depleted macrophages showed a significant decrease in the initiation and completion of HIV-1 reverse transcription, affecting subsequent events of the HIV-1 life cycle. Integration of HIV-1 cDNA as well as the formation of 2-LTR circles was notably reduced. Reporter assays showed that CD63 down regulation reduced production of the early HIV protein Tat. In agreement, CD63 silencing also inhibited production of the late protein p24. These findings suggest that CD63 plays an early post-entry role prior to or at the reverse transcription step.

  17. APOBEC3G-Augmented Stem Cell Therapy to Modulate HIV Replication: A Computational Study.

    PubMed

    Hosseini, Iraj; Mac Gabhann, Feilim

    2013-01-01

    The interplay between the innate immune system restriction factor APOBEC3G and the HIV protein Vif is a key host-retrovirus interaction. APOBEC3G can counteract HIV infection in at least two ways: by inducing lethal mutations on the viral cDNA; and by blocking steps in reverse transcription and viral integration into the host genome. HIV-Vif blocks these antiviral functions of APOBEC3G by impeding its encapsulation. Nonetheless, it has been shown that overexpression of APOBEC3G, or interfering with APOBEC3G-Vif binding, can efficiently block in vitro HIV replication. Some clinical studies have also suggested that high levels of APOBEC3G expression in HIV patients are correlated with increased CD4+ T cell count and low levels of viral load; however, other studies have reported contradictory results and challenged this observation. Stem cell therapy to replace a patient's immune cells with cells that are more HIV-resistant is a promising approach. Pre-implantation gene transfection of these stem cells can augment the HIV-resistance of progeny CD4+ T cells. As a protein, APOBEC3G has the advantage that it can be genetically encoded, while small molecules cannot. We have developed a mathematical model to quantitatively study the effects on in vivo HIV replication of therapeutic delivery of CD34+ stem cells transfected to overexpress APOBEC3G. Our model suggests that stem cell therapy resulting in a high fraction of APOBEC3G-overexpressing CD4+ T cells can effectively inhibit in vivo HIV replication. We extended our model to simulate the combination of APOBEC3G therapy with other biological activities, to estimate the likelihood of improved outcomes.

  18. The design of a peptide sequence to inhibit HIV replication: a search algorithm combining Monte Carlo and self-consistent mean field techniques.

    PubMed

    Xiao, Xingqing; Hall, Carol K; Agris, Paul F

    2014-01-01

    We developed a search algorithm combining Monte Carlo (MC) and self-consistent mean field techniques to evolve a peptide sequence that has good binding capability to the anticodon stem and loop (ASL) of human lysine tRNA species, tRNA(Lys3), with the ultimate purpose of breaking the replication cycle of human immunodeficiency virus-1. The starting point is the 15-amino-acid sequence, RVTHHAFLGAHRTVG, found experimentally by Agris and co-workers to bind selectively to hypermodified tRNA(Lys3). The peptide backbone conformation is determined via atomistic simulation of the peptide-ASL(Lys3) complex and then held fixed throughout the search. The proportion of amino acids of various types (hydrophobic, polar, charged, etc.) is varied to mimic different peptide hydration properties. Three different sets of hydration properties were examined in the search algorithm to see how this affects evolution to the best-binding peptide sequences. Certain amino acids are commonly found at fixed sites for all three hydration states, some necessary for binding affinity and some necessary for binding specificity. Analysis of the binding structure and the various contributions to the binding energy shows that: 1) two hydrophilic residues (asparagine at site 11 and the cysteine at site 12) "recognize" the ASL(Lys3) due to the VDW energy, and thereby contribute to its binding specificity and 2) the positively charged arginines at sites 4 and 13 preferentially attract the negatively charged sugar rings and the phosphate linkages, and thereby contribute to the binding affinity.

  19. Macrophage derived cystatin B/cathepsin B in HIV replication and neuropathogenesis

    PubMed Central

    Rivera, Linda E; Colón, Krystal; Cantres-Rosario, Yisel M; Zenón, Frances M; Meléndez, Loyda M

    2013-01-01

    Mononuclear phagocytes including monocytes and macrophages, are important defense components of innate immunity, but can be detrimental in HIV-1 infection by serving as the principal reservoirs of virus in brain and triggering a strong immune response. These viral reservoirs represent a challenge to HIV-1 eradication since they continue producing virus in tissue despite antiretroviral therapy. HIV-1 associated neurocognitive disorders (HAND) involves alterations to the blood-brain barrier and migration of activated HIV-1 infected monocytes to the brain with subsequent induced immune activation response. Our group recently showed that HIV replication in monocyte-derived macrophages (MDM) is associated with increased cystatin B. This cysteine protease inhibitor also inhibits the interferon-induced antiviral response by decreasing levels of tyrosine phosphorylated STAT-1. These recent discoveries reveal novel mechanisms of HIV persistence that could be targeted by new therapeutic approaches to eliminate HIV in macrophage reservoirs. However, cystatin B has been also associated with neuroprotection. Cystatin B is an inhibitor of the cysteine protease cathepsin B, a potent neurotoxin. During HIV-1 infection cystatin B and cathepsin B are upregulated in macrophages, meanwhile there is a reduction in cystatin/cathepsin interactions, leading to increased cathepsin B secretion and activity that induce neuronal apoptosis. Increased intracellular expression of both proteins was recently found in monocytes from Hispanic women with HAND. These findings provide new evidence for the role of cathepsin/cystatin system in the neuropathogenesis induced by HIV-infected macrophages. We summarize recent research on cystatin B and one of its substrates, cathepsin B, in HIV replication in macrophages and neuropathogenesis. PMID:24862331

  20. HIV-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early post-entry replication step

    SciTech Connect

    Federico, Maurizio

    2011-08-15

    The inhibitors of HIV-1 protease (PIs) have been designed to block the activity of the viral aspartyl-protease. However, it is now accepted that this family of inhibitors can also affect the activity of cell proteases. Since the replication of many virus species requires the activity of host cell proteases, investigating the effects of PIs on the life cycle of viruses other than HIV would be of interest. Here, the potent inhibition induced by saquinavir and nelfinavir on the replication of both vesicular stomatitis and influenza viruses is described. These are unrelated enveloped RNA viruses infecting target cells upon endocytosis and intracellular fusion. The PI-induced inhibition was apparently a consequence of a block at the level of the fusion between viral envelope and endosomal membranes. These findings would open the way towards the therapeutic use of PIs against enveloped RNA viruses other than HIV.

  1. Human cellular restriction factors that target HIV-1 replication

    PubMed Central

    Strebel, Klaus; Luban, Jeremy; Jeang, Kuan-Teh

    2009-01-01

    Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G), bone marrow stromal cell antigen 2 (BST-2), cyclophilin A, tripartite motif protein 5 alpha (Trim5α), and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions. PMID:19758442

  2. ATR inhibition rewires cellular signaling networks induced by replication stress.

    PubMed

    Wagner, Sebastian A; Oehler, Hannah; Voigt, Andrea; Dalic, Denis; Freiwald, Anja; Serve, Hubert; Beli, Petra

    2016-02-01

    The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated. In this study, we employed quantitative MS-based proteomics to define the cellular signaling after nucleotide depletion-induced replication stress and replication fork collapse following ATR inhibition. We demonstrate that replication stress results in increased phosphorylation of a subset of proteins, many of which are involved in RNA splicing and transcription and have previously not been associated with the cellular replication stress response. Furthermore, our data reveal the ATR-dependent phosphorylation following replication stress and discover novel putative ATR target sites on MCM6, TOPBP1, RAD51AP1, and PSMD4. We establish that ATR inhibition rewires cellular signaling networks induced by replication stress and leads to the activation of the ATM-driven double-strand break repair signaling.

  3. Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

    PubMed Central

    Vandendriessche, T; Chuah, M K; Chiang, L; Chang, H K; Ensoli, B; Morgan, R A

    1995-01-01

    Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy. PMID:7769662

  4. Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference

    PubMed Central

    Coburn, Glen A.; Cullen, Bryan R.

    2002-01-01

    Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle. PMID:12186906

  5. Factors Associated With the Control of Viral Replication and Virologic Breakthrough in a Recently Infected HIV-1 Controller.

    PubMed

    Walker-Sperling, Victoria E; Pohlmeyer, Christopher W; Veenhuis, Rebecca T; May, Megan; Luna, Krystle A; Kirkpatrick, Allison R; Laeyendecker, Oliver; Cox, Andrea L; Carrington, Mary; Bailey, Justin R; Arduino, Roberto C; Blankson, Joel N

    2017-02-01

    HIV-1 controllers are patients who control HIV-1 viral replication without antiretroviral therapy. Control is achieved very early in the course of infection, but the mechanisms through which viral replication is restricted are not fully understood. We describe a patient who presented with acute HIV-1 infection and was found to have an HIV-1 RNA level of <100copies/mL. She did not have any known protective HLA alleles, but significant immune activation of CD8+ T cells and natural killer (NK) cells was present, and both cell types inhibited viral replication. Virus cultured from this patient replicated as well in vitro as virus isolated from her partner, a patient with AIDS who was the source of transmission. Virologic breakthrough occurred 9months after her initial presentation and was associated with an increase in CD4+ T cell activation levels and a significant decrease in NK cell inhibitory capacity. Remarkably, CD8+ T cell inhibitory capacity was preserved and there were no new escape mutations in targeted Gag epitopes. These findings suggest that fully replication-competent virus can be controlled in acute HIV-1 infection in some patients without protective HLA alleles and that NK cell responses may contribute to this early control of viral replication.

  6. Suppression of HIV Replication by CD8+ Regulatory T-Cells in Elite Controllers

    PubMed Central

    Lu, Wei; Chen, Song; Lai, Chunhui; Lai, Mingyue; Fang, Hua; Dao, Hong; Kang, Jun; Fan, Jianhua; Guo, Weizhong; Fu, Linchun; Andrieu, Jean-Marie

    2016-01-01

    We previously demonstrated in the Chinese macaque model that an oral vaccine made of inactivated SIV and Lactobacillus plantarum induced CD8+ regulatory T-cells, which suppressed the activation of SIV+CD4+ T-cells, prevented SIV replication, and protected macaques from SIV challenges. Here, we sought whether a similar population of CD8+ T-regs would induce the suppression of HIV replication in elite controllers (ECs), a small population (3‰) of HIV-infected patients with undetectable HIV replication. For that purpose, we investigated the in vitro antiviral activity of fresh CD8+ T-cells on HIV-infected CD4+ T-cells taken from 10 ECs. The 10 ECs had a classical genomic profile: all of them carried the KIR3DL1 gene and 9 carried at least 1 allele of HLA-B:Bw4-80Ile (i.e., with an isoleucine residue at position 80). In the nine HLA-B:Bw4-80Ile-positive patients, we demonstrated a strong viral suppression by KIR3DL1-expressing CD8+ T-cells that required cell-to-cell contact to switch off the activation signals in infected CD4+ T-cells. KIR3DL1-expressing CD8+ T-cells withdrawal and KIR3DL1 neutralization by a specific anti-killer cell immunoglobulin-like receptor (KIR) antibody inhibited the suppression of viral replication. Our findings provide the first evidence for an instrumental role of KIR-expressing CD8+ regulatory T-cells in the natural control of HIV-1 infection. PMID:27148256

  7. Suppression of HIV-1 replication by propolis and its immunoregulatory effect.

    PubMed

    Harish, Z; Rubinstein, A; Golodner, M; Elmaliah, M; Mizrachi, Y

    1997-01-01

    In the current study we show that propolis, a non-toxic natural bee-hive product, suppresses HIV-1 replication and modulates in vitro immune responses. CEM cells were treated with propolis at nontoxic concentrations prior to or following infection with HIV-1. Propolis abolished syncytium formation at 4.5 micrograms/ml and inhibited it at lower doses in a concentration-dependent manner. Propolis decreased p24 antigen production by as much as 90-100% in a concentration-dependent manner. Furthermore, modulation of peripheral blood mononuclear cells (PBMCs) mitogenic responses upon the addition of propolis was noted, reducing the elevated responses to Concanavalin A (Con A) and enhancing suppressed mitogenic responses to pokeweed mitogen (PWM). In summary, propolis may constitute a non-toxic natural product with both anti HIV-1 and immunoregulatory effects.

  8. Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication

    PubMed Central

    Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S.; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J.

    2016-01-01

    The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280 in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. PMID:26810656

  9. Knockdown of ERM family member moesin in host cells increases HIV type 1 replication.

    PubMed

    Capalbo, Gianni; Mueller-Kuller, Thea; Markovic, Sandra; Klein, Stefan A; Dietrich, Ursula; Hoelzer, Dieter; Ottmann, Oliver G; Scheuring, Urban J

    2011-12-01

    Moesin is a member of the ERM (ezrin, radixin, moesin) family of cytoskeleton/membrane structure organizing and signal transduction proteins. Previously, we found an increased expression of moesin during HIV-1 infection. Moesin was also reported to be incorporated into HIV-1 virions. To analyze whether moesin is a host factor affecting the replication cycle of human immunodeficiency virus type 1 (HIV-1), we used small interfering RNAs (siRNAs) to evaluate the effect of moesin knockdown on HIV-1 replication in P4-CCR5 cells. Moesin's knockdown did not affect the cell viability or cell phenotype. Interestingly, we observed a marked increase in viral replication, as demonstrated by enhanced HIV-1 RNA, p24 antigen, and ß-galactosidase reporter expression. Moesin-dependent enhancement of HIV-1 replication was confirmed in lymphocytic host cells (Jurkat). These results suggest an overall rather restrictive role of moesin for HIV-1 replication in host cells in vitro.

  10. A combinational CRISPR/Cas9 gene-editing approach can halt HIV replication and prevent viral escape.

    PubMed

    Lebbink, Robert Jan; de Jong, Dorien C M; Wolters, Femke; Kruse, Elisabeth M; van Ham, Petra M; Wiertz, Emmanuel J H J; Nijhuis, Monique

    2017-02-08

    HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach. Here, we demonstrate that CRISPR/Cas9 targeting of single HIV loci, only partially inhibits HIV replication and facilitates rapid viral escape at the target site. A combinatorial approach of two strong gRNAs targeting different regions of the HIV genome can completely abrogate viral replication and prevent viral escape. Our data shows that the accelerating effect of gene-editing on viral escape can be overcome and as such gene-editing may provide a future alternative for control of HIV-infection.

  11. A combinational CRISPR/Cas9 gene-editing approach can halt HIV replication and prevent viral escape

    PubMed Central

    Lebbink, Robert Jan; de Jong, Dorien C. M.; Wolters, Femke; Kruse, Elisabeth M.; van Ham, Petra M.; Wiertz, Emmanuel J. H. J.; Nijhuis, Monique

    2017-01-01

    HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach. Here, we demonstrate that CRISPR/Cas9 targeting of single HIV loci, only partially inhibits HIV replication and facilitates rapid viral escape at the target site. A combinatorial approach of two strong gRNAs targeting different regions of the HIV genome can completely abrogate viral replication and prevent viral escape. Our data shows that the accelerating effect of gene-editing on viral escape can be overcome and as such gene-editing may provide a future alternative for control of HIV-infection. PMID:28176813

  12. Ribavirin Inhibits Parrot Bornavirus 4 Replication in Cell Culture.

    PubMed

    Musser, Jeffrey M B; Heatley, J Jill; Koinis, Anastasia V; Suchodolski, Paulette F; Guo, Jianhua; Escandon, Paulina; Tizard, Ian R

    2015-01-01

    Parrot bornavirus 4 is an etiological agent of proventricular dilatation disease, a fatal neurologic and gastrointestinal disease of psittacines and other birds. We tested the ability of ribavirin, an antiviral nucleoside analog with antiviral activity against a range of RNA and DNA viruses, to inhibit parrot bornavirus 4 replication in duck embryonic fibroblast cells. Two analytical methods that evaluate different products of viral replication, indirect immunocytochemistry for viral specific nucleoprotein and qRT-PCR for viral specific phosphoprotein gene mRNA, were used. Ribavirin at concentrations between 2.5 and 25 μg/mL inhibited parrot bornavirus 4 replication, decreasing viral mRNA and viral protein load, in infected duck embryonic fibroblast cells. The addition of guanosine diminished the antiviral activity of ribavirin suggesting that one possible mechanism of action against parrot bornavirus 4 may likely be through inosine monophosphate dehydrogenase inhibition. This study demonstrates parrot bornavirus 4 susceptibility to ribavirin in cell culture.

  13. Novel Polyanions Inhibiting Replication of Influenza Viruses

    PubMed Central

    Ciejka, Justyna; Milewska, Aleksandra; Wytrwal, Magdalena; Wojarski, Jacek; Golda, Anna; Ochman, Marek; Nowakowska, Maria

    2016-01-01

    Novel sulfonated derivatives of poly(allylamine hydrochloride) (NSPAHs) and N-sulfonated chitosan (NSCH) have been synthesized, and their activity against influenza A and B viruses has been studied and compared with that of a series of carrageenans, marine polysaccharides of well-documented anti-influenza activity. NSPAHs were found to be nontoxic and very soluble in water, in contrast to gel-forming and thus generally poorly soluble carrageenans. In vitro and ex vivo studies using susceptible cells (Madin-Darby canine kidney epithelial cells and fully differentiated human airway epithelial cultures) demonstrated the antiviral effectiveness of NSPAHs. The activity of NSPAHs was proportional to the molecular mass of the chain and the degree of substitution of amino groups with sulfonate groups. Mechanistic studies showed that the NSPAHs and carrageenans inhibit influenza A and B virus assembly in the cell. PMID:26729490

  14. Nitric oxide inhibition of coxsackievirus replication in vitro.

    PubMed Central

    Zaragoza, C; Ocampo, C J; Saura, M; McMillan, A; Lowenstein, C J

    1997-01-01

    Nitric oxide is a radical molecule with antibacterial, -parasitic, and -viral properties. We investigated the mechanism of NO inhibition of Coxsackievirus B3 (CVB3) replication in vitro by determining the effect of NO upon a single replicative cycle of CVB3 grown in HeLa cells. Transfection of inducible NO synthase cDNA into HeLa cells reduces the number of viral particles produced during a single cycle of growth. Similarly, a noncytotoxic concentration of the NO donor S-nitroso-amino-penicillamine reduces the number of viral particles in a dose-dependent manner. To explore the mechanisms by which NO exerts its antiviral effect, we assayed the attachment, replication, and translation steps of the CVB3 life cycle. NO does not affect the attachment of CVB3 to HeLa cells. However, NO inhibits CVB3 RNA synthesis, as shown by a [3H]uridine incorporation assay, reverse transcription-PCR, and Northern analysis. In addition, NO inhibits CVB3 protein synthesis, as shown by [35S]methionine protein labeling and Western blot analysis of infected cells. Thus, NO inhibits CVB3 replication in part by inhibiting viral RNA synthesis by an unknown mechanism. PMID:9312175

  15. Human cytomegalovirus function inhibits replication of herpes simplex virus

    SciTech Connect

    Cockley, K.D.; Shiraki, K.; Rapp, F.

    1988-01-01

    Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 ..mu..g/ml) for 3 or 24 h was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5/sup 0/C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with (/sup 3/H)thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection.

  16. Short Communication: High Cellular Iron Levels Are Associated with Increased HIV Infection and Replication

    PubMed Central

    Chang, Hsiang-Chun; Bayeva, Marina; Taiwo, Babafemi; Palella, Frank J.; Hope, Thomas J.

    2015-01-01

    Abstract HIV is a pandemic disease, and many cellular and systemic factors are known to alter its infectivity and replication. Earlier studies had suggested that anemia is common in HIV-infected patients; however, higher iron was also observed in AIDS patients prior to the introduction of antiretroviral therapy (ART). Therefore, the relationship between iron and viral infection is not well delineated. To address this issue, we altered the levels of cellular iron in primary CD4+ T cells and showed that higher iron is associated with increased HIV infection and replication. In addition, HIV infection alone leads to increased cellular iron, and several ART drugs increase cellular iron independent of HIV infection. Finally, HIV infection is associated with increased serum iron in HIV-positive patients regardless of treatment with ART. These results establish a relationship between iron and HIV infection and suggest that iron homeostasis may be a viable therapeutic target for HIV. PMID:25291189

  17. Short communication: high cellular iron levels are associated with increased HIV infection and replication.

    PubMed

    Chang, Hsiang-Chun; Bayeva, Marina; Taiwo, Babafemi; Palella, Frank J; Hope, Thomas J; Ardehali, Hossein

    2015-03-01

    HIV is a pandemic disease, and many cellular and systemic factors are known to alter its infectivity and replication. Earlier studies had suggested that anemia is common in HIV-infected patients; however, higher iron was also observed in AIDS patients prior to the introduction of antiretroviral therapy (ART). Therefore, the relationship between iron and viral infection is not well delineated. To address this issue, we altered the levels of cellular iron in primary CD4(+) T cells and showed that higher iron is associated with increased HIV infection and replication. In addition, HIV infection alone leads to increased cellular iron, and several ART drugs increase cellular iron independent of HIV infection. Finally, HIV infection is associated with increased serum iron in HIV-positive patients regardless of treatment with ART. These results establish a relationship between iron and HIV infection and suggest that iron homeostasis may be a viable therapeutic target for HIV.

  18. Control of HIV-1 replication in vitro by vaccine-induced human CD8+ T cells through conserved subdominant Pol epitopes

    PubMed Central

    Ahmed, Tina; Borthwick, Nicola J.; Gilmour, Jill; Hayes, Peter; Dorrell, Lucy; Hanke, Tomáš

    2016-01-01

    Objective The specificity of CD8+ T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8+ effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4+ cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication. Design CD8+ T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates. Methods Frozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells. Results We formally demonstrated that the vaccine-elicited inhibitory human CD8+ T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells. Conclusions These results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient

  19. APOBEC3G and APOBEC3F Act in Concert To Extinguish HIV-1 Replication

    PubMed Central

    Krisko, John F.; Begum, Nurjahan; Baker, Caroline E.

    2016-01-01

    ABSTRACT The multifunctional HIV-1 accessory protein Vif counters the antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F), and some Vifs counter stable alleles of APOBEC3H (A3H). Studies in humanized mice have shown that HIV-1 lacking Vif expression is not viable. Here, we look at the relative contributions of the three APOBEC3s to viral extinction. Inoculation of bone marrow/liver/thymus (BLT) mice with CCR5-tropic HIV-1JRCSF (JRCSF) expressing a vif gene inactive for A3G but not A3F degradation activity (JRCSFvifH42/43D) displayed either no or delayed replication. JRCSF expressing a vif gene mutated to inactivate A3F degradation but not A3G degradation (JRCSFvifW79S) always replicated to high viral loads with variable delays. JRCSF with vif mutated to lack both A3G and A3F degradation activities (JRCSFvifH42/43DW79S) failed to replicate, mimicking JRCSF without Vif expression (JRCSFΔvif). JRCSF and JRCSFvifH42/43D, but not JRCSFvifW79S or JRCSFvifH42/43DW79S, degraded APOBEC3D. With one exception, JRCSFs expressing mutant Vifs that replicated acquired enforced vif mutations. These mutations partially restored A3G or A3F degradation activity and fully replaced JRCSFvifH42/43D or JRCSFvifW79S by 10 weeks. Surprisingly, induced mutations temporally lagged behind high levels of virus in blood. In the exceptional case, JRCSFvifH42/43D replicated after a prolonged delay with no mutations in vif but instead a V27I mutation in the RNase H coding sequence. JRCSFvifH42/43D infections exhibited massive GG/AG mutations in pol viral DNA, but in viral RNA, there were no fixed mutations in the Gag or reverse transcriptase coding sequence. A3H did not contribute to viral extinction but, in combination with A3F, could delay JRCSF replication. A3H was also found to hypermutate viral DNA. IMPORTANCE Vif degradation of A3G and A3F enhances viral fitness, as virus with even a partially restored capacity for degradation outgrows JRCSFvifH42/43D and JRCSFvifW79S. Unexpectedly

  20. Effects of L- and D-REKR amino acid-containing peptides on HIV and SIV envelope glycoprotein precursor maturation and HIV and SIV replication.

    PubMed Central

    Bahbouhi, Bouchaib; Chazal, Nathalie; Seidah, Nabil Georges; Chiva, Cristina; Kogan, Marcelo; Albericio, Fernando; Giralt, Ernest; Bahraoui, Elmostafa

    2002-01-01

    The aim of the present study was to evaluate the capacity of synthetic l- and d-peptides encompassing the HIV-1(BRU) gp160 REKR cleavage site to interfere with HIV and simian immuno-deficiency virus (SIV) replication and maturation of the envelope glycoprotein (Env) precursors. To facilitate their penetration into cells, a decanoyl (dec) group was added at the N-terminus. The sequences synthesized included dec5d or dec5l (decREKRV), dec9d or dec9l (decRVVQREKRV) and dec14d or dec14l (TKAKRRVVQREKRV). The peptide dec14d was also prepared with a chloromethane (cmk) group as C-terminus. Because l-peptides exhibit significant cytotoxicity starting at 35 microM, further characterization was conducted mostly with d-peptides, which exhibited no cytotoxicity at concentrations higher than 70 microM. The data show that only dec14d and dec14dcmk could inhibit HIV-1(BRU), HIV-2(ROD) and SIV(mac251) replication and their syncytium-inducing capacities. Whereas peptides dec5d and dec9d were inactive, dec14dcmk was at least twice as active as peptide dec14d. At the molecular level, our data show a direct correlation between anti-viral activity and the ability of the peptides to interfere with maturation of the Env precursors. Furthermore, we show that when tested in vitro the dec14d peptide inhibited PC7 with an inhibition constant K(i)=4.6 microM, whereas the peptide dec14l preferentially inhibited furin with a K(i)=28 microM. The fact that PC7 and furin are the major prohormone convertases reported to be expressed in T4 lymphocytes, the principal cell targets of HIV, suggests that they are involved in the maturation of HIV and SIV Env precursors. PMID:12071862

  1. In Vitro Reactivation of Replication-Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors

    PubMed Central

    Banga, Riddhima; Procopio, Francesco Andrea; Cavassini, Matthias

    2015-01-01

    ABSTRACT The existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latency in vitro. Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects. IMPORTANCE The major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an

  2. Nitric Oxide Inhibits Coxiella burnetii Replication and Parasitophorous Vacuole Maturation

    PubMed Central

    Howe, Dale; Barrows, Lorraine F.; Lindstrom, Nicole M.; Heinzen, Robert A.

    2002-01-01

    Nitric oxide is a recognized cytotoxic effector against facultative and obligate intracellular bacteria. This study examined the effect of nitric oxide produced by inducible nitric oxide synthase (iNOS) up-regulated in response to cytokine stimulation, or by a synthetic nitric oxide donor, on replication of obligately intracellular Coxiella burnetii in murine L-929 cells. Immunoblotting and nitrite assays revealed that C. burnetii infection of L-929 cells augments expression of iNOS up-regulated in response to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Infection in the absence of cytokine stimulation did not result in demonstrable up-regulation of iNOS expression or in increased nitrite production. Nitrite production by cytokine-treated cells was significantly inhibited by the iNOS inhibitor S-methylisothiourea (SMT). Treatment of infected cells with IFN-γ and TNF-α or the synthetic nitric oxide donor 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NONOate) had a bacteriostatic effect on C. burnetii replication. Inhibition of replication was reversed upon addition of SMT to the culture medium of cytokine-treated cells. Microscopic analysis of infected cells revealed that nitric oxide (either cytokine induced or donor derived) inhibited formation of the mature (large) parasitophorous vacuole that is characteristic of C. burnetii infection of host cells. Instead, exposure of infected cells to nitric oxide resulted in the formation of multiple small, acidic vacuoles usually containing one C. burnetii cell. Removal of nitrosative stress resulted in the coalescence of small vacuoles to form a large vacuole harboring multiple C. burnetii cells. These experiments demonstrate that nitric oxide reversibly inhibits replication of C. burnetii and formation of the parasitophorous vacuole. PMID:12183564

  3. Inhibition of HIV-1 entry by the tricyclic coumarin GUT-70 through the modification of membrane fluidity

    SciTech Connect

    Matsuda, Kouki; Hattori, Shinichiro; Kariya, Ryusho; Komizu, Yuji; Kudo, Eriko; Goto, Hiroki; Taura, Manabu; Ueoka, Ryuichi; Kimura, Shinya; Okada, Seiji

    2015-02-13

    Membrane fusion between host cells and HIV-1 is the initial step in HIV-1 infection, and plasma membrane fluidity strongly influences infectivity. In the present study, we demonstrated that GUT-70, a natural product derived from Calophyllum brasiliense, stabilized plasma membrane fluidity, inhibited HIV-1 entry, and down-regulated the expression of CD4, CCR5, and CXCR4. Since GUT-70 also had an inhibitory effect on viral replication through the inhibition of NF-κB, it is expected to be used as a dual functional and viral mutation resistant reagent. Thus, these unique properties of GUT-70 enable the development of novel therapeutic agents against HIV-1 infection.

  4. Cyclophilin A is required for the replication of group M human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIV(CPZ)GAB but not group O HIV-1 or other primate immunodeficiency viruses.

    PubMed Central

    Braaten, D; Franke, E K; Luban, J

    1996-01-01

    The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds to cyclophilin A and incorporates this cellular peptidyl prolyl-isomerase into virions. Disruption of cyclophilin A incorporation, either by gag mutations or by cyclosporine A, inhibits virion infectivity, indicating that cyclophilin A plays an essential role in the HIV-1 life cycle. Using assays for packaging of cyclophilin A into virions and for viral replication sensitivity to cyclosporine A, as well as information gleaned from the alignment of Gag residues encoded by representative viral isolates, we demonstrate that of the five lineages of primate immunodeficiency viruses, only HIV-1 requires cyclophilin A for replication. Cloned viral isolates from clades A, B, and D of HIV-1 group M, as well as a phylogenetically related isolate from chimpanzee, all require cyclophilin A for replication. In contrast, the replication of two outlier (group O) HIV-1 isolates is unaffected by concentrations of cyclosporine A which disrupt cyclophilin A incorporation into virions, indicating that these viruses are capable of replicating independently of cyclophilin A. These studies identify the first phenotypic difference between HIV-1 group M and group O and are consistent with phylogenetic studies suggesting that the two HIV-1 groups were introduced into human populations via separate zoonotic transmission events. PMID:8676442

  5. A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

    PubMed Central

    Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

    2012-01-01

    Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop+ oriλ+ sequence. PMID:22590552

  6. Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

    NASA Astrophysics Data System (ADS)

    Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

    1994-05-01

    Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

  7. T-705 (favipiravir) inhibition of arenavirus replication in cell culture.

    PubMed

    Mendenhall, Michelle; Russell, Andrew; Juelich, Terry; Messina, Emily L; Smee, Donald F; Freiberg, Alexander N; Holbrook, Michael R; Furuta, Yousuke; de la Torre, Juan-Carlos; Nunberg, Jack H; Gowen, Brian B

    2011-02-01

    A number of New World arenaviruses (Junín [JUNV], Machupo [MACV], and Guanarito [GTOV] viruses) can cause human disease ranging from mild febrile illness to a severe and often fatal hemorrhagic fever syndrome. These highly pathogenic viruses and the Old World Lassa fever virus pose a significant threat to public health and national security. The only licensed antiviral agent with activity against these viruses, ribavirin, has had mixed success in treating severe arenaviral disease and is associated with significant toxicities. A novel pyrazine derivative currently in clinical trials for the treatment of influenza virus infections, T-705 (favipiravir), has demonstrated broad-spectrum activity against a number of RNA viruses, including arenaviruses. T-705 has also been shown to be effective against Pichinde arenavirus infection in a hamster model. Here, we demonstrate the robust antiviral activity of T-705 against authentic highly pathogenic arenaviruses in cell culture. We show that T-705 disrupts an early or intermediate stage in viral replication, distinct from absorption or release, and that its antiviral activity in cell culture is reversed by the addition of purine bases and nucleosides, but not with pyrimidines. Specific inhibition of viral replication/transcription by T-705 was demonstrated using a lymphocytic choriomeningitis arenavirus replicon system. Our findings indicate that T-705 acts to inhibit arenavirus replication/transcription and may directly target the viral RNA-dependent RNA polymerase.

  8. The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication

    PubMed Central

    Sacramento, Carolina Q.; de Melo, Gabrielle R.; de Freitas, Caroline S.; Rocha, Natasha; Hoelz, Lucas Villas Bôas; Miranda, Milene; Fintelman-Rodrigues, Natalia; Marttorelli, Andressa; Ferreira, André C.; Barbosa-Lima, Giselle; Abrantes, Juliana L.; Vieira, Yasmine Rangel; Bastos, Mônica M.; de Mello Volotão, Eduardo; Nunes, Estevão Portela; Tschoeke, Diogo A.; Leomil, Luciana; Loiola, Erick Correia; Trindade, Pablo; Rehen, Stevens K.; Bozza, Fernando A.; Bozza, Patrícia T.; Boechat, Nubia; Thompson, Fabiano L.; de Filippis, Ana M. B.; Brüning, Karin; Souza, Thiago Moreno L.

    2017-01-01

    Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV. PMID:28098253

  9. The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication.

    PubMed

    Sacramento, Carolina Q; de Melo, Gabrielle R; de Freitas, Caroline S; Rocha, Natasha; Hoelz, Lucas Villas Bôas; Miranda, Milene; Fintelman-Rodrigues, Natalia; Marttorelli, Andressa; Ferreira, André C; Barbosa-Lima, Giselle; Abrantes, Juliana L; Vieira, Yasmine Rangel; Bastos, Mônica M; de Mello Volotão, Eduardo; Nunes, Estevão Portela; Tschoeke, Diogo A; Leomil, Luciana; Loiola, Erick Correia; Trindade, Pablo; Rehen, Stevens K; Bozza, Fernando A; Bozza, Patrícia T; Boechat, Nubia; Thompson, Fabiano L; de Filippis, Ana M B; Brüning, Karin; Souza, Thiago Moreno L

    2017-01-18

    Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.

  10. Robust Suppression of HIV Replication by Intracellularly Expressed Reverse Transcriptase Aptamers Is Independent of Ribozyme Processing

    PubMed Central

    Lange, Margaret J; Sharma, Tarun K; Whatley, Angela S; Landon, Linda A; Tempesta, Michael A; Johnson, Marc C; Burke, Donald H

    2012-01-01

    RNA aptamers that bind human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) also inhibit viral replication, making them attractive as therapeutic candidates and potential tools for dissecting viral pathogenesis. However, it is not well understood how aptamer-expression context and cellular RNA pathways govern aptamer accumulation and net antiviral bioactivity. Using a previously-described expression cassette in which aptamers were flanked by two “minimal core” hammerhead ribozymes, we observed only weak suppression of pseudotyped HIV. To evaluate the importance of the minimal ribozymes, we replaced them with extended, tertiary-stabilized hammerhead ribozymes with enhanced self-cleavage activity, in addition to noncleaving ribozymes with active site mutations. Both the active and inactive versions of the extended hammerhead ribozymes increased inhibition of pseudotyped virus, indicating that processing is not necessary for bioactivity. Clonal stable cell lines expressing aptamers from these modified constructs strongly suppressed infectious virus, and were more effective than minimal ribozymes at high viral multiplicity of infection (MOI). Tertiary stabilization greatly increased aptamer accumulation in viral and subcellular compartments, again regardless of self-cleavage capability. We therefore propose that the increased accumulation is responsible for increased suppression, that the bioactive form of the aptamer is one of the uncleaved or partially cleaved transcripts, and that tertiary stabilization increases transcript stability by reducing exonuclease degradation. PMID:22948672

  11. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication

    SciTech Connect

    Di Nunzio, Francesca; Fricke, Thomas; Miccio, Annarita; Valle-Casuso, Jose Carlos; Perez, Patricio; Souque, Philippe; Rizzi, Ermanno; Severgnini, Marco; Mavilio, Fulvio; Charneau, Pierre; Diaz-Griffero, Felipe

    2013-05-25

    The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration. - Highlights: ► We studied the role of Nup98 and Nup153 in HIV-1 infection. ► Nup98 binds the HIV-1 core and is involved in HIV-1 integration. ► Nup153 binds the HIV-1 core and is involved in HIV-1 nuclear import. ► Depletion of Nup153 decreased the integration of HIV-1 in transcriptionally active sites.

  12. Adipose Tissue: Sanctuary for HIV/SIV Persistence and Replication.

    PubMed

    Pallikkuth, Suresh; Mohan, Mahesh

    2015-12-01

    This commentary highlights new findings from a recent study identifying adipose tissue as a potential HIV reservoir and a major site of inflammation during chronic human/simian immunodeficiency virus (HIV/SIV) infection. A concise discussion about upcoming challenges and new research avenues for reducing chronic adipose inflammation during HIV/SIV infection is presented.

  13. Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation.

    PubMed

    Ogram, Sushma A; Boone, Christopher D; McKenna, Robert; Flanegan, James B

    2014-09-01

    The mechanism of amiloride inhibition of Coxsackievirus B3 (CVB3) and poliovirus type 1 (PV1) RNA replication was investigated using membrane-associated RNA replication complexes. Amiloride was shown to inhibit viral RNA replication and VPgpUpU synthesis. However, the drug had no effect on polymerase elongation activity during either (-) strand or (+) strand synthesis. These findings indicated that amiloride inhibited the initiation of RNA synthesis by inhibiting VPg uridylylation. In addition, in silico binding studies showed that amiloride docks in the VPg binding site on the back of the viral RNA polymerase, 3D(pol). Since VPg binding at this site on PV1 3D(pol) was previously shown to be required for VPg uridylylation, our results suggest that amiloride inhibits VPg binding to 3D(pol). In summary, our findings are consistent with a model in which amiloride inhibits VPgpUpU synthesis and viral RNA replication by competing with VPg for binding to 3D(pol).

  14. Allosteric inhibition of HIV-1 integrase activity

    PubMed Central

    Engelman, Alan; Kessl, Jacques J.; Kvaratskhelia, Mamuka

    2013-01-01

    HIV-1 integrase is an important therapeutic target in the fight against HIV/AIDS. Integrase strand transfer inhibitors (INSTIs), which target the enzyme active site, have witnessed clinical success over the past 5 years, but the generation of drug resistance poses challenges to INSTI-based therapies moving forward. Integrase is a dynamic protein, and its ordered multimerization is critical to enzyme activity. The integrase tetramer, bound to viral DNA, interacts with host LEDGF/p75 protein to tether integration to active genes. Allosteric integrase inhibitors (ALLINIs) that compete with LEDGF/p75 for binding to integrase disrupt integrase assembly with viral DNA and allosterically inhibit enzyme function. ALLINIs display steep dose response curves and synergize with INSTIs ex vivo, highlighting this novel inhibitor class for clinical development. PMID:23647983

  15. Inhibition of adenovirus replication in vitro by trifluridine.

    PubMed

    Lennette, D A; Eiferman, R A

    1978-09-01

    At present, there is no effective chemotherapeutic agent available for the treatment of adenoviral keratoconjunctivitis. Recent evidence suggests that trifluridine (3FT) may effectively inhibit the replication of some adenovirus serotypes known to cause keratoconjunctivitis. The ability of 3FT to inhibit two reference strains of adenoviruses, type 8 and type 19, was examined using cell cultures. Two second-passage isolates of adenoviruses, identified as serotype 13, were also tested. Compared with untreated, virusinfected cell cultures, drug-treated cell cultures developed a lesser degree of cytopathic effect following infection with all three serotypes. Virus production was reduced in the drug-treated cell cultures: approximately tenfold for type 8, more than 1,000-fold for type 19, and 5,000-fold for the type 13 isolates.

  16. Gene therapeutic approaches to inhibit hepatitis B virus replication

    PubMed Central

    Gebbing, Maren; Bergmann, Thorsten; Schulz, Eric; Ehrhardt, Anja

    2015-01-01

    Acute and chronic hepatitis B virus (HBV) infections remain to present a major global health problem. The infection can be associated with acute symptomatic or asymptomatic hepatitis which can cause chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellular carcinomas. Currently available therapeutics for chronically infected individuals aim at reducing viral replication and to slow down or stop the progression of the disease. Therefore, novel treatment options are needed to efficiently combat and eradicate this disease. Here we provide a state of the art overview of gene therapeutic approaches to inhibit HBV replication. We discuss non-viral and viral approaches which were explored to deliver therapeutic nucleic acids aiming at reducing HBV replication. Types of delivered therapeutic nucleic acids which were studied since many years include antisense oligodeoxynucleotides and antisense RNA, ribozymes and DNAzymes, RNA interference, and external guide sequences. More recently designer nucleases gained increased attention and were exploited to destroy the HBV genome. In addition we mention other strategies to reduce HBV replication based on delivery of DNA encoding dominant negative mutants and DNA vaccination. In combination with available cell culture and animal models for HBV infection, in vitro and in vivo studies can be performed to test efficacy of gene therapeutic approaches. Recent progress but also challenges will be specified and future perspectives will be discussed. This is an exciting time to explore such approaches because recent successes of gene therapeutic strategies in the clinic to treat genetic diseases raise hope to find alternative treatment options for patients chronically infected with HBV. PMID:25729471

  17. Curcumin inhibits Rift Valley fever virus replication in human cells.

    PubMed

    Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

    2012-09-28

    Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.

  18. Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells*

    PubMed Central

    Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

    2012-01-01

    Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets. PMID:22847000

  19. HIVed, a knowledgebase for differentially expressed human genes and proteins during HIV infection, replication and latency

    PubMed Central

    Li, Chen; Ramarathinam, Sri H.; Revote, Jerico; Khoury, Georges; Song, Jiangning; Purcell, Anthony W.

    2017-01-01

    Measuring the altered gene expression level and identifying differentially expressed genes/proteins during HIV infection, replication and latency is fundamental for broadening our understanding of the mechanisms of HIV infection and T-cell dysfunction. Such studies are crucial for developing effective strategies for virus eradication from the body. Inspired by the availability and enrichment of gene expression data during HIV infection, replication and latency, in this study, we proposed a novel compendium termed HIVed (HIV expression database; http://hivlatency.erc.monash.edu/) that harbours comprehensive functional annotations of proteins, whose genes have been shown to be dysregulated during HIV infection, replication and latency using different experimental designs and measurements. We manually curated a variety of third-party databases for structural and functional annotations of the protein entries in HIVed. With the goal of benefiting HIV related research, we collected a number of biological annotations for all the entries in HIVed besides their expression profile, including basic protein information, Gene Ontology terms, secondary structure, HIV-1 interaction and pathway information. We hope this comprehensive protein-centric knowledgebase can bridge the gap between the understanding of differentially expressed genes and the functions of their protein products, facilitating the generation of novel hypotheses and treatment strategies to fight against the HIV pandemic. PMID:28358052

  20. Characterization of HIV replication complexes early after cell-to-cell infection.

    PubMed

    Karageorgos, L; Li, P; Burrell, C

    1993-09-01

    In this study, we have characterized the HIV DNA-containing replication complexes present in cells early after cell-to-cell infection, using sucrose gradient sedimentation and immunoprecipitation. Six hours after cell-to-cell infection, a cytoplasmic HIV replication complex sedimented as a large structure (320S). This replication complex was precipitated by antisera to three virus-coded enzymes (reverse transcriptase, integrase, protease), to the matrix protein (p17), and to cellular histones but not to the major capsid protein (p24). This replication complex was not associated with cell membranes and could not be dissociated into smaller discrete subunits, using detergents. Nuclear extracts from the same cell-to-cell infection contained a smaller (80S) complex that lacked reverse transcriptase and matrix protein (p17). Cytoplasmic replication complexes from a cell-free virus infection sedimented as 160S structures under identical conditions, as previously reported. Our results indicate that, following cell-to-cell transmission of HIV, all the HIV pol gene products, the matrix protein p17, and cellular histones are present in cytoplasmic replication complexes that are taking part in or have completed reverse transcription. Transportation of the cytoplasmic replication complex to the nucleus is associated with structural changes, including a reduction in size and altered protein composition.

  1. Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir

    PubMed Central

    Sobolewski, Michele D.; Keele, Brandon F.; Spindler, Jonathan; Wiegand, Ann; Shao, Wei; Hughes, Stephen H.; Coffin, John M.; Kearney, Mary F.; Mellors, John W.

    2017-01-01

    The major obstacle to curing HIV infection is the persistence of cells with intact proviruses that can produce replication-competent virus. This HIV reservoir is believed to exist primarily in CD4+ T-cells and is stable despite years of suppressive antiretroviral therapy. A potential mechanism for HIV persistence is clonal expansion of infected cells, but how often such clones carry replication-competent proviruses has been controversial. Here, we used single-genome sequencing to probe for identical HIV sequence matches among viruses recovered in different viral outgrowth cultures and between the sequences of outgrowth viruses and proviral or intracellular HIV RNA sequences in uncultured blood mononuclear cells from eight donors on suppressive ART with diverse proviral populations. All eight donors had viral outgrowth virus that was fully susceptible to their current ART drug regimen. Six of eight donors studied had identical near full-length HIV RNA sequences recovered from different viral outgrowth cultures, and one of the two remaining donors had identical partial viral sequence matches between outgrowth virus and intracellular HIV RNA. These findings provide evidence that clonal expansion of HIV-infected cells is an important mechanism of reservoir persistence that should be targeted to cure HIV infection. PMID:28328934

  2. Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir.

    PubMed

    Bui, John K; Sobolewski, Michele D; Keele, Brandon F; Spindler, Jonathan; Musick, Andrew; Wiegand, Ann; Luke, Brian T; Shao, Wei; Hughes, Stephen H; Coffin, John M; Kearney, Mary F; Mellors, John W

    2017-03-01

    The major obstacle to curing HIV infection is the persistence of cells with intact proviruses that can produce replication-competent virus. This HIV reservoir is believed to exist primarily in CD4+ T-cells and is stable despite years of suppressive antiretroviral therapy. A potential mechanism for HIV persistence is clonal expansion of infected cells, but how often such clones carry replication-competent proviruses has been controversial. Here, we used single-genome sequencing to probe for identical HIV sequence matches among viruses recovered in different viral outgrowth cultures and between the sequences of outgrowth viruses and proviral or intracellular HIV RNA sequences in uncultured blood mononuclear cells from eight donors on suppressive ART with diverse proviral populations. All eight donors had viral outgrowth virus that was fully susceptible to their current ART drug regimen. Six of eight donors studied had identical near full-length HIV RNA sequences recovered from different viral outgrowth cultures, and one of the two remaining donors had identical partial viral sequence matches between outgrowth virus and intracellular HIV RNA. These findings provide evidence that clonal expansion of HIV-infected cells is an important mechanism of reservoir persistence that should be targeted to cure HIV infection.

  3. Modulation of HCV replication after combination antiretroviral therapy in HCV/HIV co-infected patients.

    PubMed

    Sherman, Kenneth E; Guedj, Jeremie; Shata, Mohamed Tarek; Blackard, Jason T; Rouster, Susan D; Castro, Mario; Feinberg, Judith; Sterling, Richard K; Goodman, Zachary; Aronow, Bruce J; Perelson, Alan S

    2014-07-23

    The hepatitis C virus (HCV) is an important contributor to morbidity and mortality in patients co-infected with HIV. Co-infection results in increased HCV replication and more rapid rates of liver disease progression. The effect of HIV combination antiretroviral therapy (cART) on HCV replication has not been studied in depth. To address this issue, we enrolled a small cohort of HCV/HIV co-infected patients into a cART initiation trial and used dynamic modeling combined with evaluation of immune responses and microarray profiles to determine how effective treatment of HIV affects HCV. Treatment with cART resulted in increased HCV replication and increased alanine aminotransferase (ALT) in a subset of patients. Subjects with evidence of hepatic injury (increased ALT) were more likely to have HCV-specific immune responses directed against HCV epitopes. Over time, HCV viral loads declined. Reproducible and biologically important gene expression changes occurred in co-infected patients who underwent successful cART. The effective suppression of HIV by cART initiated a cascade of early and late events in treated patients. Early events involving down-regulation of interferon-stimulated genes may have led to transiently increased viral replication and hepatic injury. At later time points, HCV viral load declined to levels comparable to those seen in the setting of HCV monoinfection. These findings support early antiretroviral therapy in those with HCV/HIV co-infection.

  4. In Vivo validation of a bioinformatics based tool to identify reduced replication capacity in HIV-1.

    PubMed

    Kitchen, Christina M R; Krogstad, Paul; Kitchen, Scott G

    2010-01-01

    Although antiretroviral drug resistance is common in treated HIV infected individuals, it is not a consistent indicator of HIV morbidity and mortality. To the contrary, HIV resistance-associated mutations may lead to changes in viral fitness that are beneficial to infected individuals. Using a bioinformatics-based model to assess the effects of numerous drug resistance mutations, we determined that the D30N mutation in HIV-1 protease had the largest decrease in replication capacity among known protease resistance mutations. To test this in silico result in an in vivo environment, we constructed several drug-resistant mutant HIV-1 strains and compared their relative fitness utilizing the SCID-hu mouse model. We found HIV-1 containing the D30N mutation had a significant defect in vivo, showing impaired replication kinetics and a decreased ability to deplete CD4+ thymocytes, compared to the wild-type or virus without the D30N mutation. In comparison, virus containing the M184V mutation in reverse transcriptase, which shows decreased replication capacity in vitro, did not have an effect on viral fitness in vivo. Thus, in this study we have verified an in silico bioinformatics result with a biological assessment to identify a unique mutation in HIV-1 that has a significant fitness defect in vivo.

  5. HIV-1 Vpr increases HCV replication through VprBP in cell culture.

    PubMed

    Yan, Yanling; Huang, Fang; Yuan, Ting; Sun, Binlian; Yang, Rongge

    2016-09-02

    Coinfection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) occurs at a high frequency, in which HIV shows a promotion of HCV-derived liver diseases. However, the mechanism of how this occurs is not well understood. Our previous work has demonstrated that the HIV-1 accessory protein Vpr enhances HCV RNA replication in cell culture. Because Vpr performs most of its functions through host protein VprBP (DCAF1), the role of VprBP in the regulation of HCV by Vpr was investigated in this study. We found that the Vpr mutant Q65R, which is deficient in VprBP binding, could not enhance HCV replication. Furthermore, Vpr-mediated enhancement of HCV replication was severely diminished in VprBP knockdown cells. In addition, an inhibitor of Cullin RING E3 ligases, MLN4924, impaired the function of Vpr during HCV replication. Together, these results suggest that Vpr promotes HCV replication in a VprBP-dependent manner, and that the activity of Cullin RING E3 ligases is essential to this process. In conclusion, our findings demonstrate that HIV-1 Vpr makes the cellular environment more suitable for HCV replication, which might relate with the host ubiquitination system.

  6. Differential expression of lncRNAs during the HIV replication cycle: an underestimated layer in the HIV-host interplay

    PubMed Central

    Trypsteen, Wim; Mohammadi, Pejman; Van Hecke, Clarissa; Mestdagh, Pieter; Lefever, Steve; Saeys, Yvan; De Bleser, Pieter; Vandesompele, Jo; Ciuffi, Angela; Vandekerckhove, Linos; De Spiegelaere, Ward

    2016-01-01

    Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding RNAs (lncRNAs) add an extensive additional layer to the cell’s molecular network. Here, we performed transcriptome profiling throughout a primary HIV infection in vitro to investigate lncRNA expression at the different HIV replication cycle processes (reverse transcription, integration and particle production). Subsequently, guilt-by-association, transcription factor and co-expression analysis were performed to infer biological roles for the lncRNAs identified in the HIV-host interplay. Many lncRNAs were suggested to play a role in mechanisms relying on proteasomal and ubiquitination pathways, apoptosis, DNA damage responses and cell cycle regulation. Through transcription factor binding analysis, we found that lncRNAs display a distinct transcriptional regulation profile as compared to protein coding mRNAs, suggesting that mRNAs and lncRNAs are independently modulated. In addition, we identified five differentially expressed lncRNA-mRNA pairs with mRNA involvement in HIV pathogenesis with possible cis regulatory lncRNAs that control nearby mRNA expression and function. Altogether, the present study demonstrates that lncRNAs add a new dimension to the HIV-host interplay and should be further investigated as they may represent targets for controlling HIV replication. PMID:27782208

  7. Nitric oxide inhibits viral replication in murine myocarditis.

    PubMed Central

    Lowenstein, C J; Hill, S L; Lafond-Walker, A; Wu, J; Allen, G; Landavere, M; Rose, N R; Herskowitz, A

    1996-01-01

    Nitric oxide (NO) is a radical molecule that not only serves as a vasodilator and neurotransmitter but also acts as a cytotoxic effector molecule of the immune system. The inducible enzyme making NO, inducible NO synthase (iNOS), is transcriptionally activated by IFN-gamma and TNF-alpha, cytokines which are produced during viral infection. We show that iNOS is induced in mice infected with the Coxsackie B3 virus. Macrophages expressing iNOS are identified in the hearts and spleens of infected animals with an antibody raised against iNOS. Infected mice have increased titers of virus and a higher mortality when fed NOS inhibitors. Thus, viral infection induces iNOS in vivo, and NO inhibits viral replication. NO is a novel, nonspecific immune defense against viruses in vivo. PMID:8621766

  8. Extract of Scutellaria baicalensis inhibits dengue virus replication

    PubMed Central

    2013-01-01

    Background Scutellaria baicalensis (S. baicalensis) is one of the traditional Chinese medicinal herbs that have been shown to possess many health benefits. In the present study, we evaluated the in vitro antiviral activity of aqueous extract of the roots of S. baicalensis against all the four dengue virus (DENV) serotypes. Methods Aqueous extract of S. baicalensis was prepared by microwave energy steam evaporation method (MEGHE™), and the anti-dengue virus replication activity was evaluated using the foci forming unit reduction assay (FFURA) in Vero cells. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to determine the actual dengue virus RNA copy number. The presence of baicalein, a flavonoid known to inhibit dengue virus replication was determined by mass spectrometry. Results The IC50 values for the S. baicalensis extract on Vero cells following DENV adsorption ranged from 86.59 to 95.19 μg/mL for the different DENV serotypes. The IC50 values decreased to 56.02 to 77.41 μg/mL when cells were treated with the extract at the time of virus adsorption for the different DENV serotypes. The extract showed potent direct virucidal activity against extracellular infectious virus particles with IC50 that ranged from 74.33 to 95.83 μg/mL for all DENV serotypes. Weak prophylactic effects with IC50 values that ranged from 269.9 to 369.8 μg/mL were noticed when the cells were pre-treated 2 hours prior to virus inoculation. The concentration of baicalein in the S. baicalensis extract was ~1% (1.03 μg/gm dried extract). Conclusions Our study demonstrates the in vitro anti-dengue virus replication property of S. baicalensis against all the four DENV serotypes investigated. The extract reduced DENV infectivity and replication in Vero cells. The extract was rich in baicalein, and could be considered for potential development of anti-DENV therapeutics. PMID:23627436

  9. Replicating adenovirus vector prime/protein boost strategies for HIV vaccine development

    PubMed Central

    Patterson, L. Jean; Robert-Guroff, Marjorie

    2008-01-01

    Background In the last few years the HIV vaccine field has moved forward a number of promising vaccine candidates into human clinical trials. Objective In this review we briefly discuss the advances made in vaccine development and HIV pathogenesis and give an overview of the body of work our lab has generated in multiple animal models on replication-competent Ad recombinant vaccines. Methods Emphasis is placed on comparative examination of vaccine components, routes of immunization and challenge models using replicating Ad vectors. Results/conclusion The overall findings make the case that replicating Ad vectors are superior in priming multiple arms of the immune system, and in conjunction with protein boosting, have resulted in dramatic protective efficacy leading to their advancement to phase 1 trials. Implications of the recent halting of the Merck Ad5-HIV phase 2b clinical trial for our vaccine approach and other vectored vaccines are discussed. PMID:18694354

  10. Exosomes in human semen restrict HIV-1 transmission by vaginal cells and block intravaginal replication of LP-BM5 murine AIDS virus complex

    PubMed Central

    Madison, Marisa N.; Jones, Philip H.; Okeoma, Chioma M.

    2015-01-01

    Exosomes are membranous extracellular nanovesicles secreted by diverse cell types. Exosomes from healthy human semen have been shown to inhibit HIV-1 replication and to impair progeny virus infectivity. In this study, we examined the ability of healthy human semen exosomes to restrict HIV-1 and LP-BM5 murine AIDS virus transmission in three different model systems. We show that vaginal cells internalize exosomes with concomitant transfer of functional mRNA. Semen exosomes blocked the spread of HIV-1 from vaginal epithelial cells to target cells in our cell-to-cell infection model and suppressed transmission of HIV-1 across the vaginal epithelial barrier in our trans-well model. Our in vivo model shows that human semen exosomes restrict intravaginal transmission and propagation of murine AIDS virus. Our study highlights an antiretroviral role for semen exosomes that may be harnessed for the development of novel therapeutic strategies to combat HIV-1 transmission. PMID:25880110

  11. Potent inhibition of HIV type 1 infection of mononuclear phagocytes by synthetic peptide analogs of HIV type 1 protease substrates.

    PubMed

    Dukes, C S; Matthews, T J; Lambert, D M; Dreyer, G B; Petteway, S R; Weinberg, J B

    1996-06-10

    The HIV-1 genome encodes a protease that is required for viral processing of the precursor polyproteins Pr55gag and Pr160gag-pol. Interference with this process in human lymphocytes inhibits production of infectious virus. We tested the ability of several protease inhibitors to decrease replication of HIV-1BaL in human monocytes and peritoneal macrophages. The compounds tested are oligopeptide analogs of HIV-1 protease substrates in which the scissile dipeptide has been replaced by a hydroxyethylene isostere. The protease inhibitors were added only once, 1 hr prior to inoculation with virus. Every 3-5 days, half the medium was replaced with fresh medium. Inhibition of virus production was assessed by measuring reverse transcriptase (RT) activity in supernatant medium 14 days after infection. The concentration of drug required to inhibit infection by 50% (IC50) in monocytes ranged from 0.17 to 2.99 microM; IC50 values for peritoneal macrophages ranged from 0.21 to 1.9 microM. The IC50 values for these compounds were 1.1- to 10-fold higher when tested in monocytes compared to their inhibitory effect in lymphocytes, although still potently effective in the dosage range that appeared nontoxic to cells. Cell toxicity was seen only at concentrations greater than 10 microM, and varied among the drugs tested. Immunoblot analysis of two of the drugs (SB205700 and SB108922) confirmed inhibition of polyprotein processing. In control cells, 22% of viral protein pr55 was processed to p24 by 24 hr, and 51% was processed by 48 hr. In cells treated with the protease inhibitors (2 microM), Pr55 processing was inhibited 77% at 24 hr and 89% at 48 hr. Thus, these synthetic peptide analogs potently inhibit productive infection of mononuclear phagocytes by HIV-1. Drugs of this class may be useful for the treatment of HIV-1 infection in humans.

  12. Potent and highly selective human immunodeficiency virus type 1 (HIV-1) inhibition by a series of alpha-anilinophenylacetamide derivatives targeted at HIV-1 reverse transcriptase.

    PubMed Central

    Pauwels, R; Andries, K; Debyser, Z; Van Daele, P; Schols, D; Stoffels, P; De Vreese, K; Woestenborghs, R; Vandamme, A M; Janssen, C G

    1993-01-01

    In vitro evaluation of a large chemical library of pharmacologically acceptable prototype compounds in a high-capacity, cellular-based screening system has led to the discovery of another family of human immunodeficiency virus type 1 (HIV-1) inhibitors. Through optimization of a lead compound, several alpha-anilinophenylacetamide (alpha-APA) derivatives have been identified that inhibit the replication of several HIV-1 strains (IIIB/LAI, RF, NDK, MN, HE) in a variety of host cell types at concentrations that are 10,000- to 100,000-fold lower than their cytotoxic concentrations. The IC50 of the alpha-APA derivative R 89439 for HIV-1 cytopathicity in MT-4 cells was 13 nM. The median 90% inhibitory concentration (IC90) in a variety of host cells was 50-100 nM. Although these alpha-APA derivatives are active against a tetrahydroimidazo [4,5,1-jk][1,4]benzodiazepin-2(1H)-thione-(TIBO)-resistant HIV-1 strain, they do not inhibit replication of HIV-2 (strains ROD and EHO) or simian immunodeficiency virus (strains Mac251, mndGB1, and agm3). An HIV-1 strain containing the Tyr181-->Cys mutation in the reverse transcriptase region displayed reduced sensitivity. alpha-APA derivative R 89439 inhibited virion and recombinant reverse transcriptase of HIV-1 but did not inhibit that of HIV-2. Reverse transcriptase inhibition depended upon the template/primer used. The relatively uncomplicated synthesis of R 89439, its potent anti-HIV-1 activity, and its favorable pharmacokinetic profile make R 89439 a good candidate for clinical studies. PMID:7680476

  13. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    PubMed

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  14. HIV-1 Adapts To Replicate in Cells Expressing Common Marmoset APOBEC3G and BST2

    PubMed Central

    Fernández-Oliva, Alberto; Finzi, Andrés; Haim, Hillel; Menéndez-Arias, Luis; Sodroski, Joseph

    2015-01-01

    ABSTRACT Previous studies have shown that a major block to HIV-1 replication in common marmosets operates at the level of viral entry and that this block can be overcome by adaptation of the virus in tissue-cultured cells. However, our current studies indicate that HIV-1 encounters additional postentry blocks in common marmoset peripheral blood mononuclear cells. Here, we show that the common marmoset APOBEC3G (A3G) and BST2 proteins block HIV-1 in cell cultures. Using a directed-evolution method that takes advantage of the natural ability of HIV-1 to mutate during replication, we have been able to overcome these blocks in tissue-cultured cells. In the adapted viruses, specific changes were observed in gag, vif, env, and nef. The contribution of these changes to virus replication in the presence of the A3G and BST2 restriction factors was studied. We found that certain amino acid changes in Vif and Env that arise during adaptation to marmoset A3G and BST2 allow the virus to replicate in the presence of these restriction factors. The changes in Vif reduce expression levels and encapsidation of marmoset APOBEC3G, while the changes in Env increase viral fitness and discretely favor cell-to-cell transmission of the virus, allowing viral escape from these restriction factors. IMPORTANCE HIV-1 can infect only humans and chimpanzees. The main reason for this narrow tropism is the presence in many species of dominant-acting factors, known as restriction factors, that block viral replication in a species-specific way. We have been exploring the blocks to HIV-1 in common marmosets, with the ultimate goal of developing a new animal model of HIV-1 infection in these monkeys. In this study, we observed that common marmoset APOBEC3G and BST2, two known restriction factors, are able to block HIV-1 in cell cultures. We have adapted HIV-1 to replicate in the presence of these restriction factors and have characterized the mechanisms of escape. These studies can help in the

  15. Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus.

    PubMed

    Martins, Laura J; Bonczkowski, Pawel; Spivak, Adam M; De Spiegelaere, Ward; Novis, Camille L; DePaula-Silva, Ana Beatriz; Malatinkova, Eva; Typsteen, Wim; Bosque, Alberto; Vanderkerckhove, Linos; Planelles, Vicente

    2016-02-01

    HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4(+) T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput.

  16. Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus

    PubMed Central

    Martins, Laura J.; Bonczkowski, Pawel; Spivak, Adam M.; De Spiegelaere, Ward; Novis, Camille L.; DePaula-Silva, Ana Beatriz; Malatinkova, Eva; Typsteen, Wim; Vanderkerckhove, Linos

    2016-01-01

    Abstract HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4+ T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput. PMID:26171776

  17. Prepulse inhibition in HIV-associated neurocognitive disorders.

    PubMed

    Minassian, Arpi; Henry, Brook L; Woods, Steven Paul; Vaida, Florin; Grant, Igor; Geyer, Mark A; Perry, William

    2013-07-01

    Sensorimotor inhibition, or the ability to filter out excessive or irrelevant information, theoretically supports a variety of higher-level cognitive functions. Impaired inhibition may be associated with increased impulsive and risky behavior in everyday life. Individuals infected with HIV frequently show impairment on tests of neurocognitive function, but sensorimotor inhibition in this population has not been studied and may be a contributor to the profile of HIV-associated neurocognitive disorders (HAND). Thirty-seven HIV-infected individuals (15 with HAND) and 48 non-infected comparison subjects were assessed for prepulse inhibition (PPI), an eyeblink startle paradigm measuring sensorimotor gating. Although HIV status alone was not associated with PPI deficits, HIV-positive participants meeting criteria for HAND showed impaired PPI compared to cognitively intact HIV-positive subjects. In HIV-positive subjects, PPI was correlated with working memory but was not associated with antiretroviral therapy or illness factors. In conclusion, sensorimotor disinhibition in HIV accompanies deficits in higher-order cognitive functions, although the causal direction of this relationship requires investigation. Subsequent research on the role of sensorimotor gating on decision-making and risk behaviors in HIV may be indicated.

  18. Prepulse Inhibition in HIV-Associated Neurocognitive Disorders

    PubMed Central

    Minassian, Arpi; Henry, Brook L.; Woods, Steven Paul; Vaida, Florin; Grant, Igor; Geyer, Mark A.; Perry, William

    2013-01-01

    Sensorimotor inhibition, or the ability to filter out excessive or irrelevant information, theoretically supports a variety of higher-level cognitive functions. Impaired inhibition may be associated with increased impulsive and risky behavior in everyday life. Individuals infected with HIV frequently show impairment on tests of neurocognitive function, but sensorimotor inhibition in this population has not been studied and may be a contributor to the profile of HIV-associated Neurocognitive Disorders (HAND). 37 HIV-infected individuals (15 with HAND) and 48 non-infected comparison subjects were assessed for prepulse inhibition (PPI), an eyeblink startle paradigm measuring sensorimotor gating. Although HIV status alone was not associated with PPI deficits, HIV-positive participants meeting criteria for HAND showed impaired PPI compared to cognitively intact HIV-positive subjects. In HIV-positive subjects, PPI was correlated with working memory but was not associated with antiretroviral therapy or illness factors. In conclusion, sensorimotor disinhibition in HIV accompanies deficits in higher-order cognitive functions, though the causal direction of this relationship requires investigation. Subsequent research on the role of sensorimotor gating on decision-making and risk behaviors in HIV may be indicated. PMID:23552464

  19. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

    PubMed Central

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M.; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  20. A lectin isolated from bananas is a potent inhibitor of HIV replication.

    PubMed

    Swanson, Michael D; Winter, Harry C; Goldstein, Irwin J; Markovitz, David M

    2010-03-19

    BanLec is a jacalin-related lectin isolated from the fruit of bananas, Musa acuminata. This lectin binds to high mannose carbohydrate structures, including those found on viruses containing glycosylated envelope proteins such as human immunodeficiency virus type-1 (HIV-1). Therefore, we hypothesized that BanLec might inhibit HIV-1 through binding of the glycosylated HIV-1 envelope protein, gp120. We determined that BanLec inhibits primary and laboratory-adapted HIV-1 isolates of different tropisms and subtypes. BanLec possesses potent anti-HIV activity, with IC(50) values in the low nanomolar to picomolar range. The mechanism for BanLec-mediated antiviral activity was investigated by determining if this lectin can directly bind the HIV-1 envelope protein and block entry of the virus into the cell. An enzyme-linked immunosorbent assay confirmed direct binding of BanLec to gp120 and indicated that BanLec can recognize the high mannose structures that are recognized by the monoclonal antibody 2G12. Furthermore, BanLec is able to block HIV-1 cellular entry as indicated by temperature-sensitive viral entry studies and by the decreased levels of the strong-stop product of early reverse transcription seen in the presence of BanLec. Thus, our data indicate that BanLec inhibits HIV-1 infection by binding to the glycosylated viral envelope and blocking cellular entry. The relative anti-HIV activity of BanLec compared favorably to other anti-HIV lectins, such as snowdrop lectin and Griffithsin, and to T-20 and maraviroc, two anti-HIV drugs currently in clinical use. Based on these results, BanLec is a potential component for an anti-viral microbicide that could be used to prevent the sexual transmission of HIV-1.

  1. Inhibition of human immunodeficiency virus type 1 (HIV-1) nuclear import via Vpr-Importin {alpha} interactions as a novel HIV-1 therapy

    SciTech Connect

    Suzuki, Tatsunori; Yamamoto, Norio; Nonaka, Mizuho; Hashimoto, Yoshie; Matsuda, Go; Takeshima, Shin-nosuke; Matsuyama, Megumi; Igarashi, Tatsuhiko; Miura, Tomoyuki; Tanaka, Rie; Kato, Shingo; Aida, Yoko

    2009-03-20

    The development of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus (HIV) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. One such target is the interaction between Vpr, one of the accessory gene products of HIV-1 and Importin {alpha}, which is crucial, not only for the nuclear import of Vpr, but also for HIV-1 replication in macrophages. We have identified a potential parent compound, hematoxylin, which suppresses Vpr-Importin {alpha} interaction, thereby inhibiting HIV-1 replication in a Vpr-dependent manner. Analysis by real-time PCR demonstrated that hematoxylin specifically inhibited nuclear import step of pre-integration complex. Thus, hematoxylin is a new anti-HIV-1 inhibitor that targets the nuclear import of HIV-1 via the Vpr-Importin {alpha} interaction, suggesting that a specific inhibitor of the interaction between viral protein and the cellular factor may provide a new strategy for HIV-1 therapy.

  2. A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription.

    PubMed Central

    Bohjanen, P R; Colvin, R A; Puttaraju, M; Been, M D; Garcia-Blanco, M A

    1996-01-01

    Linear TAR RNA has previously been used as a decoy to inhibit HIV-1 transcription in vitro and HIV-1 replication in vivo. A 48 nucleotide circular RNA containing the stem, bulge and loop of the HIV-1 TAR element was synthesized using the self-splicing activity of a group I permuted intron-exon and was tested for its ability to function as a TAR decoy in vitro. This small circular TAR molecule was exceptionally stable in HeLa nuclear extracts, whereas a similar linear TAR molecule was rapidly degraded. The TAR circle bound specifically to Tfr38, a peptide containing the TAR-binding region of Tat. The ability of Tat to trans-activate transcription from the HIV-1 promoter in vitro was efficiently inhibited by circular TAR RNA but not by TAR circles that contained either bulge or loop mutations. TAR circles did not inhibit transactivation exclusively by binding to Tat since this inhibition was not reversed by adding excess Tat to the transcription reaction. Together, these data suggest that TAR circles act as decoys that inhibit transactivation by binding to Tat and at least one cellular factor. These data also demonstrate the utility of small circular RNA molecules as tools for biochemical studies. PMID:8871552

  3. Broad HIV-1 inhibition in vitro by vaccine-elicited CD8+ T cells in African adults

    PubMed Central

    Mutua, Gaudensia; Farah, Bashir; Langat, Robert; Indangasi, Jackton; Ogola, Simon; Onsembe, Brian; Kopycinski, Jakub T; Hayes, Peter; Borthwick, Nicola J; Ashraf, Ambreen; Dally, Len; Barin, Burc; Tillander, Annika; Gilmour, Jill; De Bont, Jan; Crook, Alison; Hannaman, Drew; Cox, Josephine H; Anzala, Omu; Fast, Patricia E; Reilly, Marie; Chinyenze, Kundai; Jaoko, Walter; Hanke, Tomáš; HIV-CORE 004 study group, the

    2016-01-01

    We are developing a pan-clade HIV-1 T-cell vaccine HIVconsv, which could complement Env vaccines for prophylaxis and be a key to HIV cure. Our strategy focuses vaccine-elicited effector T-cells on functionally and structurally conserved regions (not full-length proteins and not only epitopes) of the HIV-1 proteome, which are common to most global variants and which, if mutated, cause a replicative fitness loss. Our first clinical trial in low risk HIV-1-negative adults in Oxford demonstrated the principle that naturally mostly subdominant epitopes, when taken out of the context of full-length proteins/virus and delivered by potent regimens involving combinations of simian adenovirus and poxvirus modified vaccinia virus Ankara, can induce robust CD8+ T cells of broad specificities and functions capable of inhibiting in vitro HIV-1 replication. Here and for the first time, we tested this strategy in low risk HIV-1-negative adults in Africa. We showed that the vaccines were well tolerated and induced high frequencies of broadly HIVconsv-specific plurifunctional T cells, which inhibited in vitro viruses from four major clades A, B, C, and D. Because sub-Saharan Africa is globally the region most affected by HIV-1/AIDS, trial HIV-CORE 004 represents an important stage in the path toward efficacy evaluation of this highly rational and promising vaccine strategy. PMID:27617268

  4. A Computational Model of Inhibition of HIV-1 by Interferon-Alpha

    PubMed Central

    Browne, Edward P.; Letham, Benjamin; Rudin, Cynthia

    2016-01-01

    Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses. PMID:27010978

  5. A Computational Model of Inhibition of HIV-1 by Interferon-Alpha.

    PubMed

    Browne, Edward P; Letham, Benjamin; Rudin, Cynthia

    2016-01-01

    Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses.

  6. Replicating a Teen HIV/STD Preventive Intervention in a Multicultural City

    ERIC Educational Resources Information Center

    Morrison, Diane M.; Hoppe, Marilyn J.; Wells, Elizabeth A.; Beadnell, Blair A.; Wilsdon, Anthony; Higa, Darrel; Gillmore, Mary Rogers; Casey, Erin A.

    2007-01-01

    Although there are now several adolescent HIV and STD preventive interventions of demonstrated efficacy in the literature, little is understood about the portability of these interventions. This study replicated Stanton's Focus on Kids intervention, developed for inner city African American adolescents, in a different population, transferring it…

  7. Replicating Impact of a Primary School HIV Prevention Programme: Primary School Action for Better Health, Kenya

    ERIC Educational Resources Information Center

    Maticka-Tyndale, E.; Mungwete, R.; Jayeoba, O.

    2014-01-01

    School-based programmes to combat the spread of HIV have been demonstrated to be effective over the short-term when delivered on a small scale. The question addressed here is whether results obtained with small-scale delivery are replicable in large-scale roll-out. Primary School Action for Better Health (PSABH), a programme to train teachers to…

  8. A novel mechanism for inhibition of HIV-1 reverse transcriptase.

    PubMed

    Skillman, A Geoffrey; Maurer, Karl W; Roe, Diana C; Stauber, Margaret J; Eargle, Dolan; Ewing, Todd J A; Muscate, Angelika; Davioud-Charvet, Elisabeth; Medaglia, Maxine V; Fisher, Robert J; Arnold, Edward; Gao, Hong Qiang; Buckheit, Robert; Boyer, Paul L; Hughes, Stephen H; Kuntz, Irwin D; Kenyon, George L

    2002-12-01

    The human immunodeficiency virus (HIV) epidemic is an important medical problem. Although combination drug regimens have produced dramatic decreases in viral load, current therapies do not provide a cure for HIV infection. We have used structure-based design and combinatorial medicinal chemistry to identify potent and selective HIV-1 reverse transcriptase (RT) inhibitors that may work by a mechanism distinct from that of current HIV drugs. The most potent of these compounds (compound 4, 2-naphthalenesulfonic acid, 4-hydroxy-7-[[[[5-hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)azo], disodium salt) has an IC(50) of 90 nM for inhibition of polymerase chain extension, a K(d) of 40 nM for inhibition of DNA-RT binding, and an IC(50) of 25-100 nM for inhibition of RNaseH cleavage. The parent compound (1) was as effective against 10 nucleoside and non-nucleoside resistant HIV-1 RT mutants as it was against the wild-type enzyme. Compound 4 inhibited HIV-1 RT and murine leukemia virus (MLV) RT, but it did not inhibit T(4) DNA polymerase, T(7) DNA polymerase, or the Klenow fragment at concentrations up to 200 nM. Finally, compound 4 protected cells from HIV-1 infection at a concentration more than 40 times lower than the concentration at which it caused cellular toxicity.

  9. Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication

    PubMed Central

    Tucker, Lynne D.; Asara, John M.; Cheruiyot, Collins K.; Lu, Huafei; Wu, Zhijin J.; Newstein, Michael C.; Dooner, Mark S.; Friedman, Jennifer; Lally, Michelle A.; Ramratnam, Bharat

    2016-01-01

    A rare subset of HIV-1–infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1–infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1–infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection. PMID:27454292

  10. Role of β-catenin/TCF-4 signaling in HIV replication and pathogenesis: insights to informing novel anti-HIV molecular therapeutics.

    PubMed

    Henderson, Lisa J; Al-Harthi, Lena

    2011-06-01

    A greater understanding of the interaction between HIV and host signaling pathways that restrict virus production may lead to new methods to purge virus from latent reservoirs and enhance survival/function of cells targeted by HIV. This review highlights the role of the Wnt/β-catenin pathway as a host factor that represses HIV replication in multiple targets, especially those relevant to HIV in the central nervous system.

  11. Hydroxyurea inhibits the transactivation of the HIV-long-terminal repeat (LTR) promoter

    PubMed Central

    Calzado, M A; Macho, A; Lucena, C; Muñoz, E

    2000-01-01

    HIV-1 gene expression is regulated by the promoter/enhancer located within the U3 region of the proviral 5′ LTR that contains multiple potential cis-acting regulatory sites. Here we describe that the inhibitor of the cellular ribonucleoside reductase, hydroxyurea (HU), inhibited phorbol myristate acetate- or tumour necrosis factor-alpha-induced HIV-1-LTR transactivation in both lymphoid and non-lymphoid cells in a dose-dependent manner within the first 6 h of treatment, with a 50% inhibitory concentration of 0·5 mm. This inhibition was found to be specific for the HIV-1-LTR since transactivation of either an AP-1-dependent promoter or the CD69 gene promoter was not affected by the presence of HU. Moreover, gel-shift assays in 5.1 cells showed that HU prevented the binding of the NF-κB to the κB sites located in the HIV-1-LTR region, but it did not affect the binding of both the AP-1 and the Sp-1 transcription factors. By Western blots and cell cycle analyses we detected that HU induced a rapid dephosphorylation of the pRB, the product of the retinoblastoma tumour suppressor gene, and the cell cycle arrest was evident after 24 h of treatment. Thus, HU inhibits HIV-1 promoter activity by a novel pathway that implies an inhibition of the NF-κB binding to the LTR promoter. The present study suggests that HU may be useful as a potential therapeutic approach for inhibition of HIV-1 replication through different pathways. PMID:10792382

  12. Inhibition of HIV-1 by fusion inhibitors.

    PubMed

    Eggink, Dirk; Berkhout, Ben; Sanders, Rogier W

    2010-01-01

    The envelope glycoprotein complex (Env) is responsible for entry of the human immunodeficiency virus type 1 (HIV-1) into cells by mediating attachment to target cells and subsequent membrane fusion. Env consists of three gp120 subunits that mediate receptor and co-receptor attachment and three gp41 subunits responsible for membrane fusion. Several steps of the entry process can serve as drug targets. Receptor antagonists prevent attachment of gp120 to the receptor or co-receptor and conformational changes within gp41 required for membrane fusion can be inhibited by fusion inhibitors. Enfuvirtide (T20, Fuzeon) is a peptide based on the gp41 sequence and is the only approved fusion inhibitor. It prevents membrane fusion by competitively binding to gp41 and blocking the formation of the post-fusion structure. New generations of T20-like peptides have been developed with improved potency and stability. Besides T20 and derivatives, other fusion inhibitors have been developed that target different domains of gp41. Here we discuss the development of fusion inhibitors, their mode of action and their potential for incorporation in future drug regimens.

  13. Phospholipase D1 Couples CD4+ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication

    PubMed Central

    Taylor, Harry E.; Simmons, Glenn E.; Mathews, Thomas P.; Khatua, Atanu K.; Popik, Waldemar; Lindsley, Craig W.; D’Aquila, Richard T.; Brown, H. Alex

    2015-01-01

    Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis. PMID:26020637

  14. HIV-1 Nef Is Transferred from Expressing T Cells to Hepatocytic Cells through Conduits and Enhances HCV Replication

    PubMed Central

    Park, In-Woo; Fan, Yan; Luo, Xiaoyu; Ryou, Myoung-Gwi; Liu, Jinfeng; Green, Linden; He, Johnny J.

    2014-01-01

    HIV-1 infection enhances HCV replication and as a consequence accelerates HCV-mediated hepatocellular carcinoma (HCC). However, the precise molecular mechanism by which this takes place is currently unknown. Our data showed that infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the cell lines transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was transferred from nef-expressing T cells to hepatocytic cells through conduits, wherein up to 16% (average 10%) of the cells harbored the transferred Nef, when the hepatocytic cells were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of lipid droplets (LD), and consistently up-regulated HCV replication by 1.5∼2.5 fold in the target subgenomic replicon cells, which is remarkable in relation to the initially indolent viral replication. Nef also dramatically augmented reactive oxygen species (ROS) production and enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate HCC. Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating progression of liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay. PMID:24911518

  15. Efficient HIV-1 replication can occur in the absence of the viral matrix protein.

    PubMed Central

    Reil, H; Bukovsky, A A; Gelderblom, H R; Göttlinger, H G

    1998-01-01

    Matrix (MA), a major structural protein of retroviruses, is thought to play a critical role in several steps of the HIV-1 replication cycle, including the plasma membrane targeting of Gag, the incorporation of envelope (Env) glycoproteins into nascent particles, and the nuclear import of the viral genome in non-dividing cells. We now show that the entire MA protein is dispensable for the incorporation of HIV-1 Env glycoproteins with a shortened cytoplasmic domain. Furthermore, efficient HIV-1 replication in the absence of up to 90% of MA was observed in a cell line in which the cytoplasmic domain of Env is not required. Additional compensatory changes in Gag permitted efficient virus replication even if all of MA was replaced by a heterologous membrane targeting signal. Viruses which lacked the globular domain of MA but retained its N-terminal myristyl anchor exhibited an increased ability to form both extracellular and intracellular virus particles, consistent with a myristyl switch model of Gag membrane targeting. Pseudotyped HIV-1 particles that lacked the structurally conserved globular head of MA efficiently infected macrophages, indicating that MA is dispensable for nuclear import in terminally differentiated cells. PMID:9564051

  16. Efficient HIV-1 replication can occur in the absence of the viral matrix protein.

    PubMed

    Reil, H; Bukovsky, A A; Gelderblom, H R; Göttlinger, H G

    1998-05-01

    Matrix (MA), a major structural protein of retroviruses, is thought to play a critical role in several steps of the HIV-1 replication cycle, including the plasma membrane targeting of Gag, the incorporation of envelope (Env) glycoproteins into nascent particles, and the nuclear import of the viral genome in non-dividing cells. We now show that the entire MA protein is dispensable for the incorporation of HIV-1 Env glycoproteins with a shortened cytoplasmic domain. Furthermore, efficient HIV-1 replication in the absence of up to 90% of MA was observed in a cell line in which the cytoplasmic domain of Env is not required. Additional compensatory changes in Gag permitted efficient virus replication even if all of MA was replaced by a heterologous membrane targeting signal. Viruses which lacked the globular domain of MA but retained its N-terminal myristyl anchor exhibited an increased ability to form both extracellular and intracellular virus particles, consistent with a myristyl switch model of Gag membrane targeting. Pseudotyped HIV-1 particles that lacked the structurally conserved globular head of MA efficiently infected macrophages, indicating that MA is dispensable for nuclear import in terminally differentiated cells.

  17. Host cell factors in HIV replication: meta-analysis of genome-wide studies.

    PubMed

    Bushman, Frederic D; Malani, Nirav; Fernandes, Jason; D'Orso, Iván; Cagney, Gerard; Diamond, Tracy L; Zhou, Honglin; Hazuda, Daria J; Espeseth, Amy S; König, Renate; Bandyopadhyay, Sourav; Ideker, Trey; Goff, Stephen P; Krogan, Nevan J; Frankel, Alan D; Young, John A T; Chanda, Sumit K

    2009-05-01

    We have analyzed host cell genes linked to HIV replication that were identified in nine genome-wide studies, including three independent siRNA screens. Overlaps among the siRNA screens were very modest (<7% for any pairwise combination), and similarly, only modest overlaps were seen in pairwise comparisons with other types of genome-wide studies. Combining all genes from the genome-wide studies together with genes reported in the literature to affect HIV yields 2,410 protein-coding genes, or fully 9.5% of all human genes (though of course some of these are false positive calls). Here we report an "encyclopedia" of all overlaps between studies (available at http://www.hostpathogen.org), which yielded a more extensively corroborated set of host factors assisting HIV replication. We used these genes to calculate refined networks that specify cellular subsystems recruited by HIV to assist in replication, and present additional analysis specifying host cell genes that are attractive as potential therapeutic targets.

  18. XTT formazan widely used to detect cell viability inhibits HIV type 1 infection in vitro by targeting gp41.

    PubMed

    Zhao, Qian; Ernst, Justin T; Hamilton, Andrew D; Debnath, Asim K; Jiang, Shibo

    2002-09-20

    XTT can be metabolically reduced by mitochondrial dehydrogenase in viable cells to a water-soluble formazan product. Thus XTT has been widely used to evaluate cell viability and to screen anti-HIV agents and the cytotoxicity of these agents. The present studies demonstrated that XTT formazan derived from XTT in cell culture significantly inhibits the fusion of HIV-1-infected cells with uninfected cells. Synthetic XTT formazan effectively inhibited the replication of laboratory-adapted and primary HIV-1 isolates and cell-to-cell fusion with low cytotoxicity. It blocks the six-helix bundle formation between peptides derived from the N- and C-terminal heptad repeat regions of the gp41 ectodomain (designated N- and C-peptides, respectively). Analysis by a computer-aided docking program indicates that XTT formazan may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil of gp41. These results suggest that XTT formazan inhibits HIV-1 entry by targeting the alpha-helical coiled-coil domain of gp41. This small molecular nonpeptide antiviral compound can be used as a lead for designing more effective HIV-1 entry inhibitors targeting the fusion stage of HIV-1 infection. But because XTT formazan itself has anti-HIV-1 activity, caution should be exercised when XTT is used to evaluate HIV-1 infectivity.

  19. Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31.

    PubMed Central

    Lee-Huang, S; Huang, P L; Huang, P L; Bourinbaiar, A S; Chen, H C; Kung, H F

    1995-01-01

    MAP30 (Momordica anti-HIV protein of 30 kDa) and GAP31 (Gelonium anti-HIV protein of 31 kDa) are anti-HIV plant proteins that we have identified, purified, and cloned from the medicinal plants Momordica charantia and Gelonium multiflorum. These antiviral agents are capable of inhibiting infection of HIV type 1 (HIV-1) in T lymphocytes and monocytes as well as replication of the virus in already-infected cells. They are not toxic to normal uninfected cells because they are unable to enter healthy cells. MAP30 and GAP31 also possess an N-glycosidase activity on 28S ribosomal RNA and a topological activity on plasmid and viral DNAs including HIV-1 long terminal repeats (LTRs). LTRs are essential sites for integration of viral DNA into the host genome by viral integrase. We therefore investigated the effect of MAP30 and GAP31 on HIV-1 integrase. We report that both of these antiviral agents exhibit dose-dependent inhibition of HIV-1 integrase. Inhibition was observed in all of the three specific reactions catalyzed by the integrase, namely, 3' processing (specific cleavage of the dinucleotide GT from the viral substrate), strand transfer (integration), and "disintegration" (the reversal of strand transfer). Inhibition was studied by using oligonucleotide substrates with sequences corresponding to the U3 and U5 regions of HIV LTR. In the presence of 20 ng of viral substrate, 50 ng of target substrate, and 4 microM integrase, total inhibition was achieved at equimolar concentrations of the integrase and the antiviral proteins, with EC50 values of about 1 microM. Integration of viral DNA into the host chromosome is a vital step in the replicative cycle of retroviruses, including the AIDS virus. The inhibition of HIV-1 integrase by MAP30 and GAP31 suggests that impediment of viral DNA integration may play a key role in the anti-HIV activity of these plant proteins. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568024

  20. Modulation of HCV Replication After Combination Antiretroviral Therapy in HCV/HIV Coinfected Patients

    PubMed Central

    Sherman, Kenneth E.; Guedj, Jeremie; Shata, Mohamed Tarek; Blackard, Jason T.; Rouster, Susan D.; Castro, Mario; Feinberg, Judith; Sterling, Richard K.; Goodman, Zachary; Aronow, Bruce J.; Perelson, Alan S.

    2015-01-01

    The hepatitis C virus (HCV) is an important contributor to morbidity and mortality in patients coinfected with human immunodeficiency virus (HIV). Coinfection results in increased HCV replication and more rapid rates of liver disease progression. The effect of HIV combination antiretroviral therapy (cART) on HCV replication has not been studied in depth. To address this issue, we enrolled a small cohort of HCV/HIV coinfected patients into a cART initiation trial, and used dynamic modeling combined with evaluation of immune responses and microarray profiles to determine how effective treatment of HIV affects HCV. Treatment with cART resulted in HCV flare and alanine aminotransferase (ALT) increase (2× or more increase from baseline) in a subset of treated patients. Subjects with evidence of hepatic injury (increased ALT) were more likely to have HCV-specific immune responses directed against HCV epitopes. Over time, HCV viral loads declined. Reproducible and biologically important gene expression changes occurred in patients who underwent successful cART, particularly with respect to downregulation of genes with known antiviral roles. Our findings suggest that the effective suppression of HIV by cART initiates a cascade of early and late events in treated patients with HCV. Early events involving downregulation of interferon-stimulated genes may lead to transiently increased viral replication and hepatic injury. At later time points, HCV viral load declines to levels comparable to those seen in the setting of HCV monoinfection. These findings support early antiretroviral therapy in those with HCV/HIV coinfection. PMID:25101888

  1. Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction

    PubMed Central

    Somsouk, Ma; Hunt, Peter W.

    2017-01-01

    Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection. PMID:28241080

  2. Interference of replication between hepatitis B and C viruses in patients infected with HIV.

    PubMed

    Yang, Rong-Rong; Gui, Xien; Chen, Xue-Yuan; Zhu, Ying

    2011-07-01

    The clinical and cellular interactions between hepatitis B virus (HBV) and hepatitis C virus (HCV) were investigated in patients co-infected with the human immunodeficiency virus (HIV). One hundred ninety-nine patients followed for 6 years were evaluated to compare the level of HBV DNA and HCV RNA in patients co-infected with HIV and HBV, and patients co-infected with HIV, HBV, and HCV. A full-length HBV genome and HCV JFH1 RNA were co-transfected into HuH-7.5.1 cells in vitro to examine the impact of co-infection and dependence on the HBV PreC mutant for replication interference. Before 2',3'-dideoxy-3'-thiacytidine (3TC)-based antiretroviral therapy (ART) was initiated, HBV DNA was found in 56/123 (45.4%) patients co-infected with HIV and HBV, and in 19/76 (25.0%) patients co-infected with HIV, HBV, and HCV. After 3TC-based ART was initiated, detectable HBV DNA decreased to 7/76 (9.2%) in patients co-infected with HIV, HBV, and HCV, but HCV RNA increased from 43/76 (56.6%) to 60/76 (78.9%) (P = 0.003). In vitro HBV and HCV co-infection led to decreased replication of both viruses. The primary factors that influenced the decreased replication were the order of the HBV and HCV infection and the HBV PreC mutation.

  3. Chimpanzee GB virus C and GB virus A E2 envelope glycoproteins contain a peptide motif that inhibits human immunodeficiency virus type 1 replication in human CD4⁺ T-cells.

    PubMed

    McLinden, James H; Stapleton, Jack T; Klinzman, Donna; Murthy, Krishna K; Chang, Qing; Kaufman, Thomas M; Bhattarai, Nirjal; Xiang, Jinhua

    2013-04-01

    GB virus type C (GBV-C) is a lymphotropic virus that can cause persistent infection in humans. GBV-C is not associated with any disease, but is associated with reduced mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Related viruses have been isolated from chimpanzees (GBV-Ccpz) and from New World primates (GB virus type A, GBV-A). These viruses are also capable of establishing persistent infection. We determined the nucleotide sequence encoding the envelope glycoprotein (E2) of two GBV-Ccpz isolates obtained from the sera of captive chimpanzees. The deduced GBV-Ccpz E2 protein differed from human GBV-C by 31 % at the amino acid level. Similar to human GBV-C E2, expression of GBV-Ccpz E2 in a tet-off human CD4(+) Jurkat T-cell line significantly inhibited the replication of diverse HIV-1 isolates. This anti-HIV-replication effect of GBV-Ccpz E2 protein was reversed by maintaining cells in doxycycline to reduce E2 expression. Previously, we found a 17 aa region within human GBV-C E2 that was sufficient to inhibit HIV-1. Although GBV-Ccpz E2 differed by 3 aa differences in this region, the chimpanzee GBV-C 17mer E2 peptide inhibited HIV-1 replication. Similarly, the GBV-A peptide that aligns with this GBV-C E2 region inhibited HIV-1 replication despite sharing only 5 aa with the human GBV-C E2 sequence. Thus, despite amino acid differences, the peptide region on both the GBV-Ccpz and the GBV-A E2 protein inhibit HIV-1 replication similar to human GBV-C. Consequently, GBV-Ccpz or GBV-A infection of non-human primates may provide an animal model to study GB virus-HIV interactions.

  4. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner.

    PubMed Central

    Miller, S D; Farmer, G; Prives, C

    1995-01-01

    The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition. PMID:8524220

  5. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

    SciTech Connect

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-02-27

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

  6. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

    DOE PAGES

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-02-27

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNAmore » transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.« less

  7. The Interaction of RNA Helicase DDX3 with HIV-1 Rev-CRM1-RanGTP Complex during the HIV Replication Cycle

    PubMed Central

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-01-01

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP. PMID:25723178

  8. AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication.

    PubMed

    Booth, Laurence; Roberts, Jane L; Ecroyd, Heath; Tritsch, Sarah R; Bavari, Sina; Reid, St Patrick; Proniuk, Stefan; Zukiwski, Alexander; Jacob, Abraham; Sepúlveda, Claudia S; Giovannoni, Federico; García, Cybele C; Damonte, Elsa; González-Gallego, Javier; Tuñón, María J; Dent, Paul

    2016-10-01

    We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc.

  9. HIV-1 cellular and tissue replication patterns in infected humanized mice

    PubMed Central

    Araínga, Mariluz; Su, Hang; Poluektova, Larisa Y.; Gorantla, Santhi; Gendelman, Howard E.

    2016-01-01

    Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human. PMID:26996968

  10. Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

    PubMed Central

    König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

    2008-01-01

    Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

  11. HIV-1 replication in central nervous system increases over time on only protease inhibitor therapy.

    PubMed

    Donath, Maximilian; Wolf, Timo; Stürmer, Martin; Herrmann, Eva; Bickel, Markus; Khaykin, Pavel; Göpel, Siri; Gute, Peter; Haberl, Annette; de Leuw, Philipp; Schüttfort, Gundolf; Berger, Annemarie; Stephan, Christoph

    2016-12-01

    There are concerns about central nervous system (CNS)-replication of HIV-1 in patients on boosted protease inhibitors. Purpose of this study was to compare HIV-1 viral loads (VLs) from patients treated with only boosted dual protease inhibitor (bdPI), versus combination antiretroviral therapy (cART group), containing two nucleoside analogue reverse transcriptase inhibitors (NRTI) and a third partner. All patients from a large German HIV-treatment cohort with available medication, clinical and demographic data, including results from simultaneous HIV-1 viral load (VL) assessments in cerebrospinal fluid (CSF) and blood plasma, were retrospectively evaluated as controlled cross-sectional study. CSF had been obtained from patients with variable neurological symptoms during 2005-2014. Statistical analysis comprised nonparametric tests, regression and correlation techniques accounting for undetectable quantifications. Statistical analysis comprised nonparametric tests, regression and correlation techniques accounting for undetectable quantifications. Overall, 155 patients were evaluable (bdPI: 24; cART: 131). At time of CSF-collection, both groups were comparable in age, gender, CD4-cell counts, or primary HIV-transmission risks, though bdPI patients were clinically more advanced. The proportion of patients with undetectable HIV-1 (<50 copies/ml) in CSF was lower for bdPI group (25 vs 49.6 %; p = 0.026), but similar in plasma (46 vs 41 %). Median CSF-VL was higher in bdPI group (600 vs 50 copies/ml; p = 0.027) and similar in plasma. Mean VL CSF/plasma ratio was 342.91 for bdPI- and 54.48 for cART patients (p < 0.001). Pearson's regression analysis revealed a trend for an elevated VL-ratio over time within bdPI group. HIV-1 replication was higher and more frequently detectable in CSF from bdPI patients, indicating a worse CNS penetration effectiveness of used boosted PI. Within bdPI group, measured CNS-viral replication was increasing over time, suggesting an over

  12. Identification of a Cluster of HIV-1 Controllers Infected with Low Replicating Viruses

    PubMed Central

    Casado, Concepción; Pernas, Maria; Sandonis, Virginia; Alvaro-Cifuentes, Tamara; Olivares, Isabel; Fuentes, Rosa; Martínez-Prats, Lorena; Grau, Eulalia; Ruiz, Lidia; Delgado, Rafael; Rodríguez, Carmen; del Romero, Jorge; López-Galíndez, Cecilio

    2013-01-01

    Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70’s-early 80’s). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients. PMID:24204910

  13. Identification of a cluster of HIV-1 controllers infected with low replicating viruses.

    PubMed

    Casado, Concepción; Pernas, Maria; Sandonis, Virginia; Alvaro-Cifuentes, Tamara; Olivares, Isabel; Fuentes, Rosa; Martínez-Prats, Lorena; Grau, Eulalia; Ruiz, Lidia; Delgado, Rafael; Rodríguez, Carmen; del Romero, Jorge; López-Galíndez, Cecilio

    2013-01-01

    Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70's-early 80's). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients.

  14. siRNA and shRNA screens advance key understanding of host factors required for HIV-1 replication.

    PubMed

    Kok, Kin-Hang; Lei, Ting; Jin, Dong-Yan

    2009-08-27

    A recent RNAi screen used a genome-wide shRNA library to search for cellular factors required for HIV-1 replication. This work complements three other siRNA-based screening studies and potentially opens the door to the discovery of factors that are important for HIV-1 replication in physiological host cells such as T lymphocytes. shRNA screens can be further improved, and they could promise to unravel new pathways and new facets of virus-cell interactions.

  15. Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape

    PubMed Central

    Bar, Katharine J.; Tsao, Chun-yen; Iyer, Shilpa S.; Decker, Julie M.; Yang, Yongping; Bonsignori, Mattia; Chen, Xi; Hwang, Kwan-Ki; Montefiori, David C.; Liao, Hua-Xin; Hraber, Peter; Fischer, William; Li, Hui; Wang, Shuyi; Sterrett, Sarah; Keele, Brandon F.; Ganusov, Vitaly V.; Perelson, Alan S.; Korber, Bette T.; Georgiev, Ivelin; McLellan, Jason S.; Pavlicek, Jeffrey W.; Gao, Feng; Haynes, Barton F.; Hahn, Beatrice H.; Kwong, Peter D.; Shaw, George M.

    2012-01-01

    Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically

  16. Blocking CXCL9 Decreases HIV-1 Replication and Enhances the Activity of Prophylactic Antiretrovirals in Human Cervical Tissues

    PubMed Central

    Macura, Sherrill L.; Lathrop, Melissa J.; Gui, Jiang; Doncel, Gustavo F.; Rollenhagen, Christiane

    2016-01-01

    Objectives: The interferon-gamma–induced chemokine CXCL9 is expressed in a wide range of inflammatory conditions including those affecting the female genital tract. CXCL9 promotes immune cell recruitment, activation, and proliferation. The role of CXCL9 in modulating HIV-1 infection of cervicovaginal tissues, a main portal of viral entry, however, has not been established. We report a link between CXCL9 and HIV-1 replication in human cervical tissues and propose CXCL9 as a potential target to enhance the anti–HIV-1 activity of prophylactic antiretrovirals. Design: Using ex vivo infection of human cervical tissues as a model of mucosal HIV-1 acquisition, we described the effect of CXCL9 neutralization on HIV-1 gene expression and mucosal CD4+ T-cell activation. The anti-HIV-1 activity of tenofovir, the leading mucosal pre-exposure prophylactic microbicide, alone or in combination with CXCL9 neutralization was also studied. Methods: HIV-1 replication was evaluated by p24 ELISA. HIV-1 DNA and RNA, and CD4, CCR5, and CD38 transcription were evaluated by quantitative real-time polymerase chain reaction. Frequency of activated cervical CD4+ T cells was quantified using fluorescence-activated cell sorting. Results: Antibody blocking of CXCL9 reduced HIV-1 replication by decreasing mucosal CD4+ T-cell activation. CXCL9 neutralization in combination with suboptimal concentrations of tenofovir, possibly present in the cervicovaginal tissues of women using the drug inconsistently, demonstrated an earlier and greater decrease in HIV-1 replication compared with tissues treated with tenofovir alone. Conclusions: CXCL9 neutralization reduces HIV-1 replication and may be an effective target to enhance the efficacy of prophylactic antiretrovirals. PMID:26545124

  17. Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants▿

    PubMed Central

    Sharma, Monika; Sasvari, Zsuzsanna; Nagy, Peter D.

    2010-01-01

    The replication of plus-strand RNA viruses depends on subcellular membranes. Recent genome-wide screens have revealed that the sterol biosynthesis genes ERG25 and ERG4 affected the replication of Tomato bushy stunt virus (TBSV) in a yeast model host. To further our understanding of the role of sterols in TBSV replication, we demonstrate that the downregulation of ERG25 or the inhibition of the activity of Erg25p with an inhibitor (6-amino-2-n-pentylthiobenzothiazole; APB) leads to a 3- to 5-fold reduction in TBSV replication in yeast. In addition, the sterol biosynthesis inhibitor lovastatin reduced TBSV replication by 4-fold, confirming the importance of sterols in viral replication. We also show reduced stability for the p92pol viral replication protein as well as a decrease in the in vitro activity of the tombusvirus replicase when isolated from APB-treated yeast. Moreover, APB treatment inhibits TBSV RNA accumulation in plant protoplasts and in Nicotiana benthamiana leaves. The inhibitory effect of APB on TBSV replication can be complemented by exogenous stigmasterol, the main plant sterol, suggesting that sterols are required for TBSV replication. The silencing of SMO1 and SMO2 genes, which are orthologs of ERG25, in N. benthamiana reduced TBSV RNA accumulation but had a lesser inhibitory effect on the unrelated Tobacco mosaic virus, suggesting that various viruses show different levels of dependence on sterol biosynthesis for their replication. PMID:20015981

  18. Inhibition of HIV-1 Maturation via Small Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

    PubMed

    Wang, Weifeng; Zhou, Jing; Halambage, Upul D; Jurado, Kellie A; Jamin, Augusta V; Wang, Yujie; Engelman, Alan N; Aiken, Christopher

    2017-02-15

    The HIV-1 capsid protein is an attractive therapeutic target owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsid to HIV-1 infectivity. To date, small molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, BI compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor Bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant crosslinks in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here we show that one such compound, Compound 1, interferes with assembly of the conical viral capsid during virion maturation, and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a

  19. ACTG 5197: A Placebo Controlled Trial of Immunization of HIV-1 Infected Persons with a Replication Deficient Ad5 Vaccine Expressing the HIV-1 Core Protein

    PubMed Central

    Schooley, Robert T.; Spritzler, John; Wang, Hongying; Lederman, Michael M.; Havlir, Diane; Kuritzkes, Daniel R.; Pollard, Richard; Battaglia, Cathy; Robertson, Michael; Mehrotra, Devan; Casimiro, Danilo; Cox, Kara; Schock, Barbara

    2010-01-01

    Background HIV-1 specific cellular immunity contributes to control of HIV-1 replication. HIV-1 infected volunteers on antiretroviral therapy received a replication defective Ad5 HIV-1 gag vaccine in a randomized, blinded therapeutic vaccination study. Methods HIV-1-infected vaccine or placebo recipients underwent a 16-wk analytical treatment interruption (ATI). The log10 HIV-1 RNA at the ATI set point and time averaged area under the curve (TA-AUC) served as co-primary endpoints. Immune responses were measured by intracellular cytokine staining and CFSE dye dilution. Results Vaccine benefit trends were seen for both primary endpoints, but did not reach a pre-specified p ≤ 0.025 level of significance. The estimated shift in TA-AUC and set point were 0.24 (unadjusted p=0.04) and 0.26 (unadjusted p=0.07) log10 copies lower in the vaccine than in the placebo arm. HIV-1 gag-specific CD4+ interferon-γ producing cells were an immunologic correlate of viral control. Conclusion The vaccine was generally safe and well tolerated. Despite a trend favoring viral suppression among vaccine recipients, differences in HIV-1 RNA levels did not meet the pre-specified level of significance. Induction of HIV-1 gag-specific CD4 cells correlated with control of viral replication in vivo. Future immunogenicity studies should require a substantially higher immunogenicity threshold before an ATI is contemplated. PMID:20662716

  20. Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody.

    PubMed Central

    Lescar, J.; Brynda, J.; Rezacova, P.; Stouracova, R.; Riottot, M. M.; Chitarra, V.; Fabry, M.; Horejsi, M.; Sedlacek, J.; Bentley, G. A.

    1999-01-01

    The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity. PMID:10631984

  1. HIV-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity.

    PubMed

    Rolland, Morgane; Manocheewa, Siriphan; Swain, J Victor; Lanxon-Cookson, Erinn C; Kim, Moon; Westfall, Dylan H; Larsen, Brendan B; Gilbert, Peter B; Mullins, James I

    2013-05-01

    To overcome the problem of HIV-1 variability, candidate vaccine antigens have been designed to be composed of conserved elements of the HIV-1 proteome. Such candidate vaccines could be improved with a better understanding of both HIV-1 evolutionary constraints and the fitness cost of specific mutations. We evaluated the in vitro fitness cost of 23 mutations engineered in the HIV-1 subtype B Gag-p24 Center-of-Tree (COT) protein through fitness competition assays. While some mutations at conserved sites exacted a high fitness cost, as expected under the assumption that the most conserved residue confers the highest fitness, there was no overall strong relationship between sequence conservation and replicative capacity. By comparing sites that have evolved since the beginning of the epidemic to those that have remain unchanged, we found that sites that have evolved over time were more likely to correspond to HLA-associated sites and that their mutation had limited fitness costs. Our data showed no transcendent link between high conservation and high fitness cost, indicating that merely focusing on conserved segments of HIV-1 would not be sufficient for a successful vaccine strategy. Nonetheless, a subset of sites exacted a high fitness cost upon mutation--these sites have been under selective pressure to change since the beginning of the epidemic but have proved virtually nonmutable and could constitute preferred targets for vaccine design.

  2. HIV-1 Replication in Human Immune Cells Is Independent of TAR DNA Binding Protein 43 (TDP-43) Expression

    PubMed Central

    Nehls, Julia; Koppensteiner, Herwig; Brack-Werner, Ruth; Floss, Thomas; Schindler, Michael

    2014-01-01

    The TAR DNA binding protein (TDP-43) was originally identified as a host cell factor binding to the HIV-1 LTR and thereby suppressing HIV-1 transcription and gene expression (Ou et al., J.Virol. 1995, 69(6):3584). TDP-43 is a global regulator of transcription, can influence RNA metabolism in many different ways and is ubiquitously expressed. Thus, TDP-43 could be a major factor restricting HIV-1 replication at the level of LTR transcription and gene expression. These facts prompted us to revisit the role of TDP-43 for HIV-1 replication. We utilized established HIV-1 cell culture systems as well as primary cell models and performed a comprehensive analysis of TDP-43 function and investigated its putative impact on HIV-1 gene expression. In HIV-1 infected cells TDP-43 was neither degraded nor sequestered from the nucleus. Furthermore, TDP-43 overexpression as well as siRNA mediated knockdown did not affect HIV-1 gene expression and virus production in T cells and macrophages. In summary, our experiments argue against a restricting role of TDP-43 during HIV-1 replication in immune cells. PMID:25127017

  3. Copper inhibits the HIV-1 protease by both oxygen-dependent and oxygen-independent mechanisms

    SciTech Connect

    Karlstroem, A.R.; Levine, R.L. )

    1991-03-11

    The protease encoded by HIV-1 is essential for the processing of the viral polyproteins encoded by the gag and pol genes into mature viral proteins. Mutation or deletion of the protease gene blocks replication of the virus, making the protease an attractive target for antiviral therapy. The authors found that the HIV-1 protease is inhibited by micromolar concentrations of Cu{sup 2+}. Protease was 50% inhibited by exposure to 5 {mu}M copper for 5 min while exposure to 25 {mu}M caused complete inhibition. This inhibition was not oxygen-dependent and was not reversed by treatment with EDTA, presumably due to the slow off-rate of copper from the protease. Consistent with this interpretation, enzyme activity was recovered after denaturation and refolding of the copper exposed protease. Titration of the inactivated enzyme with Ellman's reagent demonstrated a loss of one of the two sulfhydryl groups present in the molecule, suggesting that copper inhibition was mediated through binding to a cysteine. This was confirmed in studies with a chemically synthesize, mutant protease in which the two cysteine residues were replaced by {alpha}-amino butyrate: The mutant protease was not inhibited by copper. However, both the wild-type and mutant protease were inactivated when exposed to copper, oxygen, and dithiothreitol. This inactivation required oxygen. Thus, the protease can also be inactivated by metal catalyzed oxidation (MCO), a presumably irreversible covalent modification.

  4. Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

    PubMed Central

    Bogerd, Hal P.; Kennedy, Edward M.; Whisnant, Adam W.

    2017-01-01

    ABSTRACT Analysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome. PMID:28096489

  5. Ribonucleases in HIV type 1 inhibition: effect of recombinant RNases on infection of primary T cells and immune activation-induced RNase gene and protein expression.

    PubMed

    Bedoya, Victoria I; Boasso, Adriano; Hardy, Andrew W; Rybak, Susanna; Shearer, Gene M; Rugeles, Maria T

    2006-09-01

    Ribonucleases (RNases) have therapeutic potential against cancer and viral diseases and have been reported to inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in chronically infected cell lines. The ribonuclease eosinophil-derived neurotoxin (EDN) is responsible for the anti-HIV-1 activity of a soluble factor produced in response to human alloantigens (ASF). Four recombinant RNases (EDN; a four amino acid extension of the N-terminus EDN, -4EDN; RNase A; and angiogenin) were tested for inhibition of HIV-1 replication in PHA blasts. All RNases showed anti-HIV-1 activity, irrespective of whether the RNases were added before, during, or 2 h after infection. Polyclonal antibodies against the four RNases blocked the antiviral activity. ASF inhibited HIV-1 replication in vitro if added up to 4 h after infection. We demonstrated that allostimulation induced EDN, RNase A, and angiogenin mRNA expression in peripheral blood mononuclear cells (PBMCs), although only EDN protein was detected. We identified monocytes and dendritic cells, but not macrophages or T cells, as EDN-producing cells. These findings raise the possibilities that multiple naturally occurring RNases may contribute to protection against HIV-1 infection and could be considered for utilization in HIV-1 therapy.

  6. Role of Transmitted Gag CTL Polymorphisms in Defining Replicative Capacity and Early HIV-1 Pathogenesis

    PubMed Central

    Carlson, Jonathan M.; Schaefer, Malinda; Yu, Tianwei; Lahki, Shabir; Prentice, Heather A.; Yue, Ling; Vishwanathan, Sundaram A.; Kilembe, William; Goepfert, Paul; Price, Matthew A.; Gilmour, Jill; Mulenga, Joseph; Farmer, Paul; Derdeyn, Cynthia A.; Tang, Jiaming; Heckerman, David; Kaslow, Richard A.; Allen, Susan A.; Hunter, Eric

    2012-01-01

    Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned gag sequences isolated from the earliest seroconversion plasma sample from the acutely infected recipient of 149 epidemiologically linked Zambian transmission pairs into a primary isolate, subtype C proviral vector, MJ4. We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone. PMID:23209412

  7. Replication of Human Herpesviruses Is Associated with Higher HIV DNA Levels during Antiretroviral Therapy Started at Early Phases of HIV Infection

    PubMed Central

    Anderson, Christy M.; Var, Susanna R.; Oliveira, Michelli F.; Lada, Steven M.; Vargas, Milenka V.; Little, Susan J.; Richman, Douglas D.; Strain, Matthew C.; Pérez-Santiago, Josué; Smith, Davey M.

    2016-01-01

    ABSTRACT Asymptomatic replication of human herpesviruses (HHV) is frequent in HIV-infected men and is associated with increased T-cell activation and HIV disease progression. We hypothesized that the presence of replication of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (the most frequently detected HHV) might influence HIV DNA decay during antiretroviral therapy (ART). We investigated 607 peripheral blood mononuclear cell (PBMC) samples from 107 CMV-seropositive, HIV-infected men who have sex with men, who started ART within a median of 3 months from their estimated date of infection (EDI) and were monitored for a median of 19 months thereafter. Levels of HIV, CMV, and EBV DNA and cellular HIV RNA were measured by droplet digital PCR (ddPCR) for each time point. Using a general linear mixed-effect regression model, we evaluated associations between the presence of detectable CMV DNA and EBV DNA levels and HIV DNA decay and cellular HIV RNA levels, while adjusting for peak HIV RNA, nadir CD4+ count, CD4/CD8 ratio, CMV IgG levels, time from EDI to ART initiation, time from ART initiation to virologic suppression, detectable CMV DNA pre-ART, and age. The presence of intermittent CMV DNA in PBMC during ART was significantly associated with slower decay of HIV DNA (P = 0.011) but not with increased cellular HIV RNA transcription or more detectable 2-long terminal repeat circles. Higher levels of EBV DNA were also associated with higher levels of HIV DNA (P < 0.001) and increased unspliced cellular HIV RNA transcription (P = 0.010). These observations suggest that replication of HHV may help maintain a larger HIV DNA reservoir, but the underlying mechanisms remain unclear. IMPORTANCE Over three-fourths of HIV-infected men have at least one actively replicating human herpesvirus (HHV) in their mucosal secretions at any one time. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are the most common, and although it is often asymptomatic, such CMV and EBV

  8. G3BP1 restricts HIV-1 replication in macrophages and T-cells by sequestering viral RNA.

    PubMed

    Cobos Jiménez, Viviana; Martinez, Fernando O; Booiman, Thijs; van Dort, Karel A; van de Klundert, Maarten A A; Gordon, Siamon; Geijtenbeek, Teunis B H; Kootstra, Neeltje A

    2015-12-01

    HIV-1 exploits the cellular machinery for replication and therefore several interactions with cellular factors take place, some of which are yet unknown. We identified GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) as a cellular factor that restricts HIV-1, by analyzing transcriptome profiles of in vitro-cytokine-activated macrophages that are non-permissive to HIV-1 replication. Silencing of G3BP1 by RNA interference resulted in increased HIV-1 replication in primary T-cells and macrophages, but did not affect replication of other retroviruses. G3BP1 specifically interacted with HIV-1 RNA in the cytoplasm, suggesting that it sequesters viral transcripts, thus preventing translation or packaging. G3BP1 was highly expressed in resting naïve or memory T-cells from healthy donors and HIV-1 infected patients, but significantly lower in IL-2-activated T-cells. These results strongly suggest that G3BP1 captures HIV-1 RNA transcripts and thereby restricts mRNA translation, viral protein production and virus particle formation.

  9. Pandemic Influenza A (H1N1) Virus Infection Increases Apoptosis and HIV-1 Replication in HIV-1 Infected Jurkat Cells.

    PubMed

    Wang, Xue; Tan, Jiying; Biswas, Santanu; Zhao, Jiangqin; Devadas, Krishnakumar; Ye, Zhiping; Hewlett, Indira

    2016-02-02

    Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.

  10. Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells.

    PubMed

    Ishimaru, D; Marcicano, F G; Rebello, M A

    1998-09-01

    The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 micrograms/ml PGB2 inhibited virus yield by 60%, at the same dose PGA1 suppressed virus replication by more than 90%. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2.

  11. Potential inhibition of HIV-1 encapsidation by oligoribonucleotide–dendrimer nanoparticle complexes

    PubMed Central

    Parboosing, Raveen; Chonco, Louis; de la Mata, Francisco Javier; Govender, Thavendran; Maguire, Glenn EM; Kruger, Hendrik G

    2017-01-01

    Background Encapsidation, the process during which the genomic RNA of HIV is packaged into viral particles, is an attractive target for antiviral therapy. This study explores a novel nanotechnology-based strategy to inhibit HIV encapsidation by an RNA decoy mechanism. The design of the 16-mer oligoribonucleotide (RNA) decoy is based on the sequence of stem loop 3 (SL3) of the HIV packaging signal (Ψ). Recognition of the packaging signal is essential to the encapsidation process. It is theorized that the decoy RNA, by mimicking the packaging signal, will disrupt HIV packaging if efficiently delivered into lymphocytes by complexation with a carbosilane dendrimer. The aim of the study is to measure the uptake, toxicity, and antiviral activity of the dendrimer–RNA nanocomplex. Materials and methods A dendriplex was formed between cationic carbosilane dendrimers and the RNA decoy. Uptake of the fluorescein-labeled RNA into MT4 lymphocytes was determined by flow cytometry and confocal microscopy. The cytoprotective effect (50% effective concentration [EC50]) and the effect on HIV replication were determined in vitro by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and viral load measurements, respectively. Results Flow cytometry and confocal imaging demonstrated efficient transfection of lymphocytes. The dendriplex containing the Ψ decoy showed some activity (EC50 =3.20 µM, selectivity index =8.4). However, there was no significant suppression of HIV viral load. Conclusion Oligoribonucleotide decoys containing SL3 of the packaging sequence are efficiently delivered into lymphocytes by carbosilane dendrimers where they exhibit a modest cytoprotective effect against HIV infection. PMID:28115849

  12. Control of HIV-1 in Elite Suppressors despite Ongoing Replication and Evolution in Plasma Virus▿

    PubMed Central

    O'Connell, Karen A.; Brennan, Timothy P.; Bailey, Justin R.; Ray, Stuart C.; Siliciano, Robert F.; Blankson, Joel N.

    2010-01-01

    A subset of HIV-1-infected patients known as elite controllers or suppressors (ES) control the virus naturally. We have previously demonstrated sequence discordance between proviral and plasma gag clones in ES, much of which can be attributed to selective pressure from the host (J. R. Bailey, T. M. Williams, R. F. Siliciano, and J. N. Blankson, J. Exp. Med. 203:1357-1369, 2006). However, it is not clear whether ongoing viral replication continues in ES once the control of viremia has been established or whether selective pressure impacts this evolution. The cytotoxic T-lymphocyte (CTL) response in ES often targets Gag and frequently is superior to that of HIV-1 progressors, partially due to the HLA class I alleles B*57/5801 and B*27, which are overrepresented in ES. We therefore examined longitudinal plasma and proviral gag sequences from HLA-B*57/5801 and -B*27 ES. Despite the highly conserved nature of gag, we observed clear evidence of evolution in the plasma virus, largely due to synonymous substitutions. In contrast, evolution was rare in proviral clones, suggesting that ongoing replication in ES does not permit the significant reseeding of the latent reservoir. Interestingly, there was little continual evolution in CTL epitopes, and we detected de novo CTL responses to autologous viral mutants. Thus, some ES control viremia despite ongoing replication and evolution. PMID:20444904

  13. Identification of a novel family of nucleosides that specifically inhibit HIV-1 reverse transcriptase.

    PubMed

    Chamorro, C; Lobatón, E; Bonache, M C; De Clercq, E; Balzarini, J; Velázquez, S; San-Félix, A; Camarasa, M J

    2001-12-03

    N-3-Benzyloxycarbonylmethyl- and N-3-carboxymethyl-TBDMS-substituted nucleosides were synthesized and evaluated for activity against HIV replication. It was found that the N-3-carboxymethyl-TBDMS-substituted nucleosides were specific inhibitors of HIV-1 replication. They should be considered as members of a novel and original class of NNRTIs.

  14. APOBEC3G restricts early HIV-1 replication in the cytoplasm of target cells.

    PubMed

    Anderson, Jenny L; Hope, Thomas J

    2008-05-25

    Cellular APOBEC3G (A3G) protein is packaged into human immunodeficiency virus type 1 (HIV-1) virions in producer cells yet restricts viral replication in target cells. To characterize this restriction in target cells, the effect of A3G on generating various HIV-1 cDNA products was measured by quantitative real-time PCR. A3G decreased cDNA products from Vif-deficient HIV-1, with minor effects on early reverse transcripts and larger declines in late reverse transcripts. However, the greatest decline was typically observed in nuclear 2-LTR circles. Moreover, the magnitude of these declines varied with A3G dose. Adding integration inhibitor did not stop the A3G-mediated loss in 2-LTR circles. Moreover, obstructing HIV-1 nuclear entry using vesicular stomatitis virus matrix protein did not stop the A3G-mediated decline in late reverse transcripts. Collectively, these data suggest that A3G has important restriction activity in the cytoplasm and progressively diminishes viral cytoplasmic and nuclear cDNA forms with increasing magnitude during restriction.

  15. Replicating impact of a primary school HIV prevention programme: primary school action for better health, Kenya.

    PubMed

    Maticka-Tyndale, E; Mungwete, R; Jayeoba, O

    2014-08-01

    School-based programmes to combat the spread of HIV have been demonstrated to be effective over the short-term when delivered on a small scale. The question addressed here is whether results obtained with small-scale delivery are replicable in large-scale roll-out. Primary School Action for Better Health (PSABH), a programme to train teachers to deliver HIV-prevention education in upper primary-school grades in Kenya demonstrated positive impact when tested in Nyanza Province. This article reports pre-, 10-month post- and 22-month post-training results as PSABH was delivered in five additional regions of the country. A total of 26 461 students from 110 primary schools in urban and rural, middle- and low-income settings participated in this repeated cross-sectional study. Students ranged in age from 11 to 16 years, were predominantly Christian (10% Muslim), and the majority were from five different ethnic groups. Results demonstrated positive gains in knowledge, self-efficacy related to changes in sexual behaviours and condom use, acceptance of HIV+ students, endorsement of HIV-testing and behaviours to post-pone sexual debut or decrease sexual activity. These results are as strong as or stronger than those demonstrated in the original impact evaluation conducted in Nyanza Province. They support the roll-out of the programme across Kenyan primary schools.

  16. Genital Cytomegalovirus Replication Predicts Syphilis Acquisition among HIV-1 Infected Men Who Have Sex with Men

    PubMed Central

    Gianella, Sara; Smith, Davey M.; Daar, Eric S.; Dube, Michael P.; Lisco, Andrea; Vanpouille, Christophe; Margolis, Leonid; Haubrich, Richard H.; Morris, Sheldon R.

    2015-01-01

    Objective Sexually transmitted infections (STI) are common among HIV-infected men who have sex with men (MSM). While behavioral factors are important in STI acquisition, other biological factors such as immune modulation due to chronic viral infection may further predispose to STI acquisition. Design Post Hoc analysis including data collected over 12 months of follow-up from 131 HIV-infected MSM receiving antiretroviral therapy and screened for incident bacterial STI every 3 months. Methods Genital secretions collected at baseline were used to measure herpesvirus replication and inflammatory cytokines. Baseline predictors of STI were determined using survival analysis of time to incident STI. Results All participants were seropositive for cytomegalovirus (CMV), and 52% had detectable genital CMV at baseline. Thirty-five individuals acquired STI during follow-up, sometimes with multiple pathogen (17 syphilis, 21 gonorrhea, 14 chlamydia). Syphilis acquisition was associated with genital CMV replication at baseline (19.1% CMV-shedders versus 4.8% non-shedders, p=0.03) and younger age (p=0.02). Lower seminal MCP-1 was associated with higher seminal CMV levels and with syphilis acquisition (p<0.01). For syphilis acquisition, in multivariable Cox-Proportional Hazard model adjusted hazard rates were 3.56 (95%CI:1.00–12.73) for baseline CMV replication and 2.50 (0.92–6.77) for younger age. Conclusions This post hoc analysis suggest that CMV-associated decrease in seminal MCP-1 levels might predispose HIV-infected MSM to syphilis acquisition, but not other STI. Future studies should determine underlying mechanisms and if a causal association exists. PMID:26061824

  17. Trichomonas vaginalis-Induced Epithelial Monolayer Disruption and Human Immunodeficiency Virus Type 1 (HIV-1) Replication: Implications for the Sexual Transmission of HIV-1

    PubMed Central

    Guenthner, Patricia C.; Secor, W. Evan; Dezzutti, Charlene S.

    2005-01-01

    The objective of this study was to evaluate potential mechanisms of Trichomonas vaginalis involvement in human immunodeficiency virus type 1 (HIV-1) transmission. Polarized monolayer integrity of primary cervical and prostate epithelial cells or cell lines cultured with T. vaginalis was measured by monitoring transepithelium resistance. The effect of T. vaginalis isolates on HIV-1 passage through polarized epithelial cell monolayers was evaluated for HIV-1 p24gag in the basolateral supernatants. Coincubation with T. vaginalis isolates induced disruption of monolayer integrity and resulted in passage of virus to the basolateral side of the monolayer. Furthermore, there was isolate variability in which two isolates induced greater monolayer damage and increased HIV-1 passage than did the other two isolates. Coincubation of T. vaginalis isolates with acutely HIV-1-infected peripheral blood mononuclear cells enhanced HIV-1 replication. This enhancement was associated with cellular proliferation and activation, as well as with tumor necrosis factor alpha production. In contrast to the monolayer disruption, the effect of T. vaginalis on HIV-1 replication was not isolate dependent. Thus, two mechanisms have been identified that could contribute to the epidemiologic association of trichomoniasis with the sexual transmission of HIV-1. (i) T. vaginalis disruption of urogenital epithelial monolayers could facilitate passage of HIV-1 to underlying layers. (ii) Activation of local immune cells by T. vaginalis in the presence of infectious HIV-1 might lead to increased viral replication. Collectively, these data suggest the need for more vigilant efforts in the diagnosis and treatment of T. vaginalis in women and men, especially in countries with a high prevalence of HIV-1. PMID:15972505

  18. Trichomonas vaginalis-induced epithelial monolayer disruption and human immunodeficiency virus type 1 (HIV-1) replication: implications for the sexual transmission of HIV-1.

    PubMed

    Guenthner, Patricia C; Secor, W Evan; Dezzutti, Charlene S

    2005-07-01

    The objective of this study was to evaluate potential mechanisms of Trichomonas vaginalis involvement in human immunodeficiency virus type 1 (HIV-1) transmission. Polarized monolayer integrity of primary cervical and prostate epithelial cells or cell lines cultured with T. vaginalis was measured by monitoring transepithelium resistance. The effect of T. vaginalis isolates on HIV-1 passage through polarized epithelial cell monolayers was evaluated for HIV-1 p24gag in the basolateral supernatants. Coincubation with T. vaginalis isolates induced disruption of monolayer integrity and resulted in passage of virus to the basolateral side of the monolayer. Furthermore, there was isolate variability in which two isolates induced greater monolayer damage and increased HIV-1 passage than did the other two isolates. Coincubation of T. vaginalis isolates with acutely HIV-1-infected peripheral blood mononuclear cells enhanced HIV-1 replication. This enhancement was associated with cellular proliferation and activation, as well as with tumor necrosis factor alpha production. In contrast to the monolayer disruption, the effect of T. vaginalis on HIV-1 replication was not isolate dependent. Thus, two mechanisms have been identified that could contribute to the epidemiologic association of trichomoniasis with the sexual transmission of HIV-1. (i) T. vaginalis disruption of urogenital epithelial monolayers could facilitate passage of HIV-1 to underlying layers. (ii) Activation of local immune cells by T. vaginalis in the presence of infectious HIV-1 might lead to increased viral replication. Collectively, these data suggest the need for more vigilant efforts in the diagnosis and treatment of T. vaginalis in women and men, especially in countries with a high prevalence of HIV-1.

  19. Amphotericin B Inhibits Enterovirus 71 Replication by Impeding Viral Entry

    PubMed Central

    Xu, Fengwen; Zhao, Xiaoxiao; Hu, Siqi; Li, Jian; Yin, Lijuan; Mei, Shan; Liu, Tingting; Wang, Ying; Ren, Lili; Cen, Shan; Zhao, Zhendong; Wang, Jianwei; Jin, Qi; Liang, Chen; Ai, Bin; Guo, Fei

    2016-01-01

    Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. There is thus an urgent need to find new treatments to control EV71 infection. In this study, we report potent inhibition of EV71 by a polyene antibiotic Amphotericin B. Amphotericin B profoundly diminished the expression of EV71 RNA and viral proteins in the RD cells and the HEK293 cells. As a result, EV71 production was inhibited by Amphotericin B with an EC50 (50% effective concentration) of 1.75 μM in RD cells and 0.32 μM in 293 cells. In addition to EV71, EV68 was also strongly inhibited by Amphotericin B. Results of mechanistic studies revealed that Amphotericin B targeted the early stage of EV71 infection through impairing the attachment and internalization of EV71 by host cells. As an effective anti-fungi drug, Amphotericin B thus holds the promise of formulating a novel therapeutic to treat EV71 infection. PMID:27608771

  20. Inhibition of ganciclovir-resistant human cytomegalovirus replication by Kampo (Japanese herbal medicine).

    PubMed

    Murayama, Tsugiya; Yamaguchi, Nobuo; Iwamoto, Kozo; Eizuru, Yoshito

    2006-01-01

    We examined the effect of Kampo on the replication of ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) in the human embryonic fibroblast cell line MRC-5. Treatment of HCMV-infected cells with Sho-seiryu-to (SST; Xiao-Qing-Long-Tang in Chinese) resulted in the inhibition of viral replication without affecting the cell growth. SST treatment decreased the synthesis of viral DNA, but had no virucidal effect on cell-free HCMV. However, the inhibitory effect of SST on HCMV replication was ablated by anti-interferon-beta (IFN-beta) antibody suggesting that SST inhibits the replication of GCV-resistant HCMV through the induction of IFN-beta. These results suggest that SST is a novel compund with potential as an anti-HCMV.

  1. Autophagy induction by histone deacetylase inhibitors inhibits HIV type 1.

    PubMed

    Campbell, Grant R; Bruckman, Rachel S; Chu, Yen-Lin; Spector, Stephen A

    2015-02-20

    Histone deacetylase inhibitors (HDACi) are being evaluated in a "shock-and-kill" therapeutic approach to reverse human immunodeficiency virus type-1 (HIV) latency from CD4(+) T cells. Using this approach, HDACi have induced HIV RNA synthesis in latently infected cells from some patients. The hope is that the increase in viral production will lead to killing of the infected cell either by the virus itself or by the patient's immune system, a "sterilizing cure." Although administered within the context of combination antiretroviral therapy, the infection of bystander cells remains a concern. In this study, we investigated the effect of HDACi (belinostat, givinostat, panobinostat, romidepsin, and vorinostat) on the productive infection of macrophages. We demonstrate that the HDACi tested do not alter the initial susceptibility of macrophages to HIV infection. However, we demonstrate that HDACi decrease HIV release from macrophages in a dose-dependent manner (belinostat < givinostat < vorinostat < panobinostat < romidepsin) via degradation of intracellular HIV through the canonical autophagy pathway. This mechanism involves unc-51-like autophagy-activating kinase 1 (ULK1) and the inhibition of the mammalian target of rapamycin and requires the formation of autophagosomes and their maturation into autolysosomes in the absence of increased cell death. These data provide further evidence in support of a role for autophagy in the control of HIV infection and suggest that careful consideration of off-target effects will be essential if HDACi are to be a component of a multipronged approach to eliminate latently infected cells.

  2. [Replication process of HIV: with a central focus on the viral genome].

    PubMed

    Sakuragi, Jun-Ichi

    2013-01-01

    It has been 30 years passed since the discovery of HIVs as the agents of AIDS. During the period, many energetic research works about this gigantic menace have been performed globally and many outcomes have been applied to intercept the epidemic. Because of a brilliant progress of the therapeutic strategy, it is said that AIDS is no longer the deadly disease, but one of the mere chronic disease nowadays. On the other hand, giving an eye to the virus itself, many dark gaps are found in a superficially good-looking story of the viral replication. Thus, we are still far from fundamental understanding of the virus. In this review, I especially pick up the viral genome RNA as a central player of the story and give an introduction about various steps of viral replication. With several recent reports, I will exposit well-known and/or unclear events around virus.

  3. Inhibition of HSV-1 Replication by Gene Editing Strategy

    PubMed Central

    Roehm, Pamela C.; Shekarabi, Masoud; Wollebo, Hassen S.; Bellizzi, Anna; He, Lifan; Salkind, Julian; Khalili, Kamel

    2016-01-01

    HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases. PMID:27064617

  4. Inhibition of HSV-1 Replication by Gene Editing Strategy.

    PubMed

    Roehm, Pamela C; Shekarabi, Masoud; Wollebo, Hassen S; Bellizzi, Anna; He, Lifan; Salkind, Julian; Khalili, Kamel

    2016-04-11

    HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.

  5. Structural basis for inhibition of DNA replication by aphidicolin

    DOE PAGES

    Baranovskiy, A. G.; Babayeva, N. D.; Suwa, Y.; ...

    2014-11-27

    Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with anmore » RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.« less

  6. Structural basis for inhibition of DNA replication by aphidicolin

    SciTech Connect

    Baranovskiy, A. G.; Babayeva, N. D.; Suwa, Y.; Gu, J.; Pavlov, Y. I.; Tahirov, T. H.

    2014-11-27

    Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.

  7. Estramustine phosphate reversibly inhibits an early stage during adenovirus replication.

    PubMed

    Everitt, E; Ekstrand, H; Boberg, B; Hartley-Asp, B

    1990-01-01

    Estramustine phosphate, an estradiol-mustard conjugate, was shown to reversibly inhibit a stage during the first hour of productive adenovirus 2 infection of HeLa cells. This drug, employed in the therapy of advanced prostatic cancer, specifically interacts with microtubule-associated proteins (MAPs) of the cytoskeleton. The results obtained under physiological conditions in vivo suggest a MAPs-interference with the microtubule-mediated vectorial migration of the virus inoculum to the nucleus. Virus attachment, uncoating kinetics and the appearance of established uncoating intermediates were not affected.

  8. Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells

    SciTech Connect

    Clark, J.M.; Hanawalt, P.C.

    1982-07-01

    The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with /sup 3/H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA.

  9. Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis.

    PubMed

    Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E

    2013-03-01

    Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I-II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.

  10. Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii

    PubMed Central

    Jeffers, Victoria; Gao, Hongyu; Checkley, Lisa A.; Liu, Yunlong; Ferdig, Michael T.

    2016-01-01

    Lysine acetylation is a critical posttranslational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasite Toxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product garcinol was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5 family KAT known to be required for Toxoplasma tachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed transcriptome sequencing (RNA-seq), which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previously reported chromatin immunoprecipitation assay with microarray technology (ChIP-chip) analysis. The dysregulated gene expression induced by garcinol significantly inhibits Toxoplasma tachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibits Plasmodium falciparum asexual replication with a 50% inhibitory concentration (IC50) similar to that for Toxoplasma. Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication. PMID:26810649

  11. The JAK2 inhibitor AZD1480 inhibits hepatitis A virus replication in Huh7 cells.

    PubMed

    Jiang, Xia; Kanda, Tatsuo; Nakamoto, Shingo; Saito, Kengo; Nakamura, Masato; Wu, Shuang; Haga, Yuki; Sasaki, Reina; Sakamoto, Naoya; Shirasawa, Hiroshi; Okamoto, Hiroaki; Yokosuka, Osamu

    2015-03-20

    The JAK2 inhibitor AZD1480 has been reported to inhibit La protein expression. We previously demonstrated that the inhibition of La expression could inhibit hepatitis A virus (HAV) internal ribosomal entry-site (IRES)-mediated translation and HAV replication in vitro. In this study, we analyzed the effects of AZD1480 on HAV IRES-mediated translation and replication. HAV IRES-mediated translation in COS7-HAV-IRES cells was inhibited by 0.1-1 μM AZD1480, a dosage that did not affect cell viability. Results showed a significant reduction in intracellular HAV HA11-1299 genotype IIIA RNA levels in Huh7 cells treated with AZD1480. Furthermore, AZD1480 inhibited the expression of phosphorylated-(Tyr-705)-signal transducer and activator of transcription 3 (STAT3) and La in Huh7 cells. Therefore, we propose that AZD1480 can inhibit HAV IRES activity and HAV replication through the inhibition of the La protein.

  12. Alkaloids from the Sponge Stylissa carteri Present Prospective Scaffolds for the Inhibition of Human Immunodeficiency Virus 1 (HIV-1).

    PubMed

    O'Rourke, Aubrie; Kremb, Stephan; Bader, Theresa Maria; Helfer, Markus; Schmitt-Kopplin, Philippe; Gerwick, William H; Brack-Werner, Ruth; Voolstra, Christian R

    2016-02-04

    The sponge Stylissa carteri is known to produce a number of secondary metabolites displaying anti-fouling, anti-inflammatory, and anti-cancer activity. However, the anti-viral potential of metabolites produced by S. carteri has not been extensively explored. In this study, an S. carteri extract was HPLC fractionated and a cell based assay was used to evaluate the effects of HPLC fractions on parameters of Human Immunodeficiency Virus (HIV-1) infection and cell viability. Candidate HIV-1 inhibitory fractions were then analyzed for the presence of potential HIV-1 inhibitory compounds by mass spectrometry, leading to the identification of three previously characterized compounds, i.e., debromohymenialdisine (DBH), hymenialdisine (HD), and oroidin. Commercially available purified versions of these molecules were re-tested to assess their antiviral potential in greater detail. Specifically, DBH and HD exhibit a 30%-40% inhibition of HIV-1 at 3.1 μM and 13 μM, respectively; however, both exhibited cytotoxicity. Conversely, oroidin displayed a 50% inhibition of viral replication at 50 μM with no associated toxicity. Additional experimentation using a biochemical assay revealed that oroidin inhibited the activity of the HIV-1 Reverse Transcriptase up to 90% at 25 μM. Taken together, the chemical search space was narrowed and previously isolated compounds with an unexplored anti-viral potential were found. Our results support exploration of marine natural products for anti-viral drug discovery.

  13. Alkaloids from the Sponge Stylissa carteri Present Prospective Scaffolds for the Inhibition of Human Immunodeficiency Virus 1 (HIV-1)

    PubMed Central

    O’Rourke, Aubrie; Kremb, Stephan; Bader, Theresa Maria; Helfer, Markus; Schmitt-Kopplin, Philippe; Gerwick, William H.; Brack-Werner, Ruth; Voolstra, Christian R.

    2016-01-01

    The sponge Stylissa carteri is known to produce a number of secondary metabolites displaying anti-fouling, anti-inflammatory, and anti-cancer activity. However, the anti-viral potential of metabolites produced by S. carteri has not been extensively explored. In this study, an S. carteri extract was HPLC fractionated and a cell based assay was used to evaluate the effects of HPLC fractions on parameters of Human Immunodeficiency Virus (HIV-1) infection and cell viability. Candidate HIV-1 inhibitory fractions were then analyzed for the presence of potential HIV-1 inhibitory compounds by mass spectrometry, leading to the identification of three previously characterized compounds, i.e., debromohymenialdisine (DBH), hymenialdisine (HD), and oroidin. Commercially available purified versions of these molecules were re-tested to assess their antiviral potential in greater detail. Specifically, DBH and HD exhibit a 30%–40% inhibition of HIV-1 at 3.1 μM and 13 μM, respectively; however, both exhibited cytotoxicity. Conversely, oroidin displayed a 50% inhibition of viral replication at 50 μM with no associated toxicity. Additional experimentation using a biochemical assay revealed that oroidin inhibited the activity of the HIV-1 Reverse Transcriptase up to 90% at 25 μM. Taken together, the chemical search space was narrowed and previously isolated compounds with an unexplored anti-viral potential were found. Our results support exploration of marine natural products for anti-viral drug discovery. PMID:26861355

  14. Colorectal mucus binds DC-SIGN and inhibits HIV-1 trans-infection of CD4+ T-lymphocytes.

    PubMed

    Stax, Martijn J; Mouser, Emily E I M; van Montfort, Thijs; Sanders, Rogier W; de Vries, Henry J C; Dekker, Henk L; Herrera, Carolina; Speijer, Dave; Pollakis, Georgios; Paxton, William A

    2015-01-01

    Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1-3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.

  15. Quantitative Assessment of HIV Replication and Variation in Vivo: Relevance to Disease Pathogenesis and Response to Therapy

    DTIC Science & Technology

    1994-07-20

    Chamot E, Hirschel B, Perrin LH. Quantitation of human immunodeficiency provirus and circulating virus: relationship with immunologic parameters. J...Science 1986;234:1563-66. 34. Walker CM, Levy JA. A diffusible lymphokine produced by CD8& T lymphocytes suppresses HIV replication. Immunology 1989;66...49. Urdea MS. Synthesis and characterization of branched DNA (bDNA) for the direct and quantitative detection of CMV, HBV, HCV , and HIV. Clin Chem

  16. Novel genetic locus implicated for HIV-1 acquisition with putative regulatory links to HIV replication and infectivity: a genome-wide association study.

    PubMed

    Johnson, Eric O; Hancock, Dana B; Gaddis, Nathan C; Levy, Joshua L; Page, Grier; Novak, Scott P; Glasheen, Cristie; Saccone, Nancy L; Rice, John P; Moreau, Michael P; Doheny, Kimberly F; Romm, Jane M; Brooks, Andrew I; Aouizerat, Bradley E; Bierut, Laura J; Kral, Alex H

    2015-01-01

    Fifty percent of variability in HIV-1 susceptibility is attributable to host genetics. Thus identifying genetic associations is essential to understanding pathogenesis of HIV-1 and important for targeting drug development. To date, however, CCR5 remains the only gene conclusively associated with HIV acquisition. To identify novel host genetic determinants of HIV-1 acquisition, we conducted a genome-wide association study among a high-risk sample of 3,136 injection drug users (IDUs) from the Urban Health Study (UHS). In addition to being IDUs, HIV-controls were frequency-matched to cases on environmental exposures to enhance detection of genetic effects. We tested independent replication in the Women's Interagency HIV Study (N=2,533). We also examined publicly available gene expression data to link SNPs associated with HIV acquisition to known mechanisms affecting HIV replication/infectivity. Analysis of the UHS nominated eight genetic regions for replication testing. SNP rs4878712 in FRMPD1 met multiple testing correction for independent replication (P=1.38x10(-4)), although the UHS-WIHS meta-analysis p-value did not reach genome-wide significance (P=4.47x10(-7) vs. P<5.0x10(-8)) Gene expression analyses provided promising biological support for the protective G allele at rs4878712 lowering risk of HIV: (1) the G allele was associated with reduced expression of FBXO10 (r=-0.49, P=6.9x10(-5)); (2) FBXO10 is a component of the Skp1-Cul1-F-box protein E3 ubiquitin ligase complex that targets Bcl-2 protein for degradation; (3) lower FBXO10 expression was associated with higher BCL2 expression (r=-0.49, P=8x10(-5)); (4) higher basal levels of Bcl-2 are known to reduce HIV replication and infectivity in human and animal in vitro studies. These results suggest new potential biological pathways by which host genetics affect susceptibility to HIV upon exposure for follow-up in subsequent studies.

  17. Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus.

    PubMed

    Li, Anatoliy; Blow, J Julian

    2005-01-26

    In late mitosis and G1, Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other cell cycle stages, licensing is inhibited, thus ensuring that origins fire only once per cell cycle. Three additional factors--the origin recognition complex, Cdc6 and Cdt1--are required for origin licensing. We examine here how licensing is regulated in Xenopus egg extracts. We show that Cdt1 is downregulated late in the cell cycle by two different mechanisms: proteolysis, which occurs in part due to the activity of the anaphase-promoting complex (APC/C), and inhibition by a protein called geminin. If both these regulatory mechanisms are abrogated, extracts undergo uncontrolled re-licensing and re-replication. The extent of re-replication is limited by checkpoint kinases that are activated as a consequence of re-replication itself. These results allow us to build a comprehensive model of how re-replication of DNA is prevented in Xenopus, with Cdt1 regulation being the key feature. The results also explain the original experiments that led to the proposal of a replication licensing factor.

  18. Inhibition of nuclear factor kappa B activation reduces Coxsackievirus B3 replication in lymphoid cells.

    PubMed

    Sobotta, Katharina; Wilsky, Steffi; Althof, Nadine; Wiesener, Nadine; Wutzler, Peter; Henke, Andreas

    2012-02-01

    Interactions between viral replication machineries and host cell metabolism display interesting information how certain viruses capitalize cellular pathways to support progeny production. Among those pathogens, Coxsackievirus B3 (CVB3) has been identified to manipulate intracellular signaling very comprehensively. Next to others, this human pathogenic virus causes acute and chronic forms of myocarditis, pancreatitis, and meningitis. Here, activation of nuclear factor kappa B (NFκB) signaling appears to be involved in successful infection. Viral replication is not restricted to solid organs but involves susceptible immune cells as well. In the present study, p65 phosphorylation as one aspect of NFκB activation and inhibition via BAY 11-7085 administration was analyzed in the context of CVB3 replication in lymphoid cells. During CVB3 infection, an up-regulation of p65 translation is detectable, which is accompanied by noticeable phosphorylation. Inhibition of NFκB signaling reduces viral replication in a dose- and time-dependent manner. Taken together, these results indicate that during CVB3 replication in human and murine lymphoid cells, NFκB signaling is activated and facilitates viral replication. Therefore, antiviral strategies to target such central cellular signaling pathways may represent potential possibilities for the development of new virostatica.

  19. Replication of Many Human Viruses Is Refractory to Inhibition by Endogenous Cellular MicroRNAs

    PubMed Central

    Bogerd, Hal P.; Skalsky, Rebecca L.; Kennedy, Edward M.; Furuse, Yuki; Whisnant, Adam W.; Flores, Omar; Schultz, Kimberly L. W.; Putnam, Nicole; Barrows, Nicholas J.; Sherry, Barbara; Scholle, Frank; Garcia-Blanco, Mariano A.; Griffin, Diane E.

    2014-01-01

    ABSTRACT The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of

  20. Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs.

    PubMed

    Bogerd, Hal P; Skalsky, Rebecca L; Kennedy, Edward M; Furuse, Yuki; Whisnant, Adam W; Flores, Omar; Schultz, Kimberly L W; Putnam, Nicole; Barrows, Nicholas J; Sherry, Barbara; Scholle, Frank; Garcia-Blanco, Mariano A; Griffin, Diane E; Cullen, Bryan R

    2014-07-01

    The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. Importance: Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of

  1. Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition.

    PubMed

    Berti, Matteo; Ray Chaudhuri, Arnab; Thangavel, Saravanabhavan; Gomathinayagam, Shivasankari; Kenig, Sasa; Vujanovic, Marko; Odreman, Federico; Glatter, Timo; Graziano, Simona; Mendoza-Maldonado, Ramiro; Marino, Francesca; Lucic, Bojana; Biasin, Valentina; Gstaiger, Matthias; Aebersold, Ruedi; Sidorova, Julia M; Monnat, Raymond J; Lopes, Massimo; Vindigni, Alessandro

    2013-03-01

    Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal through a process that requires poly(ADP-ribose) polymerase (PARP) activity. Whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown. We have combined biochemical and EM approaches with single-molecule DNA fiber analysis to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition that is not shared by other human RecQ proteins. We show that the poly(ADP-ribosyl)ation activity of PARP1 stabilizes forks in the regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

  2. Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition

    PubMed Central

    Berti, Matteo; Chaudhuri, Arnab Ray; Thangavel, Saravanabhavan; Gomathinayagam, Shivasankari; Kenig, Sasa; Vujanovic, Marko; Odreman, Federico; Glatter, Timo; Graziano, Simona; Mendoza-Maldonado, Ramiro; Marino, Francesca; Lucic, Bojana; Biasin, Valentina; Gstaiger, Matthias; Aebersold, Ruedi; Sidorova, Julia M.; Monnat, Raymond J.; Lopes, Massimo; Vindigni, Alessandro

    2014-01-01

    SUMMARY Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal, in a process that requires PARP activity. Whether regressed forks can efficiently restart and the factors required to restart fork progression after fork reversal are still unknown. Here we combined biochemical and electron microscopy approaches with single-molecule DNA fiber analysis, to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition, not shared by other human RecQ proteins. We show that the poly(ADPribosyl)ation activity of PARP1 stabilizes forks in their regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition. PMID:23396353

  3. Drug-eluting fibers for HIV-1 inhibition and contraception.

    PubMed

    Ball, Cameron; Krogstad, Emily; Chaowanachan, Thanyanan; Woodrow, Kim A

    2012-01-01

    Multipurpose prevention technologies (MPTs) that simultaneously prevent sexually transmitted infections (STIs) and unintended pregnancy are a global health priority. Combining chemical and physical barriers offers the greatest potential to design effective MPTs, but integrating both functional modalities into a single device has been challenging. Here we show that drug-eluting fiber meshes designed for topical drug delivery can function as a combination chemical and physical barrier MPT. Using FDA-approved polymers, we fabricated nanofiber meshes with tunable fiber size and controlled degradation kinetics that facilitate simultaneous release of multiple agents against HIV-1, HSV-2, and sperm. We observed that drug-loaded meshes inhibited HIV-1 infection in vitro and physically obstructed sperm penetration. Furthermore, we report on a previously unknown activity of glycerol monolaurate (GML) to potently inhibit sperm motility and viability. The application of drug-eluting nanofibers for HIV-1 prevention and sperm inhibition may serve as an innovative platform technology for drug delivery to the lower female reproductive tract.

  4. Drug-Eluting Fibers for HIV-1 Inhibition and Contraception

    PubMed Central

    Ball, Cameron; Krogstad, Emily; Chaowanachan, Thanyanan; Woodrow, Kim A.

    2012-01-01

    Multipurpose prevention technologies (MPTs) that simultaneously prevent sexually transmitted infections (STIs) and unintended pregnancy are a global health priority. Combining chemical and physical barriers offers the greatest potential to design effective MPTs, but integrating both functional modalities into a single device has been challenging. Here we show that drug-eluting fiber meshes designed for topical drug delivery can function as a combination chemical and physical barrier MPT. Using FDA-approved polymers, we fabricated nanofiber meshes with tunable fiber size and controlled degradation kinetics that facilitate simultaneous release of multiple agents against HIV-1, HSV-2, and sperm. We observed that drug-loaded meshes inhibited HIV-1 infection in vitro and physically obstructed sperm penetration. Furthermore, we report on a previously unknown activity of glycerol monolaurate (GML) to potently inhibit sperm motility and viability. The application of drug-eluting nanofibers for HIV-1 prevention and sperm inhibition may serve as an innovative platform technology for drug delivery to the lower female reproductive tract. PMID:23209601

  5. Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.

    PubMed Central

    Robertson, A T; Stout, E R; Bates, R C

    1984-01-01

    Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle. Images PMID:6422050

  6. Salicylates Inhibit Flavivirus Replication Independently of Blocking Nuclear Factor Kappa B Activation

    PubMed Central

    Liao, Ching-Len; Lin, Yi-Ling; Wu, Bi-Ching; Tsao, Chang-Huei; Wang, Mei-Chuan; Liu, Chiu-I; Huang, Yue-Ling; Chen, Jui-Hui; Wang, Jia-Pey; Chen, Li-Kuang

    2001-01-01

    Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-κB), and salicylates have been shown to inhibit NF-κB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-κB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IκBα-ΔN, a dominant-negative mutant that antagonizes NF-κB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-κB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IκBα-ΔN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-κB pathway. PMID:11483726

  7. HIV Tat protein inhibits hERG K+ channels: a potential mechanism of HIV infection induced LQTs.

    PubMed

    Bai, Yun-Long; Liu, Hui-Bin; Sun, Bo; Zhang, Ying; Li, Qi; Hu, Chao-Wei; Zhu, Jiu-Xin; Gong, Dong-Mei; Teng, Xue; Zhang, Qin; Yang, Bao-Feng; Dong, De-Li

    2011-11-01

    HIV-infected patients have a high prevalence of long QT syndrome (LQTs). hERG K(+) channel encoded by human ether-a-go-go related gene contributes to IKr K(+) currents responsible for the repolarization of cardiomyocytes. Inhibition of hERG K(+) channels leads to LQTs. HIV Tat protein, the virus transactivator protein, plays a pivotal role in AIDS. The aim of the present study is to examine the effects of HIV Tat protein on hERG K(+) channels stably expressed in HEK293 cells. The hERG K(+) currents were recorded by whole-cell patch-clamp technique and the hERG channel expression was measured by real-time PCR and Western blot techniques. HIV Tat protein at 200 ng/ml concentration showed no acute effect on hERG currents, but HIV Tat protein (200 ng/ml) incubation for 24 h significantly inhibited hERG currents. In HIV Tat incubated cells, the inactivation and the recovery time from inactivation of hERG channels were significantly changed. HIV Tat protein incubation (200 ng/ml) for 24h had no effect on the hERG mRNA expression, but dose-dependently inhibited hERG protein expression. The MTT assay showed that HIV Tat protein at 50 ng/ml and 200 ng/ml had no effect on the cell viability. HIV Tat protein increased reactive oxygen species (ROS) generation and the inhibition of hERG channel protein expression by HIV Tat protein was prevented by antioxidant tempol. HIV Tat protein in vivo treatment reduced IKr currents and prolonged action potential duration of guinea pig cardiomyocytes. We conclude that HIV Tat protein inhibits hERG K(+) currents through the inhibition of hERG protein expression, which might be the potential mechanism of HIV infection induced LQTs.

  8. Inhibition of fatty acid synthase by amentoflavone reduces coxsackievirus B3 replication.

    PubMed

    Wilsky, Steffi; Sobotta, Katharina; Wiesener, Nadine; Pilas, Johanna; Althof, Nadine; Munder, Thomas; Wutzler, Peter; Henke, Andreas

    2012-02-01

    Coxsackievirus B3 (CVB3) is a human pathogen that causes acute and chronic infections, but an antiviral drug to treat these diseases has not yet been developed for clinical use. Several intracellular pathways are altered to assist viral transcription, RNA replication, and progeny release. Among these, fatty acid synthase (FAS) expression is increased. In order to test the potential of FAS inhibition as an anti-CVB3 strategy, several experiments were performed, including studies on the correlation of CVB3 replication and FAS expression in human Raji cells and an analysis of the time and dose dependence of the antiviral effect of FAS inhibition due to treatment with amentoflavone. The results demonstrate that CVB3 infection induces an up-regulation of FAS expression already at 1 h postinfection (p.i.). Incubation with increasing concentrations of amentoflavone inhibited CVB3 replication significantly up to 8 h p.i. In addition, suppression of p38 MAP kinase activity by treatment with SB239063 decreased FAS expression as well as viral replication. These data provide evidence that FAS inhibition via amentoflavone administration might present a target for anti-CVB3 therapy.

  9. Interferon-Inducible Cholesterol-25-Hydroxylase Inhibits Hepatitis C Virus Replication via Distinct Mechanisms

    PubMed Central

    Chen, Yongzhi; Wang, Shanshan; Yi, Zhaohong; Tian, Huabin; Aliyari, Roghiyh; Li, Yanhua; Chen, Gang; Liu, Ping; Zhong, Jin; Chen, Xinwen; Du, Peishuang; Su, Lishan; Qin, F. Xiao-Feng; Deng, Hongyu; Cheng, Genhong

    2014-01-01

    Cholesterol 25-hydroxylase (CH25H) as an interferon-stimulated gene (ISG) has recently been shown to exert broad antiviral activity through the production of 25-hydroxycholesterol (25HC), which is believed to inhibit the virus-cell membrane fusion during viral entry. However, little is known about the function of CH25H on HCV infection and replication and whether antiviral function of CH25H is exclusively mediated by 25HC. In the present study, we have found that although 25HC produced by CH25H can inhibit HCV replication, CH25H mutants lacking the hydroxylase activity still carry the antiviral activity against HCV but not other viruses such as MHV-68. Further studies have revealed that CH25H can interact with the NS5A protein of HCV and inhibit its dimer formation, which is essential for HCV replication. Thus, our work has uncovered a novel mechanism by which CH25H restricts HCV replication, suggesting that CH25H inhibits viral infection through both 25HC-dependent and independent events. PMID:25467815

  10. Suppression of autophagy by mycophenolic acid contributes to inhibition of HCV replication in human hepatoma cells

    PubMed Central

    Fang, Shoucai; Su, Jinming; Liang, Bingyu; Li, Xu; Li, Yu; Jiang, Junjun; Huang, Jiegang; Zhou, Bo; Ning, Chuanyi; Li, Jieliang; Ho, Wenzhe; Li, Yiping; Chen, Hui; Liang, Hao; Ye, Li

    2017-01-01

    Previous studies have shown that mycophenolic acid (MPA) has an anti-HCV activity. However, the mechanism of MPA-mediated inhibition of HCV replication remains to be determined. This study investigated whether MPA has an effect on autophagy, a cellular machinery required for HCV replication, thereby, inhibits HCV replication in Huh7 cells. MPA treatment of Huh7 cells could suppress autophagy, evidenced by decreased LC3B-II level and conversion of LC3B-I to LC3B-II, decreased autophagosome formation, and increased p62 level compared to MPA-untreated cells. Tunicamycin treatment or HCV infection could induce cellular autophagy, however, MPA also exhibited its inhibitory effect on tunicamycin- or HCV infection-induced autophagy. The expression of three autophagy-related genes, Atg3, Atg5, and Atg7 were identified to be inhibited by MPA treatment. Over-expression of these genes could partly recover HCV replication inhibited by MPA; however, silencing their expression by siRNAs could enhance the inhibitory effect of MPA on HCV. Collectively, these results reveal that suppression of autophagy by MPA plays a role in its anti-HCV activity. Down-regulating the expression of three autophagy-related genes by MPA involves in its antiviral mechanism. PMID:28276509

  11. ACH-806, an NS4A antagonist, inhibits hepatitis C virus replication by altering the composition of viral replication complexes.

    PubMed

    Yang, Wengang; Sun, Yongnian; Hou, Xiaohong; Zhao, Yongsen; Fabrycki, Joanne; Chen, Dawei; Wang, Xiangzhu; Agarwal, Atul; Phadke, Avinash; Deshpande, Milind; Huang, Mingjun

    2013-07-01

    Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

  12. Inhibition of Feline leukemia virus replication by the integrase inhibitor Raltegravir.

    PubMed

    Cattori, Valentino; Weibel, Beatrice; Lutz, Hans

    2011-08-26

    The oncogenic gammaretrovirus Feline leukemia virus (FeLV) has been the leading cause of death among domestic cats until the introduction of efficient diagnostics and vaccines in the late 1980s. So far, no efficient treatment for viremic animals is available. Hence, use of the FeLV model to evaluate antiretroviral therapies applied to HIV is a timely task. The efficacy of the integrase inhibitor Raltegravir, which is widely used for the treatment of HIV in humans, has been assessed in vitro for the FeLV-A/Glasgow-1 strain. EC(50) values for FeLV-A inhibition in feline cell lines are in the range of that observed for HIV and xenotropic murine leukemia virus-related gammaretrovirus. Therefore, Raltegravir may be a potential therapeutical agent for felids with progressive FeLV infection.

  13. HSV-2- and HIV-1- permissive cell lines co-infected by HSV-2 and HIV-1 co-replicate HSV-2 and HIV-1 without production of HSV-2/HIV-1 pseudotype particles

    PubMed Central

    LeGoff, Jérôme; Bouhlal, Hicham; Lecerf, Maxime; Klein, Christophe; Hocini, Hakim; Si-Mohamed, Ali; Muggeridge, Martin; Bélec, Laurent

    2007-01-01

    Background Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1. Methods We evaluated in vitro the production of HSV-2/HIV-1 pseudotypes in mononuclear CEM cells and epithelial HT29 and P4P cells. We analyzed the incorporation into the HIV-1 membrane of HSV-2 gB and gD, two major HSV-2 glycoproteins required for HSV-2 fusion with the cell membrane, in co-infected cells and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD. Results We show that HSV-2 and HIV-1 co-replicated in dually infected cells, and gB and gD were co-localized with gp160. However, HIV-1 particles, produced in HIV-1-infected cells expressing gB or gD after transfection or HSV-2 superinfection, did not incorporate either gB or gD in the viral membrane, and did not have the capacity to infect cells normally non-permissive for HIV-1, such as epithelial cells. Conclusion Our results do not support the hypothesis of HSV-2/HIV-1 pseudotype formation and involvement in the synergistic genital interactions between HIV-1 and HSV-2. PMID:17207276

  14. T cells detect intracellular DNA but fail to induce type I IFN responses: implications for restriction of HIV replication.

    PubMed

    Berg, Randi K; Rahbek, Stine H; Kofod-Olsen, Emil; Holm, Christian K; Melchjorsen, Jesper; Jensen, David G; Hansen, Anne Louise; Jørgensen, Louise B; Ostergaard, Lars; Tolstrup, Martin; Larsen, Carsten S; Paludan, Søren R; Jakobsen, Martin R; Mogensen, Trine H

    2014-01-01

    HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1.

  15. [Expression and immunity of multi-HIV B'/C subype genes in replicating DNA vaccines].

    PubMed

    Gao, Ying-ying; Deng, Yao; Qi, Xiang-rong; Zhang, Xiang-min; Meng, Xin; Wang, Hui-juan; Tan, Wen-jie; Ruan, Li

    2010-05-01

    To understand the effect of various gene structures of HIV B'/C subtype on the gene expression and immunity in DNA vaccine, replicating DNA vector pSCK2 was used to construct seven DNA vaccines carrying one or more of HIV B'/C subtype genes: gagpol, gp160 and rtn (rev, tat and nef fusion gene). Immunofluorescence staining indicated that Gag, Gp160, Rev, Tat and Nef could be expressed from the seven DNA vaccines. Stronger expression was observed with the gene in single-gene expression plasmid or with the gene located at upper-IRES in double- or multi-gene expression plasmid. ELISA test showed that Gag induced higher antibody response, but the antibody titers stimulated by Gp160, Pol, or RTN were very low. Both Gag single-gene expression plasmid and Gag-RTN double-gene expression plasmid separately inoculating induced stronger antibody response against Gag than Gag-Gp160 double-gene expression plasmid and Gagpol-Gp160-RTN multi-gene expression plasmid or combined inoculation of Gag and Gp160 single-gene expression plasmids did. ELISPOT detection showed that all the seven DNA vaccines could stimulate cellular immune response against Gag, Pol, Gp160, Tat, and Nef, respectively. Gagpol or Gp160 single-gene expression plasmid separately inoculating stimulated the strongest cellular immune response. Tat and Nef expressed in all the plasmids induced similar immune response. These results indicated that HIV B'/C subtype genes gagpol, gp160 and rtn could be efficiently expressed in the replicating DNA vaccine vector, single-gene expression plasmid had the higher gene expression level and induced stronger immune response; combined immunization of Gagpol and Gp160 had dramatically lower immunity than Gagpol or Gp160 separated immunization did. Immunity of RTN had no difference between combined and separated immunizations. Therefore, in case of immunization with DNA vaccines containing different HIV genes, it is necessary to optimize the combined immunization procedure

  16. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins.

    PubMed

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-06-24

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

  17. Identification of personal lubricants that can cause rectal epithelial cell damage and enhance HIV type 1 replication in vitro.

    PubMed

    Begay, Othell; Jean-Pierre, Ninochka; Abraham, Ciby J; Chudolij, Anne; Seidor, Samantha; Rodriguez, Aixa; Ford, Brian E; Henderson, Marcus; Katz, David; Zydowsky, Thomas; Robbiani, Melissa; Fernández-Romero, José A

    2011-09-01

    Over-the-counter personal lubricants are used frequently during vaginal and anal intercourse, but they have not been extensively tested for biological effects that might influence HIV transmission. We evaluated the in vitro toxicity anti-HIV-1 activity and osmolality of popular lubricants. A total of 41 lubricants were examined and compared to Gynol II and Carraguard as positive and negative controls for toxicity, respectively. Cytotoxicity was assessed using the XTT assay. The MAGI assay with R5 and X4 HIV-1 laboratory strains was used to evaluate antiviral activity. The effect of the lubricants on differentiated Caco-2 cell monolayers (transepithelial electrical resistance, TEER) was also measured. None of the lubricants tested showed significant activity against HIV-1. Surprisingly, four of them, Astroglide Liquid, Astroglide Warming Liquid, Astroglide Glycerin & Paraben-Free Liquid, and Astroglide Silken Secret, significantly enhanced HIV-1 replication (p<0.0001). A common ingredient in three of these preparations is polyquaternium-15. In vitro testing of a chemically related compound (MADQUAT) confirmed that this similarly augmented HIV-1 replication. Most of the lubricants were found to be hyperosmolar and the TEER value dropped approximately 60% 2 h after exposure to all lubricants tested. Cells treated with Carraguard, saline, and cell controls maintained about 100% initial TEER value after 2-6 h. We have identified four lubricants that significantly increase HIV-1 replication in vitro. In addition, the epithelial damage caused by these and many other lubricants may have implications for enhancing HIV transmission in vivo. These data emphasize the importance of performing more rigorous safety testing on these products.

  18. Identification of Personal Lubricants That Can Cause Rectal Epithelial Cell Damage and Enhance HIV Type 1 Replication in Vitro

    PubMed Central

    Begay, Othell; Jean-Pierre, Ninochka; Abraham, Ciby J.; Chudolij, Anne; Seidor, Samantha; Rodriguez, Aixa; Ford, Brian E.; Henderson, Marcus; Katz, David; Zydowsky, Thomas; Robbiani, Melissa

    2011-01-01

    Abstract Over-the-counter personal lubricants are used frequently during vaginal and anal intercourse, but they have not been extensively tested for biological effects that might influence HIV transmission. We evaluated the in vitro toxicity anti-HIV-1 activity and osmolality of popular lubricants. A total of 41 lubricants were examined and compared to Gynol II and Carraguard as positive and negative controls for toxicity, respectively. Cytotoxicity was assessed using the XTT assay. The MAGI assay with R5 and X4 HIV-1 laboratory strains was used to evaluate antiviral activity. The effect of the lubricants on differentiated Caco-2 cell monolayers (transepithelial electrical resistance, TEER) was also measured. None of the lubricants tested showed significant activity against HIV-1. Surprisingly, four of them, Astroglide Liquid, Astroglide Warming Liquid, Astroglide Glycerin & Paraben-Free Liquid, and Astroglide Silken Secret, significantly enhanced HIV-1 replication (p<0.0001). A common ingredient in three of these preparations is polyquaternium-15. In vitro testing of a chemically related compound (MADQUAT) confirmed that this similarly augmented HIV-1 replication. Most of the lubricants were found to be hyperosmolar and the TEER value dropped approximately 60% 2 h after exposure to all lubricants tested. Cells treated with Carraguard, saline, and cell controls maintained about 100% initial TEER value after 2–6 h. We have identified four lubricants that significantly increase HIV-1 replication in vitro. In addition, the epithelial damage caused by these and many other lubricants may have implications for enhancing HIV transmission in vivo. These data emphasize the importance of performing more rigorous safety testing on these products. PMID:21309617

  19. Morphine inhibits intrahepatic interferon- alpha expression and enhances complete hepatitis C virus replication.

    PubMed

    Li, Yuan; Ye, Li; Peng, Jin-Song; Wang, Chuan-Qing; Luo, Guang-Xiang; Zhang, Ting; Wan, Qi; Ho, Wen-Zhe

    2007-09-01

    Heroin addicts are a high-risk group for hepatitis C virus (HCV) infection and the development of chronic HCV disease. We thus examined whether morphine, the active metabolite of heroin, has the ability to inhibit intrahepatic interferon (IFN)- alpha expression, facilitating HCV replication in human hepatocytes. Morphine inhibited intrahepatic IFN- alpha expression, which was associated with an increase in HCV replication in hepatocytes. Moreover, morphine compromised the anti-HCV effect of recombinant IFN- alpha . Investigation of the mechanism responsible for the morphine action revealed that morphine inhibited expression of IFN regulatory factor 5 in the hepatocytes. In addition, morphine suppressed the expression of p38, an important signal-transducing molecule involved in IFN- alpha -mediated anti-HCV activity. These findings indicate that morphine plays a cofactor role in facilitating HCV persistence in human hepatocytes.

  20. Effective Inhibition of Human Immunodeficiency Virus 1 Replication by Engineered RNase P Ribozyme

    PubMed Central

    Trang, Phong; Vu, Gia-Phong; Lu, Sangwei; Wu, Jianguo; Liu, Fenyong

    2012-01-01

    Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G83 -> U83 and G340 -> A340) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application. PMID:23300569

  1. Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication.

    PubMed Central

    Croen, K D

    1993-01-01

    Nitric oxide (NO) has antimicrobial activity against a wide spectrum of infectious pathogens, but an antiviral effect has not been reported. The impact of NO, from endogenous and exogenous sources, on herpes simplex virus type 1 (HSV 1) replication was studied in vitro. HSV 1 replication in RAW 264.7 macrophages was reduced 1,806-fold in monolayers induced to make NO by activation with gamma IFN and LPS. A competitive and a noncompetitive inhibitor of nitric oxide synthetase substantially reduced the antiviral effect of activated RAW macrophages. S-nitroso-L-acetyl penicillamine (SNAP) is a donor of NO and was added to the media of infected monolayers to assess the antiviral properties of NO in the absence of gamma IFN and LPS. A single dose of S-nitroso-L-acetyl penicillamine 3 h after infection inhibited HSV 1 replication in Vero, HEp2, and RAW 264.7 cells in a dose-dependent manner. Neither virucidal nor cytocidal effects of NO were observed under conditions that inhibited HSV 1 replication. Nitric oxide had inhibitory effects, comparable to that of gamma IFN/LPS, on protein and DNA synthesis as well as on cell replication. This report demonstrates that, among its diverse properties, NO has an antiviral effect. PMID:8390481

  2. A dual-targeting approach to inhibit Brucella abortus replication in human cells

    PubMed Central

    Czyż, Daniel M.; Jain-Gupta, Neeta; Shuman, Howard A.; Crosson, Sean

    2016-01-01

    Brucella abortus is an intracellular bacterial pathogen and an etiological agent of the zoonotic disease known as brucellosis. Brucellosis can be challenging to treat with conventional antibiotic therapies and, in some cases, may develop into a debilitating and life-threatening chronic illness. We used multiple independent assays of in vitro metabolism and intracellular replication to screen a library of 480 known bioactive compounds for novel B. abortus anti-infectives. Eighteen non-cytotoxic compounds specifically inhibited B. abortus replication in the intracellular niche, which suggests these molecules function by targeting host cell processes. Twenty-six compounds inhibited B. abortus metabolism in axenic culture, thirteen of which are non-cytotoxic to human host cells and attenuate B. abortus replication in the intracellular niche. The most potent non-cytotoxic inhibitors of intracellular replication reduce B. abortus metabolism in axenic culture and perturb features of mammalian cellular biology including mitochondrial function and receptor tyrosine kinase signaling. The efficacy of these molecules as inhibitors of B. abortus replication in the intracellular niche suggests “dual-target” compounds that coordinately perturb host and pathogen are promising candidates for development of improved therapeutics for intracellular infections. PMID:27767061

  3. Efficient inhibition of human immunodeficiency virus replication using novel modified microRNA-30a targeting 3′-untranslated region transcripts

    PubMed Central

    NEJATI, AHMAD; SHAHMAHMOODI, SHOHREH; AREFIAN, EHSAN; SHOJA, ZABIHOLLAH; MARASHI, SAYED-MAHDI; TABATABAIE, HAMIDEH; MOLLAEI-KANDELOUS, YAGHOUB; SOLEIMANI, MASOUD; NATEGH, RAKHSHANDEH

    2016-01-01

    RNA interference (RNAi)-based gene therapy is currently considered to be a combinatorial anti-human immunodeficiency virus-1 (HIV-1) therapy. Although artificial polycistronic microRNAs (miRs) can reduce HIV-1 escape mutant variants, this approach may increase the risk of side effects. The present study aimed to optimize the efficiency of anti-HIV RNAi gene therapy in order to reduce the cell toxicity induced by multi-short hairpin RNA expression. An artificial miR-30a-3′-untranslated region (miR-3-UTR) obtained from a single RNA polymerase II was used to simultaneously target all viral transcripts. The results of the present study demonstrated that HIV-1 replication was significantly inhibited in the cells with the miR-3-UTR construct, suggesting that miR-3′-UTR may serve as a promising tool for RNAi-based gene therapy in the treatment of HIV-1. PMID:27168813

  4. Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2.

    PubMed

    Furuya, Andrea Kinga Marias; Sharifi, Hamayun J; Jellinger, Robert M; Cristofano, Paul; Shi, Binshan; de Noronha, Carlos M C

    2016-04-01

    Marburg virus, the Kaposi's sarcoma-associated herpesvirus (KSHV) and Dengue virus all activate, and benefit from, expression of the transcription regulator nuclear erythroid 2-related factor 2 (Nrf2). The impact of Nrf2 activation on human immunodeficiency virus (HIV) infection has not been tested. Sulforaphane (SFN), produced in cruciferous vegetables after mechanical damage, mobilizes Nrf2 to potently reprogram cellular gene expression. Here we show for the first time that SFN blocks HIV infection in primary macrophages but not in primary T cells. Similarly SFN blocks infection in PMA-differentiated promonocytic cell lines, but not in other cell lines tested. siRNA-mediated depletion of Nrf2 boosted HIV infectivity in primary macrophages and reduced the anti-viral effects of SFN treatment. This supports a model in which anti-viral activity is mediated through Nrf2 after it is mobilized by SFN. We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles. Interestingly however, neither SAMHD1 nor MX2 were upregulated. This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition.

  5. Dynamic mechanism for encapsulating two HIV replication inhibitor peptides with carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Chen, Bao-Dong; Yang, Chuan-Lu; Wang, Mei-Shan; Ma, Xiao-Guang

    2012-08-01

    Encapsulation of biomolecules inside a carbon nanotube (CNT) has attracted great interest because it could enable the delivery of nanoscale pharmaceutical drugs with CNT-based devices. Using a molecular dynamics simulation, we investigate the dynamic process by which human immunodeficiency virus (HIV) replication inhibitor peptides (HRIPs) are encapsulated in a water solution contained inside a CNT. The van der Waals attraction between the HRIPs and the CNT and the root-mean-square deviation are used to analyse the evolution of the encapsulation. It is found that the interaction between the HRIPs and the CNT is the main driving force for the encapsulation process, which does not cause an obvious conformational change to the HRIPs.

  6. HIV-1 and Its gp120 Inhibits the Influenza A(H1N1)pdm09 Life Cycle in an IFITM3-Dependent Fashion

    PubMed Central

    Mesquita, Milene; Fintelman-Rodrigues, Natalia; Sacramento, Carolina Q.; Abrantes, Juliana L.; Costa, Eduardo; Temerozo, Jairo R.; Siqueira, Marilda M.; Bou-Habib, Dumith Chequer; Souza, Thiago Moreno L.

    2014-01-01

    HIV-1-infected patients co-infected with A(H1N1)pdm09 surprisingly presented benign clinical outcome. The knowledge that HIV-1 changes the host homeostatic equilibrium, which may favor the patient resistance to some co-pathogens, prompted us to investigate whether HIV-1 infection could influence A(H1N1)pdm09 life cycle in vitro. We show here that exposure of A(H1N1)pdm09-infected epithelial cells to HIV-1 viral particles or its gp120 enhanced by 25% the IFITM3 content, resulting in a decrease in influenza replication. This event was dependent on toll-like receptor 2 and 4. Moreover, knockdown of IFITM3 prevented HIV-1 ability to inhibit A(H1N1)pdm09 replication. HIV-1 infection also increased IFITM3 levels in human primary macrophages by almost 100%. Consequently, the arrival of influenza ribonucleoproteins (RNPs) to nucleus of macrophages was inhibited, as evaluated by different approaches. Reduction of influenza RNPs entry into the nucleus tolled A(H1N1)pdm09 life cycle in macrophages earlier than usual, limiting influenza's ability to induce TNF-α. As judged by analysis of the influenza hemagglutin (HA) gene from in vitro experiments and from samples of HIV-1/A(H1N1)pdm09 co-infected individuals, the HIV-1-induced reduction of influenza replication resulted in delayed viral evolution. Our results may provide insights on the mechanisms that may have attenuated the clinical course of Influenza in HIV-1/A(H1N1)pdm09 co-infected patients during the recent influenza form 2009/2010. PMID:24978204

  7. Inhibition of Mayaro virus replication by cerulenin in Aedes albopictus cells.

    PubMed

    Pereira, H S; Rebello, M A

    1998-12-01

    The antibiotic cerulenin, an inhibitor of lipid synthesis, was shown to suppress Mayaro virus replication in Aedes albopictus cells at non-cytotoxic doses. Cerulenin blocked the incorporation of [3H]glycerol into lipids when present at any time post infection (p.i.). Cerulenin added at the beginning of infection inhibited the synthesis of virus proteins. However, when this antibiotic was added at later stages of infection, it had only a mild effect on the virus protein synthesis. The possibility that cerulenin acts by blocking an initial step in the Mayaro virus replication after virus entry and before late viral translation is discussed.

  8. Cyclophilin A binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly.

    PubMed

    Kovalev, Nikolay; Nagy, Peter D

    2013-12-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.

  9. TOE1 is an inhibitor of HIV-1 replication with cell-penetrating capability

    PubMed Central

    Sperandio, Sabina; Barat, Corinne; Cabrita, Miguel A.; Gargaun, Ana; Berezovski, Maxim V.; Tremblay, Michel J.; de Belle, Ian

    2015-01-01

    Target of Egr1 (TOE1) is a nuclear protein localized primarily in nucleoli and Cajal bodies that was identified as a downstream target of the immediate early gene Egr1. TOE1 displays a functional deadenylation domain and has been shown to participate in spliceosome assembly. We report here that TOE1 can function as an inhibitor of HIV-1 replication and show evidence that supports a direct interaction of TOE1 with the viral specific transactivator response element as part of the inhibitory mechanism. In addition, we show that TOE1 can be secreted by activated CD8+ T lymphocytes and can be cleaved by the serine protease granzyme B, one of the main components of cytotoxic granules. Both full-length and cleaved TOE1 can spontaneously cross the plasma membrane and penetrate cells in culture, retaining HIV-1 inhibitory activity. Antiviral potency of TOE1 and its cell-penetrating capability have been identified to lie within a 35-amino-acid region containing the nuclear localization sequence. PMID:26056259

  10. Endogenous CD317/Tetherin limits replication of HIV-1 and murine leukemia virus in rodent cells and is resistant to antagonists from primate viruses.

    PubMed

    Goffinet, Christine; Schmidt, Sarah; Kern, Christian; Oberbremer, Lena; Keppler, Oliver T

    2010-11-01

    Human CD317 (BST-2/tetherin) is an intrinsic immunity factor that blocks the release of retroviruses, filoviruses, herpesviruses, and arenaviruses. It is unclear whether CD317 expressed endogenously in rodent cells has the capacity to interfere with the replication of the retroviral rodent pathogen murine leukemia virus (MLV) or, in the context of small-animal model development, contributes to the well-established late-phase restriction of human immunodeficiency virus type 1 (HIV-1). Here, we show that small interfering RNA (siRNA)-mediated knockdown of CD317 relieved a virion release restriction and markedly enhanced the egress of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) in rat cells, including primary macrophages. Moreover, rodent CD317 potently inhibited MLV release, and siRNA-mediated depletion of CD317 in a mouse T-cell line resulted in the accelerated spread of MLV. Several virus-encoded antagonists have recently been reported to overcome the restriction imposed by human or monkey CD317, including HIV-1 Vpu, envelope glycoproteins of HIV-2 and Ebola virus, Kaposi's sarcoma-associated herpesvirus K5, and SIV Nef. In contrast, both rat and mouse CD317 showed a high degree of resistance to these viral antagonists. These data suggest that CD317 is a broadly acting and conserved mediator of innate control of retroviral infection and pathogenesis that restricts the release of retroviruses and lentiviruses in rodents. The high degree of resistance of the rodent CD317 restriction factors to antagonists from primate viruses has implications for HIV-1 small-animal model development and may guide the design of novel antiviral interventions.

  11. Allo-immunization elicits CCR5 antibodies, SDF-1 chemokines, and CD8-suppressor factors that inhibit transmission of R5 and X4 HIV-1 in women.

    PubMed

    Wang, Y; Underwood, J; Vaughan, R; Harmer, A; Doyle, C; Lehner, T

    2002-09-01

    Studies in humans suggest that allo-immunization induces CC-chemokines, CD8-suppressor factors (SF) and anti-HIV immunity. Here we report that allo-immunization with unmatched leucocytes from partners of women with recurrent spontaneous abortion elicits specific antibodies to the CCR5 receptor. Such antibodies inhibit replication of M-tropic HIV-1 (R5) and MIP-1beta-mediated chemotaxis. These CCR5 antibodies were also found in the sera of multiparous women that were naturally immunized by semi-allogeneic fetal antigens. The specificity of these antibodies was demonstrated by adsorption with CCR5 transfected HEK-293 cells, a baculovirus CCR5 preparation and a peptide of the 2nd extra-cellular loop of CCR5. Allo-immunization also stimulated increased concentrations of the CXC chemokine, SDF-1alpha and CD8-SF that inhibit T-tropic HIV-1 (X4) replication. We suggest that allo- immunization may elicit (a) CC chemokines, CCR5 antibodies and CD8-SF that inhibit M-tropic HIV-1 infection and (b) the CXC chemokine SDF-1alpha and CD8-SF that inhibit T-tropic HIV-1 infection.

  12. Expression of the zinc-finger antiviral protein inhibits alphavirus replication.

    PubMed

    Bick, Matthew J; Carroll, John-William N; Gao, Guangxia; Goff, Stephen P; Rice, Charles M; MacDonald, Margaret R

    2003-11-01

    The rat zinc-finger antiviral protein (ZAP) was recently identified as a host protein conferring resistance to retroviral infection. We analyzed ZAP's ability to inhibit viruses from other families and found that ZAP potently inhibits the replication of multiple members of the Alphavirus genus within the Togaviridae, including Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. However, expression of ZAP did not induce a broad-spectrum antiviral state as some viruses, including vesicular stomatitis virus, poliovirus, yellow fever virus, and herpes simplex virus type 1, replicated to normal levels in ZAP-expressing cells. We determined that ZAP expression inhibits Sindbis virus replication after virus penetration and entry, but before the amplification of newly synthesized plus strand genomic RNA. Using a temperature-sensitive Sindbis virus mutant expressing luciferase, we further showed that translation of incoming viral RNA is blocked by ZAP expression. Elucidation of the antiviral mechanism by which ZAP inhibits Sindbis virus translation may lead to the development of agents with broad activity against alphaviruses.

  13. IL-27 inhibits HIV-1 infection in human macrophages by down-regulating host factor SPTBN1 during monocyte to macrophage differentiation.

    PubMed

    Dai, Lue; Lidie, Kristy B; Chen, Qian; Adelsberger, Joseph W; Zheng, Xin; Huang, DaWei; Yang, Jun; Lempicki, Richard A; Rehman, Tauseef; Dewar, Robin L; Wang, Yanmei; Hornung, Ronald L; Canizales, Kelsey A; Lockett, Stephen J; Lane, H Clifford; Imamichi, Tomozumi

    2013-03-11

    The susceptibility of macrophages to HIV-1 infection is modulated during monocyte differentiation. IL-27 is an anti-HIV cytokine that also modulates monocyte activation. In this study, we present new evidence that IL-27 promotes monocyte differentiation into macrophages that are nonpermissive for HIV-1 infection. Although IL-27 treatment does not affect expression of macrophage differentiation markers or macrophage biological functions, it confers HIV resistance by down-regulating spectrin β nonerythrocyte 1 (SPTBN1), a required host factor for HIV-1 infection. IL-27 down-regulates SPTBN1 through a TAK-1-mediated MAPK signaling pathway. Knockdown of SPTBN1 strongly inhibits HIV-1 infection of macrophages; conversely, overexpression of SPTBN1 markedly increases HIV susceptibility of IL-27-treated macrophages. Moreover, we demonstrate that SPTBN1 associates with HIV-1 gag proteins. Collectively, our results underscore the ability of IL-27 to protect macrophages from HIV-1 infection by down-regulating SPTBN1, thus indicating that SPTBN1 is an important host target to reduce HIV-1 replication in one major element of the viral reservoir.

  14. The multi-targeted kinase inhibitor sorafenib inhibits human cytomegalovirus replication.

    PubMed

    Michaelis, Martin; Paulus, Christina; Löschmann, Nadine; Dauth, Stephanie; Stange, Elisabeth; Doerr, Hans Wilhelm; Nevels, Michael; Cinatl, Jindrich

    2011-03-01

    Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised individuals. Here, non-toxic concentrations of the anti-cancer kinase inhibitor sorafenib were shown to inhibit replication of different HCMV strains (including a ganciclovir-resistant strain) in different cell types. In contrast to established anti-HCMV drugs, sorafenib inhibited HCMV major immediate early promoter activity and HCMV immediate early antigen (IEA) expression. Sorafenib is known to inhibit Raf. Comparison of sorafenib with the MEK inhibitor U0126 suggested that sorafenib inhibits HCMV IEA expression through inhibition of Raf but independently of signaling through the Raf downstream kinase MEK 1/2. In concordance, siRNA-mediated depletion of Raf but not of MEK-reduced IEA expression. In conclusion, sorafenib diminished HCMV replication in clinically relevant concentrations and inhibited HCMV IEA expression, a pathophysiologically relevant event that is not affected by established anti-HCMV drugs. Moreover, we demonstrated for the first time that Raf activation is involved in HCMV IEA expression.

  15. Mechanism of inhibition of HSV-1 replication by tumor necrosis factor and interferon gamma.

    PubMed

    Feduchi, E; Carrasco, L

    1991-02-01

    Tumor necrosis factor (TNF) synergizes with interferon (IFN gamma) in the blockade of HSV-1 replication. Antibodies against IFN beta block this synergism, implying a role of IFN beta in the antiviral activity of TNF plus IFN gamma. IFN beta 1 added exogenously to Hep-2 cells shows antiviral activity against HSV-1 only at high concentrations, whereas IFN beta 2 (also known as IL-6) alone has no effect on the replication of VSV or HSV-1 even when 1,000 U/ml are present. Our results are in accordance with the idea that TNF induces IFN beta 1 and that both cytokines must be present in the culture medium to synergize with IFN gamma in order to inhibit HSV-1 replication.

  16. Identification and characterization of interferon-induced proteins that inhibit alphavirus replication.

    PubMed

    Zhang, Yugen; Burke, Crystal W; Ryman, Kate D; Klimstra, William B

    2007-10-01

    Alpha/beta interferon (IFN-alpha/beta) produces antiviral effects through upregulation of many interferon-stimulated genes (ISGs) whose protein products are effectors of the antiviral state. Previous data from our laboratory have shown that IFN-alpha/beta can limit Sindbis virus (SB) replication through protein kinase R (PKR)-dependent and PKR-independent mechanisms and that one PKR-independent mechanism inhibits translation of the infecting virus genome (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). Further, using Affymetrix microarray technology, we identified 44 genes as candidates for PKR/RNase L-independent IFN-induced antiviral activities. In the current studies, we have begun analyzing these gene products for antialphavirus activity using three techniques: (i) overexpression of the protein from SB vectors and assessment of virulence attenuation in mice; (ii) overexpression of the proteins in a stable tetracycline-inducible murine fibroblast culture system and assessment of effects upon SB replication; and (iii) small interfering RNA-mediated knockdown of gene mRNA in fibroblast cultures followed by SB replication assessment as above. Tested proteins included those we hypothesized had potential to affect virus genome translation and included murine ISG20, ISG15, the zinc finger antiviral protein (ZAP), viperin, p56, p54, and p49. Interestingly, the pattern of antiviral activity for some gene products was different between in vitro and in vivo assays. Viperin and ZAP attenuated virulence most profoundly in mice. However, ISG20 and ZAP potently inhibited SB replication in vitro, whereas and viperin, p56, and ISG15 exhibited modest replication inhibition in vitro. In contrast, p54 and p49 had little to no effect in any assay.

  17. Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells

    PubMed Central

    Sinha, Namita; Kumar, Anil; Kumar, Santosh

    2016-01-01

    While cigarette smoking is prevalent amongst HIV-infected patients, the effects of cigarette smoke constituents in cells of myeloid lineage are poorly known. Recently, we have shown that nicotine induces oxidative stress through cytochrome P450 (CYP) 2A6-mediated pathway in U937 monocytic cells. The present study was designed to examine the effect of cigarette smoke condensate (CSC), which contains majority of tobacco constituents, on oxidative stress, cytotoxicity, expression of CYP1A1, and/or HIV-1 replication in HIV-infected (U1) and uninfected U937 cells. The effects of CSC on induction of CYP1 enzymes in HIV-infected primary macrophages were also analyzed. The results showed that the CSC-mediated increase in production of reactive oxygen species (ROS) in U937 cells is dose- and time-dependent. Moreover, CSC treatment was found to induce cytotoxicity in U937 cells through the apoptotic pathway via activation of caspase-3. Importantly, pretreatment with vitamin C blocked the CSC-mediated production of ROS and induction of caspase-3 activity. In U1 cells, acute treatment of CSC increased ROS production at 6H (>2-fold) and both ROS (>2 fold) and HIV-1 replication (>3-fold) after chronic treatment. The CSC mediated effects were associated with robust induction in the expression of CYP1A1 mRNA upon acute CSC treatment of U937 and U1 cells (>20-fold), and upon chronic CSC treatment to U1 cells (>30-fold). In addition, the CYP1A1 induction in U937 cells was mediated through the aromatic hydrocarbon receptor pathway. Lastly, CSC, which is known to increase viral replication in primary macrophages, was also found to induce CYP1 enzymes in HIV-infected primary macrophages. While mRNA levels of both CYP1A1 and CYP1B1 were elevated following CSC treatment, only CYP1B1 protein levels were increased in HIV-infected primary macrophages. In conclusion, these results suggest a possible association between oxidative stress, CYP1 expression, and viral replication in CSC

  18. Aqueous Extracts of the Marine Brown Alga Lobophora variegata Inhibit HIV-1 Infection at the Level of Virus Entry into Cells

    PubMed Central

    Kremb, Stephan; Helfer, Markus; Kraus, Birgit; Wolff, Horst; Wild, Christian; Schneider, Martha; Voolstra, Christian R.; Brack-Werner, Ruth

    2014-01-01

    In recent years, marine algae have emerged as a rich and promising source of molecules with potent activities against various human pathogens. The widely distributed brown alga Lobophora variegata that is often associated with tropical coral reefs exerts strong antibacterial and antiprotozoal effects, but so far has not been associated with specific anti-viral activities. This study investigated potential HIV-1 inhibitory activity of L. variegata collected from different geographical regions, using a cell-based full replication HIV-1 reporter assay. Aqueous L. variegata extracts showed strong inhibitory effects on several HIV-1 strains, including drug-resistant and primary HIV-1 isolates, and protected even primary cells (PBMC) from HIV-1-infection. Anti-viral potency was related to ecological factors and showed clear differences depending on light exposition or epiphyte growth. Assays addressing early events of the HIV-1 replication cycle indicated that L. variegata extracts inhibited entry of HIV-1 into cells at a pre-fusion step possibly by impeding mobility of virus particles. Further characterization of the aqueous extract demonstrated that even high doses had only moderate effects on viability of cultured and primary cells (PBMCs). Imaging-based techniques revealed extract effects on the plasma membrane and actin filaments as well as induction of apoptosis at concentrations exceeding EC50 of anti-HIV-1 activity by more than 400 fold. In summary, we show for the first time that L. variegata extracts inhibit HIV-1 entry, thereby suggesting this alga as promising source for the development of novel HIV-1 inhibitors. PMID:25144758

  19. Aqueous extracts of the marine brown alga Lobophora variegata inhibit HIV-1 infection at the level of virus entry into cells.

    PubMed

    Kremb, Stephan; Helfer, Markus; Kraus, Birgit; Wolff, Horst; Wild, Christian; Schneider, Martha; Voolstra, Christian R; Brack-Werner, Ruth

    2014-01-01

    In recent years, marine algae have emerged as a rich and promising source of molecules with potent activities against various human pathogens. The widely distributed brown alga Lobophora variegata that is often associated with tropical coral reefs exerts strong antibacterial and antiprotozoal effects, but so far has not been associated with specific anti-viral activities. This study investigated potential HIV-1 inhibitory activity of L. variegata collected from different geographical regions, using a cell-based full replication HIV-1 reporter assay. Aqueous L. variegata extracts showed strong inhibitory effects on several HIV-1 strains, including drug-resistant and primary HIV-1 isolates, and protected even primary cells (PBMC) from HIV-1-infection. Anti-viral potency was related to ecological factors and showed clear differences depending on light exposition or epiphyte growth. Assays addressing early events of the HIV-1 replication cycle indicated that L. variegata extracts inhibited entry of HIV-1 into cells at a pre-fusion step possibly by impeding mobility of virus particles. Further characterization of the aqueous extract demonstrated that even high doses had only moderate effects on viability of cultured and primary cells (PBMCs). Imaging-based techniques revealed extract effects on the plasma membrane and actin filaments as well as induction of apoptosis at concentrations exceeding EC50 of anti-HIV-1 activity by more than 400 fold. In summary, we show for the first time that L. variegata extracts inhibit HIV-1 entry, thereby suggesting this alga as promising source for the development of novel HIV-1 inhibitors.

  20. Pegylated Interferon Alfa-2a Monotherapy Results in Suppression of HIV Type 1 Replication and Decreased Cell-Associated HIV DNA Integration

    PubMed Central

    Azzoni, Livio; Foulkes, Andrea S.; Papasavvas, Emmanouil; Mexas, Angela M.; Lynn, Kenneth M.; Mounzer, Karam; Tebas, Pablo; Jacobson, Jeffrey M.; Frank, Ian; Busch, Michael P.; Deeks, Steven G.; Carrington, Mary; O'Doherty, Una; Kostman, Jay; Montaner, Luis J.

    2013-01-01

    Background. Antiretroviral therapy (ART)–mediated immune reconstitution fails to restore the capacity of the immune system to spontaneously control human immunodeficiency virus (HIV) replication. Methods. A total of 23 HIV type 1 (HIV-1)–infected, virologically suppressed subjects receiving ART (CD4+ T-cell count, >450 cells/μL) were randomly assigned to have 180 μg/week (for arm A) or 90 μg/week (for arm B) of pegylated (Peg) interferon alfa-2a added to their current ART regimen. After 5 weeks, ART was interrupted, and Peg–interferon alfa-2a was continued for up to 12 weeks (the primary end point), with an option to continue to 24 weeks. End points included virologic failure (viral load, ≥400 copies/mL) and adverse events. Residual viral load and HIV-1 DNA integration were also assessed. Results. At week 12 of Peg–interferon alfa-2a monotherapy, viral suppression was observed in 9 of 20 subjects (45%), a significantly greater proportion than expected (arm A, P = .0088; arm B, P = .0010; combined arms, P < .0001). Over 24 weeks, both arms had lower proportions of subjects who had viral load, compared with the proportion of subjects in a historical control group (arm A, P = .0046; arm B, P = .0011). Subjects who had a sustained viral load of <400 copies/mL had decreased levels of integrated HIV DNA (P = .0313) but increased residual viral loads (P = .0078), compared with subjects who experienced end-point failure. Conclusions. Peg–interferon alfa-2a immunotherapy resulted in control of HIV replication and decreased HIV-1 integration, supporting a role for immunomediated approaches in HIV suppression and/or eradication. Clinical Trials Registration. NCT00594880. PMID:23105144

  1. Silencing E1A mRNA by RNA interference inhibits adenovirus replication.

    PubMed

    Chung, Y-S; Kim, M-K; Lee, W-J; Kang, C

    2007-01-01

    The adenovirus family contains 51 human serotypes, and most human adenoviruses cause widespread respiratory tract infections. Adenovirus infections can result in severe complications in some cases, such as in adenovirus type 11 infection in immunocompromised patients. However, effective treatment methods for adenovirus infections are currently unavailable. This prompted the search for antiviral agents effective against adenovirus infections. In the present study, adenovirus E1A was targeted by RNA interference (RNAi) using synthetic small interfering RNAs (siRNAs) in an attempt to inhibit viral replication, since adenovirus E1A proteins are known to be involved in the transcriptional activation of the viral and cellular genes necessary for controlling the cell cycle and viral replication. The results indicated that the siRNAs effectively reduced the amount of adenovirus E1A mRNA and the levels of replicative intermediates. Additionally, siRNA-mediated gene silencing inhibited adenovirus replication by suppressing the E1A mRNA. These results suggest that the RNAi-mediated targeting of adenovirus E1A may have a potentially therapeutic effect in controlling adenovirus infections.

  2. Oleanane triterpenes from the flowers of Camellia japonica inhibit porcine epidemic diarrhea virus (PEDV) replication.

    PubMed

    Yang, Jun-Li; Ha, Thi-Kim-Quy; Dhodary, Basanta; Pyo, Euisun; Nguyen, Ngoc Hieu; Cho, Hyomoon; Kim, Eunhee; Oh, Won Keun

    2015-02-12

    Porcine epidemic diarrhea virus (PEDV) infections have resulted in a severe economic loss in the swine industry in many countries due to no effective treatment approach. Fifteen oleanane triterpenes (1-15), including nine new ones (1-4 and 10-14), were isolated from the flowers of Camellia japonica, and their molecular structures were determined by extensive spectroscopic methods. These compounds were evaluated for their antiviral activity against PEDV replication, and the structure-activity relationships (SARs) were discussed. Compounds 6, 9, 11, and 13 showed most potent inhibitory effects on PEDV replication. They were found to inhibit PEDV genes encoding GP6 nucleocapsid, GP2 spike, and GP5 membrane protein synthesis based on RT-PCR data. Western blot analysis also demonstrated their inhibitory effects on PEDV GP6 nucleocapsid and GP2 spike protein synthesis during viral replication. The present study suggested the potential of compounds 6, 9, 11, and 13 as promising scaffolds for treating PEDV infection via inhibiting viral replication.

  3. Oseltamivir inhibits influenza virus replication and transmission following ocular-only aerosol inoculation of ferrets.

    PubMed

    Belser, Jessica A; Maines, Taronna R; Creager, Hannah M; Katz, Jacqueline M; Tumpey, Terrence M

    2015-10-01

    Ocular exposure to influenza virus represents an alternate route of virus entry capable of establishing a respiratory infection in mammals, but the effectiveness of currently available antiviral treatments to limit virus replication within ocular tissue or inhibit virus spread from ocular sites to the respiratory tract is poorly understood. Using an inoculation method that delivers an aerosol inoculum exclusively to the ocular surface, we demonstrate that oral oseltamivir administration following ocular-only aerosol inoculation with multiple avian and human influenza viruses protected ferrets from a fatal and systemic infection, reduced clinical signs and symptoms of illness, and decreased virus transmissibility to susceptible contacts when a respiratory infection was initiated. The presence of oseltamivir further inhibited influenza virus replication in primary human corneal epithelial cells. These findings provide critical experimental evidence supporting the use of neuraminidase inhibitors during outbreaks of influenza virus resulting in ocular disease or following ocular exposure.

  4. Gene- and protein-delivered zinc finger-staphylococcal nuclease hybrid for inhibition of DNA replication of human papillomavirus.

    PubMed

    Mino, Takashi; Mori, Tomoaki; Aoyama, Yasuhiro; Sera, Takashi

    2013-01-01

    Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

  5. Rabies viruses leader RNA interacts with host Hsc70 and inhibits virus replication.

    PubMed

    Zhang, Ran; Liu, Chuangang; Cao, Yunzi; Jamal, Muhammad; Chen, Xi; Zheng, Jinfang; Li, Liang; You, Jing; Zhu, Qi; Liu, Shiyong; Dai, Jinxia; Cui, Min; Fu, Zhen F; Cao, Gang

    2017-03-23

    Viruses have been shown to be equipped with regulatory RNAs to evade host defense system. It has long been known that rabies virus (RABV) transcribes a small regulatory RNA, leader RNA (leRNA), which mediates the transition from viral RNA transcription to replication. However, the detailed molecular mechanism remains enigmatic. In the present study, we determined the genetic architecture of RABV leRNA and demonstrated its inhibitory effect on replication of wild-type rabies, DRV-AH08. The RNA immunoprecipitation results suggest that leRNA inhibits RABV replication via interfering the binding of RABV nucleoprotein with genomic RNA. Furthermore, we identified heat shock cognate 70 kDa protein (Hsc70) as a leRNA host cellular interacting protein, of which the expression level was dynamically regulated by RABV infection. Notably, our data suggest that Hsc70 was involved in suppressing RABV replication by leader RNA. Finally, our experiments imply that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis. Together, this study suggested leRNA in concert with its host interacting protein Hsc70, dynamically down-regulate RABV replication.

  6. Development of a novel anti-HIV-1 agent from within: Effect of chimeric Vpr-containing protease cleavage site residues on virus replication

    PubMed Central

    Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

    1997-01-01

    Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag–Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection. PMID:9096396

  7. Abasic Phosphorothioate Oligomers Inhibit HIV-1 Reverse Transcription and Block Virus Transmission across Polarized Ectocervical Organ Cultures

    PubMed Central

    Fraietta, Joseph A.; Mueller, Yvonne M.; Lozenski, Karissa L.; Ratner, Deena; Boesteanu, Alina C.; Hancock, Aidan S.; Lackman-Smith, Carol; Zentner, Isaac J.; Chaiken, Irwin M.; Chung, Suhman; LeGrice, Stuart F. J.; Snyder, Beth A.; Mankowski, Marie K.; Jones, Natalie M.; Hope, Jennifer L.; Gupta, Phalguni; Anderson, Sharon H.; Wigdahl, Brian

    2014-01-01

    In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2′ deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1. PMID:25224013

  8. Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication

    PubMed Central

    Albert, Benjamin; Hafner, Lukas; Lezaja, Aleksandra; Costanzo, Michael; Boone, Charlie; Shore, David

    2016-01-01

    The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common. PMID:27820830

  9. Effects of varying concentrations of bleach on in vitro HIV-1 replication and the relevance to injection drug use.

    PubMed

    Contoreggi, C; Jones, S; Simpson, P; Lange, W R; Meyer, W A

    2000-01-01

    The use of bleach (hypochlorite) as a disinfectant for drug injection equipment in the intravenous-drug-using population was recommended early in the HIV-1/AIDS epidemic. Epidemiological studies have challenged the use of bleach as an effective measure to prevent HIV-1 transmission. However, in vitro HIV-1 coculture studies have shown that a high concentration of bleach is an effective cytotoxic and potentially virucidal agent. In this study, we demonstrate that HIV-1 peripheral blood mononuclear cell cocultures containing low concentrations of hypochlorite in the media showed earlier conversion to HIV-1 positivity, as measured by the presence of p24 antigen. HIV-1 cocultures with high concentrations of hypochlorite in the culture media, which appeared to be highly cytotoxic, and HIV-1 cocultures without bleach in the media did not exhibit this early p24 antigen positivity. Hypochlorite chemically disinfects by releasing free chlorine that is a potent oxidant. In injection drug equipment, a low residual concentration of bleach is likely to remain in cleaned equipment despite rinsing with water. Low concentrations of oxidants have been shown to enhance tissue inflammation, in vivo, as well as HIV-1 replication in vitro. Previous studies have shown that despite vigorous cleaning of blood-contaminated injection syringes with bleach followed by water, microaggregates of residual blood remained in bleach-cleaned blood-contaminated syringes. Hypothetically, oxidant effects of the residual bleach in the bleach-cleaned syringes could enhance the possibility of infection by remaining HIV-1 contained in a contaminated syringe. We suggest that the likelihood of an injection drug user contracting HIV-1 through the sharing of a bleach-cleaned blood-contaminated syringe may be increased by the cotransmission of residual bleach and its localized tissue-inflammatory effects; however, this has not been statistically proven in epidemiological studies.

  10. The Interplay Between Host Genetic Variation, Viral Replication, and Microbial Translocation in Untreated HIV-Infected Individuals

    PubMed Central

    Perkins, Molly R.; Bartha, Istvan; Timmer, J. Katherina; Liebner, Julia C.; Wollinsky, David; Günthard, Huldrych F.; Hauser, Christoph; Bernasconi, Enos; Hoffmann, Matthias; Calmy, Alexandra; Battegay, Manuel; Telenti, Amalio; Douek, Daniel C.; Fellay, Jacques; Aubert, V.; Battegay, M.; Bernasconi, E.; Böni, J.; Bucher, H.C.; Burton-Jeangros, C.; Calmy, A.; Cavassini, M.; Dollenmaier, G.; Egger, M.; Elzi, L.; Fehr, J.; Fellay, J.; Furrer, H.; Fux, C.A.; Gorgievski, M.; Günthard, H.; Haerry, D.; Hasse, B.; Hirsch, H.H.; Hoffmann, M.; Hösli, I.; Kahlert, C.; Kaiser, L.; Keiser, O.; Klimkait, T.; Kouyos, R.; Kovari, H.; Ledergerber, B.; Martinetti, G.; Martinez de Tejada, B.; Metzner, K.; Müller, N.; Nadal, D.; Nicca, D.; Pantaleo, G.; Rauch, A.; Regenass, S.; Rickenbach, M.; Rudin, C.; Schöni-Affolter, F.; Schmid, P.; Schüpbach, J.; Speck, R.; Tarr, P.; Telenti, A.; Trkola, A.; Vernazza, P.; Weber, R.; Yerly, S.

    2015-01-01

    Systemic immune activation, a major determinant of human immunodeficiency virus (HIV) disease progression, is the result of a complex interplay between viral replication, dysregulation of the immune system, and microbial translocation due to gut mucosal damage. Although human genetic variants influencing HIV load have been identified, it is unknown how much the host genetic background contributes to interindividual differences in other determinants of HIV pathogenesis such as gut damage and microbial translocation. Using samples and data from 717 untreated participants in the Swiss HIV Cohort Study and a genome-wide association study design, we searched for human genetic determinants of plasma levels of intestinal fatty acid–binding protein (I-FABP/FABP2), a marker of gut damage, and of soluble CD14 (sCD14), a marker of lipopolysaccharide bioactivity and microbial translocation. We also assessed the correlations between HIV load, sCD14, and I-FABP. Although we found no genome-wide significant determinant of the tested plasma markers, we observed strong associations between sCD14 and both HIV load and I-FABP, shedding new light on the relationships between processes that drive progression of untreated HIV infection. PMID:25701868

  11. A Truncated Nef Peptide from SIVcpz Inhibits the Production of HIV-1 Infectious Progeny

    PubMed Central

    Sabino Cunha, Marcela; Lima Sampaio, Thatiane; Peterlin, B. Matija; Jesus da Costa, Luciana

    2016-01-01

    Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity. PMID:27399760

  12. Protein grafting of an HIV-1-inhibiting epitope

    NASA Astrophysics Data System (ADS)

    Sia, Samuel K.; Kim, Peter S.

    2003-08-01

    Protein grafting, the transfer of a binding epitope of one ligand onto the surface of another protein, is a potentially powerful technique for presenting peptides in preformed and active three-dimensional conformations. Its utility, however, has been limited by low biological activity of the designed ligands and low tolerance of the protein scaffolds to surface substitutions. Here, we graft the complete binding epitope (19 nonconsecutive amino acids with a solvent-accessible surface area of >2,000 Å2) of an HIV-1 C-peptide, which is derived from the C-terminal region of HIV-1 gp41 and potently inhibits HIV-1 entry into cells, onto the surface of a GCN4 leucine zipper. The designed peptide, named C34coil, displays a potent antiviral activity approaching that of the native ligand. Moreover, whereas the linear C-peptide is unstructured and sensitive to degradation by proteases, C34coil is well structured, conformationally stable, and exhibits increased resistance to proteolytic degradation compared with the linear peptide. In addition to being a structured antiviral inhibitor, C34coil may also serve as the basis for the development of an alternative class of immunogens. This study demonstrates that "one-shot" protein grafting, without subsequent rounds of optimization, can be used to create ligands with structural conformations and improved biomedical properties.

  13. Inhibition of HIV-1 enhancer-controlled transcription by artificial enhancer-binding peptides derived from bacteriophage 434 repressor.

    PubMed

    Caderas, G; Klauser, S; Liu, N; Bienz, A; Gutte, B

    1999-12-01

    An artificial HIV-1 enhancer-binding 42-residue peptide (R42) that had been derived from bacteriophage 434 repressor inhibited the cell-free in vitro transcription of HIV-1 enhancer-containing plasmids [Hehlgans, T., Stolz, M., Klauser, S., Cui, T., Salgam, P., Brenz Verca, S., Widmann, M., Leiser, A., Städler, K. & Gutte, B. (1993) FEBS Lett. 315, 51-55; Caderas, G. (1997) PhD Thesis, University of Zürich]. Here we show that, after N-terminal extension of R42 with a viral nuclear localization signal, the resulting nucR42 peptide was active in intact cells. NucR42 could be detected immunologically in nuclear extracts and produced a 60-70% reduction of the rate of transcription of an HIV-1 enhancer-carrying plasmid in COS-1 cells that had been cotransfected with the HIV enhancer plasmid, an expression plasmid for nucR42, and a control. NucR42 was also synthesized chemically and the synthetic product characterized by HPLC, mass spectrometry, and quantitative amino acid analysis. Band shift, footprint, and in vitro transcription assays in the presence of exogenous NF-kappaBp50 indicated that the binding sites of nucR42 and NF-kappaB on the HIV enhancers overlapped and that a relatively small excess of nucR42 sufficed to displace NF-kappaBp50. Band shift and in vitro transcription experiments showed also that exchange of the 434 repressor-derived nine-residue recognition helix of nucR42 for four glycines abolished the HIV enhancer binding specificity whereas leucine zipper- or retro-leucine zipper-mediated dimerization of R42 analogues increased it suggesting the potential application of such dimeric HIV enhancer-binding peptides as intracellular inhibitors of HIV replication.

  14. Inhibition of T-tropic HIV Strains by Selective Antagonization of the Chemokine Receptor CXCR4

    PubMed Central

    Schols, Dominique; Struyf, Sofie; Damme, Jo Van; Esté, José A.; Henson, Geoffrey; Clercq, Erik De

    1997-01-01

    Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1α or MIP-1β, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca2+ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell–derived factor 1α, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca2+ flux induced by the CC-chemokines MIP-1α, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca2+ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists. PMID:9334378

  15. Inhibition of T-tropic HIV strains by selective antagonization of the chemokine receptor CXCR4.

    PubMed

    Schols, D; Struyf, S; Van Damme, J; Esté, J A; Henson, G; De Clercq, E

    1997-10-20

    Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1alpha or MIP-1beta, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca2+ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell-derived factor 1alpha, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca2+ flux induced by the CC-chemokines MIP-1alpha, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca2+ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists.

  16. Consensus siRNA for inhibition of HCV genotype-4 replication

    PubMed Central

    Zekri, Abdel Rahman N; Bahnassy, Abeer A; El-Din, Hanaa M Alam; Salama, Hosny M

    2009-01-01

    Background HCV is circulating as a heterogeneous group of quasispecies. It has been addressed that siRNA can inhibit HCV replication in-vitro using HCV clone and/or replicon which have only one genotype. The current study was conducted to assess whether siRNA can inhibit different HCV genotypes with many quasispecies and to assess whether consensus siRNA have the same effect as regular siRNA. Methods We generated two chemically synthesized consensus siRNAs (Z3 and Z5) which cover most known HCV genotype sequences and quasispecies using Ambium system. Highly positive HCV patient's serum with nine quasispecies was transfected in-vitro to Huh-7 cell line which supports HCV genotype-4 replication. siRNA (Z3&Z5) were transfected according to Qiagen Porta-lipid technique and subsequently cultured for eight days. HCV replication was monitored by RT-PCR for detection of plus and minus strands. Real-time PCR was used for quantification of HCV, whereas detection of the viral core protein was performed by western blot. Results HCV RNA levels decreased 18-fold (P = 0.001) and 25-fold (P = 0.0005) in cells transfected with Z3 and Z5, respectively, on Day 2 post transfection and continued for Day 3 by Z3 and Day 7 by Z5. Reduction of core protein expression was reported at Day 2 post Z3 siRNA transfection and at Day 1 post Z5 siRNA, which was persistent for Day 4 for the former and for Day 6 for the latter. Conclusion Consensus siRNA could be used as a new molecular target therapy to effectively inhibit HCV replication in the presence of more than one HCV quasispecies. PMID:19173711

  17. Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells.

    PubMed

    Wang, Hua; Blair, Carol D; Olson, Ken E; Clem, Rollie J

    2008-11-01

    Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either pro-apoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

  18. Toll-Like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication

    PubMed Central

    Bhargavan, Biju; Woollard, Shawna M.; Kanmogne, Georgette D.

    2016-01-01

    TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetics modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests TLR3 can acts as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects. PMID:26569339

  19. Flos Farfarae Inhibits Enterovirus 71-Induced Cell Injury by Preventing Viral Replication and Structural Protein Expression.

    PubMed

    Chiang, Ya Wen; Yeh, Chia Feng; Yen, Ming Hong; Lu, Chi Yu; Chiang, Lien Chai; Shieh, Den En; Chang, Jung San

    2017-02-23

    Enterovirus 71 (EV71) infection can cause airway symptoms, brainstem encephalitis, neurogenic shock, and neurogenic pulmonary edema with high morbidity and mortality. There is no proven therapeutic modality. Flos Farfarae is the dried flower bud of Tussilago farfara L. that has been used to manage airway illnesses for thousands of years. It has neuro-protective activity and has been used to manage neuro-inflammatory diseases. However, it is unknown whether Flos Farfarae has activity against EV71-induced neuropathy. The current study used both human foreskin fibroblast (CCFS-1/KMC) and human rhabdomyosarcoma (RD) cell lines to test the hypothesis that a hot water extract of Flos Farfarae could effectively inhibit EV71 infection. The authenticity of Flos Farfarae was confirmed by HPLC-UV fingerprint. Through plaque reduction assays and flow cytometry, Flos Farfarae was found to inhibit EV71 infection ([Formula: see text]). Inhibition of viral replication and protein expression were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR), and western blot, respectively. The estimated IC[Formula: see text]s were 106.3[Formula: see text][Formula: see text]g/mL in CCFS-1/KMC, and 15.0[Formula: see text][Formula: see text]g/mL in RD cells. Therefore, Flos Farfarae could be beneficial to inhibit EV71 infection by preventing viral replication and structural protein expression.

  20. Impact of HIV Infection and Kaposi Sarcoma on Human Herpesvirus-8 Mucosal Replication and Dissemination in Uganda

    PubMed Central

    Johnston, Christine; Orem, Jackson; Okuku, Fred; Kalinaki, Mary; Saracino, Misty; Katongole-Mbidde, Edward; Ronald, Allan; McAdam, Keith; Huang, Meei-Li; Drolette, Linda; Selke, Stacy; Wald, Anna; Corey, Lawrence; Casper, Corey

    2009-01-01

    Introduction Kaposi sarcoma (KS) is the leading cause of cancer in Uganda and occurs in people with and without HIV. Human herpesvirus-8 (HHV-8) replication is important both in transmission of HHV-8 and progression to KS. We characterized the sites and frequency of HHV-8 detection in Ugandans with and without HIV and KS. Methods Participants were enrolled into one of four groups on the basis of HIV and KS status (HIV negative/KS negative, HIV positive/KS negative, HIV negative/KS positive, and HIV positive/KS positive). Participants collected oral swabs daily and clinicians collected oral swabs, anogenital swabs, and plasma samples weekly over 4 weeks. HHV-8 DNA at each site was quantified by polymerase chain reaction (PCR). Results 78 participants collected a total of 2063 orals swabs and 358 plasma samples. Of these, 428 (21%) oral swabs and 96 (27%) plasma samples had detectable HHV-8 DNA. HHV-8 was detected more frequently in both the oropharynx of persons with KS (24 (57%) of 42 persons with KS vs. 8 (22%) of 36 persons without, p = 0.002) and the peripheral blood (30 (71%) of 42 persons with KS vs. 8 (22%) of 36 persons without, p<0.001). In a multivariate model, HHV-8 viremia was more frequent among men (IRR = 3.3, 95% CI = 1.7–6.2, p<0.001), persons with KS (IRR = 3.9, 95% CI = 1.7–9.0, p = 0.001) and persons with HIV infection (IRR = 1.7, 95% CI = 1.0–2.7, p = 0.03). Importantly, oral HHV-8 detection predicted the subsequent HHV-8 viremia. HHV-8 viremia was significantly more common when HHV-8 DNA was detected from the oropharynx during the week prior than when oral HHV-8 was not detected (RR = 3.3, 95% CI = 1.8–5.9 p<0.001). Genital HHV-8 detection was rare (9 (3%) of 272 swabs). Conclusions HHV-8 detection is frequent in the oropharynx and peripheral blood of Ugandans with endemic and epidemic KS. Replication at these sites is highly correlated, and viremia is increased in men and those with HIV. The

  1. The Mitochondrial Translocator Protein, TSPO, Inhibits HIV-1 Envelope Glycoprotein Biosynthesis via the Endoplasmic Reticulum-Associated Protein Degradation Pathway

    PubMed Central

    Zhou, Tao; Dang, Ying

    2014-01-01

    ABSTRACT The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4+ T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. IMPORTANCE The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral

  2. Casp8p41: The Protean Mediator of Death in CD4 T-cells that Replicate HIV

    PubMed Central

    Sampath, Rahul; Cummins, Nathan W.; Badley, Andrew D.

    2016-01-01

    HIV cure is now the focus of intense research after Timothy Ray Brown (the Berlin patient) set the precedent of being the first and only person cured. A major barrier to achieving this goal on a meaningful scale is an elimination of the latent reservoir, which is thought to comprise CD4-positive cells that harbor integrated, replication-competent HIV provirus. These cells do not express viral proteins, are indistinguishable from uninfected CD4 cells, and are thought to be responsible for HIV viral rebound—that occurs within weeks of combination anti retroviral therapy (cART) interruption. Modalities to engineer transcriptional stimulation (reactivation) of this dormant integrated HIV provirus, leading to expression of cytotoxic viral proteins, are thought to be a specific way to eradicate the latently infected CD4 pool and are becoming increasingly relevant in the era of HIV cure. HIV protease is one such protein produced after HIV reactivation that cleaves procaspase-8 to generate a novel protein Casp8p41. Casp8p41 then binds to the BH3 domain of BAK, leading to BAK oligomerization, mitochondrial depolarization, and apoptosis. In central memory T cells (TCMs) from HIV-infected patients, an elevated Bcl-2/procaspase-8 ratio was observed, and Casp8p41 binding to Bcl-2 was associated with a lack of reactivation-induced cell death. This was reversed by priming cells with a specific Bcl-2 antagonist prior to reactivation, resulting in increased cell death and decreased HIV DNA in a Casp8p41-dependent pathway. This review describes the biology, clinical relevance, and implications of Casp8p41 for a potential cure. PMID:27721655

  3. M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner

    PubMed Central

    Wang, Qin; Zhang, Xiaolin; Han, Yuling; Wang, Xinlu; Gao, Guangxia

    2016-01-01

    M2BP (also called 90K) is an interferon-stimulated gene product that is upregulated in HIV-1 infection. A recent study revealed that M2BP reduces the infectivity of HIV-1 by inhibiting the processing of the viral envelope protein. Here we report that in addition to reducing viral infectivity, M2BP inhibits HIV-1 virion production. We provide evidence showing that M2BP inhibits HIV-1 Gag trafficking to the plasma membrane in a vimentin-dependent manner. When vimentin filaments were collapsed by treating cells with acrylamide or by overexpression of a dominant-negative mutant of vimentin, M2BP inhibition of HIV-1 virion production was significantly relieved. We further show that M2BP interacts with both HIV-1 Gag and vimentin and thereby mediates their interactions. We propose that M2BP traps HIV-1 Gag to vimentin filaments to inhibit the transportation of HIV-1 Gag to the plasma membrane. These findings uncover a novel mechanism by which a host antiviral factor inhibits HIV-1 virion production. PMID:27604950

  4. IL-9 Inhibits Viral Replication in Coxsackievirus B3-Induced Myocarditis

    PubMed Central

    Yu, Miao; Long, Qi; Li, Huan-Huan; Liang, Wei; Liao, Yu-Hua; Yuan, Jing; Cheng, Xiang

    2016-01-01

    Myocardial injuries in viral myocarditis (VMC) are caused by viral infection and related autoimmune disorders. Recent studies suggest that IL-9 mediated both antimicrobial immune and autoimmune responses in addition to allergic diseases. However, the role of IL-9 in viral infection and VMC remains controversial and uncertain. In this study, we infected Balb/c mice with Coxsackievirus B3 (CVB3), and found that IL-9 was enriched in the blood and hearts of VMC mice on days 5 and 7 after virus infection. Most of IL-9 was secreted by CD8+ T cells on day 5 and CD4+ T cells on day 7 in the myocardium. Further, IL-9 knockout exacerbated cardiac damage following CVB3 infection, along with a sharp increase in viral replication and IL-17a expression, as well as a decrease in TGF-β. In contrast, the repletion of IL-9 in Balb/c mice with CVB infection induced the opposite effect. Studies in vitro further revealed that IL-9 directly inhibited viral replication in cardiomyocytes by reducing coxsackie and adenovirus receptor expression, which might be associated with upregulation of TGF-β autocrine effect in these cells. However, IL-9 had no direct effect on apoptosis in cardiomyocytes. Our data indicated that IL-9 played a protective role in disease progression by inhibiting CVB3 replication in the early stages of VMC. PMID:27766098

  5. Inhibition of porcine reproductive and respiratory syndrome virus replication by flavaspidic acid AB.

    PubMed

    Yang, Qian; Gao, Li; Si, Jianyong; Sun, Yipeng; Liu, Jinhua; Cao, Li; Feng, Wen-Hai

    2013-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) represents a significant challenge to the swine industry worldwide. Current control strategies against PRRSV are still inadequate and there is an urgent need for new antiviral therapies. Flavaspidic acid AB (FA-AB) is a compound derived from Dryopteris crassirhizoma, a traditional antiviral Chinese medicine. Here, we first identified its anti-PRRSV activity through targeting multiple stages in PRRSV infection in vitro. Our studies demonstrated that FA-AB could inhibit the internalization and cell-to-cell spreading of PRRSV, but not block PRRSV binding to cells. By monitoring the kinetics of PRRSV replication, we showed that FA-AB significantly suppressed PRRSV replication when treatment was initiated 24h after virus infection. Furthermore, we confirmed that FA-AB was able to significantly induce IFN-α, IFN-β, and IL1-β expression in porcine alveolar macrophages, suggesting that induction of antiviral cytokines by FA-AB could contribute to FA-AB induced inhibition of PRRSV replication. In conclusion, we provide a foundation for the possibility to develop a new therapeutic agent to control PRRSV infection.

  6. Inhibition of hepatitis C virus replication by intracellular delivery of multiple siRNAs by nanosomes.

    PubMed

    Chandra, Partha K; Kundu, Anup K; Hazari, Sidhartha; Chandra, Sruti; Bao, Lili; Ooms, Tara; Morris, Gilbert F; Wu, Tong; Mandal, Tarun K; Dash, Srikanta

    2012-09-01

    Sustained antiviral responses of chronic hepatitis C virus (HCV) infection have improved recently by the use of direct-acting antiviral agents along with interferon (IFN)-α and ribavirin. However, the emergence of drug-resistant variants is expected to be a major problem. We describe here a novel combinatorial small interfering RNA (siRNA) nanosome-based antiviral approach to clear HCV infection. Multiple siRNAs targeted to the highly conserved 5'-untranslated region (UTR) of the HCV genome were synthesized and encapsulated into lipid nanoparticles called nanosomes. We show that siRNA can be repeatedly delivered to 100% of cells in culture using nanosomes without toxicity. Six siRNAs dramatically reduced HCV replication in both the replicon and infectious cell culture model. Repeated treatments with two siRNAs were better than a single siRNA treatment in minimizing the development of an escape mutant, resulting in rapid inhibition of viral replication. Systemic administration of combinatorial siRNA-nanosomes is well tolerated in BALB/c mice without liver injury or histological toxicity. As a proof-of-principle, we showed that systemic injections of siRNA nanosomes significantly reduced HCV replication in a liver tumor-xenotransplant mouse model of HCV. Our results indicate that systemic delivery of combinatorial siRNA nanosomes can be used to minimize the development of escape mutants and inhibition of HCV infection.

  7. Geminin Inhibits a Late Step in the Formation of Human Pre-replicative Complexes*

    PubMed Central

    Wu, Min; Lu, Wenyan; Santos, Ruth E.; Frattini, Mark G.; Kelly, Thomas J.

    2014-01-01

    The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2–7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2–7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2–7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2–7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication. PMID:25231993

  8. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    NASA Astrophysics Data System (ADS)

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-07-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  9. Association between HIV replication and serum leptin levels: an observational study of a cohort of HIV-1-infected South African women

    PubMed Central

    2010-01-01

    Background Advanced HIV infection can result in lipoatrophy and wasting, even in the absence of ongoing opportunistic infections, suggesting that HIV may directly affect adipose tissue amount and distribution. Methods We assessed the relationship of fat (measured using anthropometry, DEXA, MRI scans) or markers related to glucose and lipid metabolism with viral load in a cross-sectional sample of 83 antiretroviral-naïve HIV-1-infected South African women. A multivariable linear model was fitted to log10VL to assess the combined effect of these variables. Results In addition to higher T cell activation, women with viral load greater than the population median had lower waist circumference, body mass index and subcutaneous abdominal fat, as well as lower serum leptin. We demonstrate that leptin serum levels are inversely associated with viral replication, independent of the amount of adipose tissue. This association is maintained after adjusting for multiple variables associated with disease progression (i.e., cellular activation and innate immunity effector levels). Conclusions Our results demonstrate that serum leptin levels are inversely associated with viral replication, independent of disease progression: we postulate that leptin may affect viral replication. PMID:20822522

  10. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    SciTech Connect

    Li Guiqiu; Gu Hongxi . E-mail: hxgu2432@163.com; Li Di; Xu Weizhen

    2007-04-06

    The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies.

  11. The Acyclic Retinoid Peretinoin Inhibits Hepatitis C Virus Replication and Infectious Virus Release in Vitro

    NASA Astrophysics Data System (ADS)

    Shimakami, Tetsuro; Honda, Masao; Shirasaki, Takayoshi; Takabatake, Riuta; Liu, Fanwei; Murai, Kazuhisa; Shiomoto, Takayuki; Funaki, Masaya; Yamane, Daisuke; Murakami, Seishi; Lemon, Stanley M.; Kaneko, Shuichi

    2014-04-01

    Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

  12. Activation of COX-2/PGE2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production.

    PubMed

    Alfajaro, Mia Madel; Choi, Jong-Soon; Kim, Deok-Song; Seo, Ja-Young; Kim, Ji-Yun; Park, Jun-Gyu; Soliman, Mahmoud; Baek, Yeong-Bin; Cho, Eun-Hyo; Kwon, Joseph; Kwon, Hyung-Jun; Park, Su-Jin; Lee, Woo Song; Kang, Mun-Il; Hosmillo, Myra; Goodfellow, Ian; Cho, Kyoung-Oh

    2017-02-01

    Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product, prostaglandin E2 (PGE2), are known to play important roles in the modulation of both the host response to infection and the replicative cycles of several viruses. However, the precise mechanism(s) by which the COX/PGE2 pathway regulates sapovirus replication remains largely unknown. In this study, infection with porcine sapovirus (PSaV) strain Cowden, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 h postinfection (hpi), with only a transient increase in COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors, such as nonsteroidal anti-inflammatory drugs or small interfering RNAs (siRNAs) against COX-1 and COX-2, significantly reduced PGE2 production, as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-l-methyl-arginine ester. This study identified a pivotal role for the COX/PGE2 pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of this poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection.

  13. Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

    PubMed

    Xu, Cheng; Guo, Tz-Chun; Mutoloki, Stephen; Haugland, Øyvind; Marjara, Inderjit S; Evensen, Øystein

    2010-09-01

    Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-alpha)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-gamma in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-alpha [rIFN-alpha] and rIFN-gamma) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-alpha and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-alpha induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-alpha is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-alpha results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-gamma has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-alpha, while postinfection antiviral responses or postinfection treatment with rIFN-alpha is not able to limit viral replication.

  14. A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

    PubMed

    Fun, Axel; Mok, Hoi Ping; Wills, Mark R; Lever, Andrew M

    2017-02-24

    Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4(+) T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4(+) T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.

  15. A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir

    PubMed Central

    Fun, Axel; Mok, Hoi Ping; Wills, Mark R.; Lever, Andrew M.

    2017-01-01

    Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4+ T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4+ T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity. PMID:28233807

  16. Validation, replication, and sensitivity testing of Heckman-type selection models to adjust estimates of HIV prevalence.

    PubMed

    Clark, Samuel J; Houle, Brian

    2014-01-01

    A recent study using Heckman-type selection models to adjust for non-response in the Zambia 2007 Demographic and Health Survey (DHS) found a large correction in HIV prevalence for males. We aim to validate this finding, replicate the adjustment approach in other DHSs, apply the adjustment approach in an external empirical context, and assess the robustness of the technique to different adjustment approaches. We used 6 DHSs, and an HIV prevalence study from rural South Africa to validate and replicate the adjustment approach. We also developed an alternative, systematic model of selection processes and applied it to all surveys. We decomposed corrections from both approaches into rate change and age-structure change components. We are able to reproduce the adjustment approach for the 2007 Zambia DHS and derive results comparable with the original findings. We are able to replicate applying the approach in several other DHSs. The approach also yields reasonable adjustments for a survey in rural South Africa. The technique is relatively robust to how the adjustment approach is specified. The Heckman selection model is a useful tool for assessing the possibility and extent of selection bias in HIV prevalence estimates from sample surveys.

  17. Nuclear localization of Vpr is crucial for the efficient replication of HIV-1 in primary CD4{sup +} T cells

    SciTech Connect

    Iijima, Sayuki; Nitahara-Kasahara, Yuko; Kimata, Kiyonori; Wen Zhongzhuang; Kamata, Masakazu; Isogai, Maya; Miwa, Masanao; Tsunetsugu-Yokota, Yasuko; Aida, Yoko . E-mail: aida@riken.jp

    2004-10-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr appears to make a substantial contribution to the replication of HIV-1 in established T cell lines when HIV-1 is present at very low multiplicities of infection. However, the role of Vpr in viral replication in primary CD4{sup +} T cells remains to be clarified. In this study, we generated a panel of viruses that encoded mutant forms of Vpr that lacked either the ability to accumulate in the nucleus and induce G{sub 2} arrest or the ability to induce apoptosis, which has been shown to occur independently of G{sub 2} arrest of the cell cycle. We demonstrate here that the nuclear localization of Vpr and consequent G{sub 2} arrest but not the induction of apoptosis by Vpr are important for viral replication in primary CD4{sup +} T cells at both high and low multiplicities of infection. Viruses that encoded mutant forms of Vpr that failed to be imported into the nucleus in the presence of cytoplasmic extracts from primary CD4{sup +} T cells in an in vitro nuclear import assay replicated at drastically reduced rates. Thus, Vpr might be a key regulator of the viral nuclear import process during infection in primary CD4{sup +} T cells. By contrast, a mutant form of Vpr that exhibited diffuse cytosolic staining exclusively in an immunofluorescence assay of HeLa cells and was not imported into nucleus by the cytosol from HeLa cells was effectively imported into the nucleus by cytosol from primary CD4{sup +} T cells. This Vpr mutant virus replicated well in primary CD4{sup +} T cells, indicating that cellular factors in primary CD4{sup +} T cells are indispensable for the accumulation of Vpr in the nucleus and, thus, for viral replication. Our results suggest that the nuclear import of Vpr might be a good target in efforts to block the early stages of replication of HIV-1.

  18. HIV-1 Vpr accelerates viral replication during acute infection by exploitation of proliferating CD4+ T cells in vivo.

    PubMed

    Sato, Kei; Misawa, Naoko; Iwami, Shingo; Satou, Yorifumi; Matsuoka, Masao; Ishizaka, Yukihito; Ito, Mamoru; Aihara, Kazuyuki; An, Dong Sung; Koyanagi, Yoshio

    2013-01-01

    The precise role of viral protein R (Vpr), an HIV-1-encoded protein, during HIV-1 infection and its contribution to the development of AIDS remain unclear. Previous reports have shown that Vpr has the ability to cause G2 cell cycle arrest and apoptosis in HIV-1-infected cells in vitro. In addition, vpr is highly conserved in transmitted/founder HIV-1s and in all primate lentiviruses, which are evolutionarily related to HIV-1. Although these findings suggest an important role of Vpr in HIV-1 pathogenesis, its direct evidence in vivo has not been shown. Here, by using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrated that Vpr causes G2 cell cycle arrest and apoptosis predominantly in proliferating CCR5(+) CD4(+) T cells, which mainly consist of regulatory CD4(+) T cells (Tregs), resulting in Treg depletion and enhanced virus production during acute infection. The Vpr-dependent enhancement of virus replication and Treg depletion is observed in CCR5-tropic but not CXCR4-tropic HIV-1-infected mice, suggesting that these effects are dependent on the coreceptor usage by HIV-1. Immune activation was observed in CCR5-tropic wild-type but not in vpr-deficient HIV-1-infected humanized mice. When humanized mice were treated with denileukin diftitox (DD), to deplete Tregs, DD-treated humanized mice showed massive activation/proliferation of memory T cells compared to the untreated group. This activation/proliferation enhanced CCR5 expression in memory CD4(+) T cells and rendered them more susceptible to CCR5-tropic wild-type HIV-1 infection than to vpr-deficient virus. Taken together, these results suggest that Vpr takes advantage of proliferating CCR5(+) CD4(+) T cells for enhancing viremia of CCR5-tropic HIV-1. Because Tregs exist in a higher cycling state than other T cell subsets, Tregs appear to be more vulnerable to exploitation by Vpr during acute HIV-1 infection.

  19. Broadly neutralizing antibodies developed by an HIV-positive elite neutralizer exact a replication fitness cost on the contemporaneous virus.

    PubMed

    Sather, D Noah; Carbonetti, Sara; Kehayia, Jenny; Kraft, Zane; Mikell, Iliyana; Scheid, Johannes F; Klein, Florian; Stamatatos, Leonidas

    2012-12-01

    Approximately 1% of those infected with HIV-1 develop broad and potent serum cross-neutralizing antibody activities. It is unknown whether or not the development of such immune responses affects the replication of the contemporaneous autologous virus. Here, we defined a pathway of autologous viral escape from contemporaneous potent and broad serum neutralizing antibodies developed by an elite HIV-1-positive (HIV-1(+)) neutralizer. These antibodies potently neutralize diverse isolates from different clades and target primarily the CD4-binding site (CD4-BS) of the viral envelope glycoprotein. Viral escape required mutations in the viral envelope glycoprotein which limited the accessibility of the CD4-binding site to the autologous broadly neutralizing anti-CD4-BS antibodies but which allowed the virus to infect cells by utilizing CD4 receptors on their surface. The acquisition of neutralization resistance, however, resulted in reduced cell entry potential and slower viral replication kinetics. Our results indicate that in vivo escape from autologous broadly neutralizing antibodies exacts fitness costs to HIV-1.

  20. CD8+ T cells from HLA-B*57 elite suppressors effectively suppress replication of HIV-1 escape mutants

    PubMed Central

    2013-01-01

    Background Elite Controllers or Suppressors (ES) are HIV-1 positive individuals who maintain plasma viral loads below the limit of detection of standard clinical assays without antiretroviral therapy. Multiple lines of evidence suggest that the control of viral replication in these patients is due to a strong and specific cytotoxic T lymphocyte (CTL) response. The ability of CD8+ T cells to control HIV-1 replication is believed to be impaired by the development of escape mutations. Surprisingly, viruses amplified from the plasma of ES have been shown to contain multiple escape mutations, and it is not clear how immunologic control is maintained in the face of virologic escape. Results We investigated the effect of escape mutations within HLA*B-57-restricted Gag epitopes on the CD8+ T cell mediated suppression of HIV-1 replication. Using site directed mutagenesis, we constructed six NL4-3 based viruses with canonical escape mutations in one to three HLA*B-57-restricted Gag epitopes. Interestingly, similar levels of CTL-mediated suppression of replication in autologous primary CD4+ T cells were observed for all of the escape mutants. Intracellular cytokine staining was performed in order to determine the mechanisms involved in the suppression of the escape variants. While low baseline CD8+ T cells responses to wild type and escape variant peptides were seen, stimulation of PBMC with either wild type or escape variant peptides resulted in increased IFN-γ and perforin expression. Conclusions These data presented demonstrate that CD8+ T cells from ES are capable of suppressing replication of virus harboring escape mutations in HLA-B*57-restricted Gag epitopes. Additionally, our data suggest that ES CD8+ T cells are capable of generating effective de novo responses to escape mutants. PMID:24330837

  1. IFN-λ Inhibits Drug-Resistant HIV Infection of Macrophages

    PubMed Central

    Wang, Xu; Wang, He; Liu, Man-Qing; Li, Jie-Liang; Zhou, Run-Hong; Zhou, Yu; Wang, Yi-Zhong; Zhou, Wang; Ho, Wen-Zhe

    2017-01-01

    Type III interferons (IFN-λs) have been demonstrated to inhibit a number of viruses, including HIV. Here, we further examined the anti-HIV effect of IFN-λs in macrophages. We found that IFN-λs synergistically enhanced anti-HIV activity of antiretrovirals [azidothymidine (AZT), efavirenz, indinavir, and enfuvirtide] in infected macrophages. Importantly, IFN-λs could suppress HIV infection of macrophages with the drug-resistant strains, including AZT-resistant virus (A012) and reverse transcriptase inhibitor-resistant virus (TC49). Mechanistically, IFN-λs were able to induce the expression of several important anti-HIV cellular factors, including myxovirus resistance 2 (Mx2), a newly identified HIV post-entry inhibitor and tetherin, a restriction factor that blocks HIV release from infected cells. These observations provide additional evidence to support the potential use of IFN-λs as therapeutics agents for the treatment of HIV infection. PMID:28321215

  2. Apigenin inhibits enterovirus 71 replication through suppressing viral IRES activity and modulating cellular JNK pathway.

    PubMed

    Lv, Xiaowen; Qiu, Min; Chen, Deyan; Zheng, Nan; Jin, Yu; Wu, Zhiwei

    2014-09-01

    Enterovirus 71 (EV71) is a member of genus Enterovirus in Picornaviridae family, which is one of the major causative agents for hand, foot and mouth disease (HFMD), and sometimes associated with severe central nervous system diseases in children. Currently there are no effective therapeutic medicines or vaccines for the disease. In this report, we found that apigenin and luteolin, two flavones that differ only in the number of hydroxyl groups could inhibit EV71-mediated cytopathogenic effect (CPE) and EV71 replication with low cytotoxicity. Both molecules also showed inhibitory effect on the viral polyprotein expression. They prevented EV71-induced cell apoptosis, intracellular reactive oxygen species (ROS) generation and cytokines up-regulation. Time-of-drug addition study demonstrated that apigenin and luteolin acted after viral entry. We examined the effect of apigenin and luteolin on 2A(pro) and 3C(pro) activity, two viral proteases responsible for viral polyprotein processing, and found that they showed less inhibitory activity on 2A(pro) or 3C(pro). Further studies demonstrated that apigenin, but not luteolin could interfere with viral IRES activity. Also, apigenin inhibited EV71-induced c-Jun N-terminal kinase (JNK) activation which is critical for viral replication, in contrast to luteolin that did not. This study demonstrated that apigenin may inhibit EV71 replication through suppressing viral IRES activity and modulating cellular JNK pathway. It also provided evidence that one hydroxyl group difference in the B ring between apigenin and luteolin resulted in the distinct antiviral mechanisms. This study will provide the basis for better drug development and further identification of potential drug targets.

  3. Prostaglandin A1 inhibits replication of Mayaro virus in Aedes albopictus cells.

    PubMed

    Barbosa, J A; Rebello, M A

    1995-01-01

    Prostaglandin A1 (PGA1) reduced Mayaro virus replication in Aedes albopictus (mosquito) cells in culture. The highest nontoxic dose of PGA1, 7.5 microM, decreased virus production by 90%. In Mayaro virus-infected cells, PGA1 inhibited virus-specific protein synthesis. However, in mock-infected cells the presence of PGA1 stimulated the synthesis of several proteins with molecular masses of 70, 57 and 23 kDa, respectively. The data obtained from this study show that PGA1 plays a role in the metabolic regulation of Aedes albopictus cells, blocking the synthesis of Mayaro virus and inducing the synthesis of cellular polypeptides.

  4. RASSF4 promotes EV71 replication to accelerate the inhibition of the phosphorylation of AKT.

    PubMed

    Zhang, Fengfeng; Liu, Yongjuan; Chen, Xiong; Dong, Lanlan; Zhou, Bingfei; Cheng, Qingqing; Han, Song; Liu, Zhongchun; Peng, Biwen; He, Xiaohua; Liu, Wanhong

    2015-03-20

    Enterovirus 71 (EV71) is a neurotropic virus that causes hand, foot and mouth disease (HFMD), occasionally leading to death. As a member of the RAS association domain family (RASSFs), RASSF4 plays important roles in cell death, tumor development and signal transduction. However, little is known about the relationship between RASSF4 and EV71. Our study reveals for the first time that RASSF4 promotes EV71 replication and then accelerates AKT phosphorylation inhibition in EV71-infected 293T cells, suggesting that RASSF4 may be a potential new target for designing therapeutic measures to prevent and control EV71 infection.

  5. Inhibition of HIV-1 Reverse Transcriptase Dimerization by Small Molecules.

    PubMed

    Tintori, Cristina; Corona, Angela; Esposito, Francesca; Brai, Annalaura; Grandi, Nicole; Ceresola, Elisa Rita; Clementi, Massimo; Canducci, Filippo; Tramontano, Enzo; Botta, Maurizio

    2016-04-15

    Because HIV-1 reverse transcriptase is an enzyme whose catalytic activity depends on its heterodimeric structure, this system could be a target for inhibitors that perturb the interactions between the protein subunits, p51 and p66. We previously demonstrated that the small molecule MAS0 reduced the association of the two RT subunits and simultaneously inhibited both the polymerase and ribonuclease H activities. In this study, some analogues of MAS0 were rationally selected by docking studies and evaluated in vitro for their ability to disrupt dimeric assembly. Two inhibitors were identified with improved activity compared to MAS0. This study lays the basis for the rational design of more potent inhibitors of RT dimerization.

  6. High-throughput sequencing identifies HIV-1-replication- and latency-related miRNAs in CD4(+) T cell lines.

    PubMed

    Lu, Xiangyun; Yang, Jin; Wu, Haibo; Yang, Zongxing; Jin, Changzhong; Wang, Juan; Cheng, Linfang; Peng, Xiaorong; Liu, Fumin; Peng, Xiuming; Ji, Sujing; Ou, Huilin; Xie, Tiansheng; Yao, Hangping; Wu, Nanping

    2017-03-16

    MicroRNAs are potent gene expression regulators involved in regulating various biological processes, including host-pathogen interactions. In this study, we used high-throughput sequencing to investigate cellular miRNA signatures related to HIV-1 replication and latent infection in CD4(+) T cell lines, which included HIV-1-replicating H9/HTLV-IIIB, HIV-1-latently-infected CEM-Bru cells, and their parental uninfected H9 and CEM-SS cells. Relatively few miRNAs were found to be modulated by HIV-1 replication or latent infection, while the cell-lineage-specific miRNA difference was more pronounced, irrespective of HIV-1 infection. In silico analysis showed that some of our HIV-1 infection-regulated miRNA profiles echoed previous studies, while others were novel. In addition, some of the miRNAs that were differentially expressed between the productively and latently infected cells seemed to participate in shaping the differential infection state. Thus, the newly identified miRNA profiles related to HIV-1 replication and latency provide information about the interplay between HIV-1 and its host.

  7. Ga(III) Nanoparticles Inhibit Growth of both Mycobacterium tuberculosis and HIV and Release of Interleukin-6 (IL-6) and IL-8 in Coinfected Macrophages

    PubMed Central

    Choi, Seoung-ryoung; Britigan, Bradley E.

    2017-01-01

    ABSTRACT Treatment of individuals coinfected with human immunodeficiency virus (HIV) type 1 and Mycobacterium tuberculosis is challenging due to the prolonged treatment requirements, drug toxicity, and emergence of drug resistance. Mononuclear phagocytes (MP; macrophages) are one of the natural reservoirs for both HIV and M. tuberculosis. Here, the treatment of HIV and M. tuberculosis coinfection was studied by preloading human macrophages with MP-targeted gallium (Ga) nanoparticles to limit subsequent simultaneous infection with both HIV and M. tuberculosis. Ga nanoparticles provided sustained drug release for 15 days and significantly inhibited the replication of both HIV and M. tuberculosis. Addition of Ga nanoparticles to MP already infected with M. tuberculosis or HIV resulted in a significant decrease in the magnitude of these infections, but the magnitude was less than that achieved with nanoparticle preloading of the MP. In addition, macrophages that were coinfected with HIV and M. tuberculosis and that were loaded with Ga nanoparticles reduced the levels of interleukin-6 (IL-6) and IL-8 secretion for up to 15 days after drug loading. Ga nanoparticles also reduced the levels of IL-6 and IL-8 secretion by ionomycin- and lipopolysaccharide-induced macrophages, likely by modulating the IκB kinase-β/NF-κB pathway. Delivery of Ga nanoparticles to macrophages is a potent long-acting approach for suppressing HIV and M. tuberculosis coinfection of macrophages in vitro and sets the stage for the development of new approaches to the treatment of these important infections. PMID:28167548

  8. Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative.

    PubMed

    Sanchez-Cespedes, Javier; Moyer, Crystal L; Whitby, Landon R; Boger, Dale L; Nemerow, Glen R

    2014-08-01

    The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections.

  9. Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative

    PubMed Central

    Sanchez-Cespedes, Javier; Moyer, Crystal L.; Whitby, Landon R.; Boger, Dale L.; Nemerow, Glen R.

    2014-01-01

    The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections. PMID:24907427

  10. Inhibition of Mayaro virus replication by prostaglandin A(1) in Vero cells.

    PubMed

    Burlandy, F M; Rebello, M A

    2001-01-01

    Prostaglandins exhibit antiviral activity against a wide variety of RNA and DNA viruses. In the present report, we describe the effect of cyclopentenone prostaglandin A(1) (PGA(1)) on Mayaro virus replication in Vero cells. Virus yield was significantly reduced at nontoxic concentrations which did not suppress DNA, RNA or protein synthesis in uninfected or infected cells. Antiviral action decreased if PGA(1) was added at later times after infection. In Mayaro virus-infected cells, PGA(1) inhibited the synthesis of virus proteins. This effect is accompanied by the induction of heat shock proteins (HSPs). Actinomycin D treatment not only inhibited the induction of HSPs but also partially prevented PGA(1) antiviral activity.

  11. Inhibition of respiratory syncytial virus replication and virus-induced p38 kinase activity by berberine.

    PubMed

    Shin, Han-Bo; Choi, Myung-Soo; Yi, Chae-Min; Lee, Jun; Kim, Nam-Jung; Inn, Kyung-Soo

    2015-07-01

    Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection and poses a major public health threat worldwide. No effective vaccines or therapeutics are currently available; berberine, an isoquinoline alkaloid from various medicinal plants, has been shown to exert antiviral and several other biological effects. Recent studies have shown that p38 mitogen-activated protein kinase (MAPK) activity is implicated in infection by and replication of viruses such as RSV and the influenza virus. Because berberine has previously been implicated in modulating the activity of p38 MAPK, its effects on RSV infection and RSV-mediated p38 MAPK activation were examined. Replication of RSV in epithelial cells was significantly reduced by treatment with berberine. Berberine treatment caused decrease in viral protein and mRNA syntheses. Similar to previously reported findings, RSV infection caused phosphorylation of p38 MAPK at a very early time point of infection, and phosphorylation was dramatically reduced by berberine treatment. In addition, production of interleukin-6 mRNA upon RSV infection was significantly suppressed by treatment with berberine, suggesting the anti-inflammatory role of berberine during RSV infection. Taken together, we showed that berberine, a natural compound already proven to be safe for human consumption, suppresses the replication of RSV. In addition, the current study suggests that inhibition of RSV-mediated early p38 MAPK activation, which has been implicated as an early step in viral infection, as a potential molecular mechanism.

  12. Zinc finger antiviral protein inhibits coxsackievirus B3 virus replication and protects against viral myocarditis.

    PubMed

    Li, Min; Yan, Kepeng; Wei, Lin; Yang, Jie; Lu, Chenyu; Xiong, Fei; Zheng, Chunfu; Xu, Wei

    2015-11-01

    The host Zinc finger antiviral protein (ZAP) has been reported exhibiting antiviral activity against positive-stranded RNA viruses (Togaviridae), negative-stranded RNA viruses (Filoviridae) and retroviruses (Retroviridae). However, whether ZAP restricts the infection of enterovirus and the development of enterovirus mediated disease remains unknown. Here, we reported the antiviral properties of ZAP against coxsackievirus B3 (CVB3), a single-stranded RNA virus of the Enterovirus genus within the Picornaviridae as a major causative agent of viral myocarditis (VMC). We found that the expression of ZAP was significantly induced after CVB3 infection in heart tissues of VMC mice. ZAP potently inhibited CVB3 replication in cells after infection, while overexpression of ZAP in mice significantly increased the resistance to CVB3 replication and viral myocarditis by significantly reducing cardiac inflammatory cytokine production. The ZAP-responsive elements (ZREs) were mapped to the 3'UTR and 5'UTR of viral RNA. Taken together, ZAP confers resistance to CVB3 infection via directly targeting viral RNA and protects mice from acute myocarditis by suppressing viral replication and cardiac inflammatory cytokine production. Our finding further expands ZAP's range of viral targets, and suggests ZAP as a potential therapeutic target for viral myocarditis caused by CVB3.

  13. Schisandrin A inhibits dengue viral replication via upregulating antiviral interferon responses through STAT signaling pathway

    PubMed Central

    Yu, Jung-Sheng; Wu, Yu-Hsuan; Tseng, Chin-Kai; Lin, Chun-Kuang; Hsu, Yao-Chin; Chen, Yen-Hsu; Lee, Jin-Ching

    2017-01-01

    Dengue virus (DENV) infects 400 million people worldwide annually. Infection of more than one serotype of DENV highly corresponds to dengue hemorrhagic fever and dengue shock syndrome, which are the leading causes of high mortality. Due to lack of effective vaccines and unavailable therapies against DENV, discovery of anti-DENV agents is urgently needed. We first characterize that Schisandrin A can inhibit the replication of four serotypes of DENV in a concentration- and time-dependent manner, with an effective half-maximal effective concentration 50% (EC50) value of 28.1 ± 0.42 μM against DENV serotype type 2 without significant cytotoxicity. Furthermore, schisandrin A can effectively protect mice from DENV infection by reducing disease symptoms and mortality of DENV-infected mice. We demonstrate that STAT1/2-mediated antiviral interferon responses contribute to the action of schisandrin A against DENV replication. Schisandrin A represents a potential antiviral agent to block DENV replication in vitro and in vivo. In conclusion, stimulation of STAT1/2-mediated antiviral interferon responses is a promising strategy to develop antiviral drug. PMID:28338050

  14. Nanoparticles inhibit DNA replication by binding to DNA: modeling and experimental validation.

    PubMed

    Li, Kungang; Zhao, Xiaonan; K Hammer, Brian; Du, Songyan; Chen, Yongsheng

    2013-11-26

    Predictive models are beneficial tools for researchers to use in prioritizing nanoparticles (NPs) for toxicological tests, but experimental evaluation can be time-consuming and expensive, and thus, priority should be given to tests that identify the NPs most likely to be harmful. For characterization of NPs, the physical binding of NPs to DNA molecules is important to measure, as interference with DNA function may be one cause of toxicity. Here, we determined the interaction energy between 12 types of NPs and DNA based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) model and then predicted the affinity of the NPs for DNA. Using the single-molecule imaging technique known as atomic force microscopy (AFM), we experimentally determined the binding affinity of those NPs for DNA. Theoretical predictions and experimental observations of the binding affinity agreed well. Furthermore, the effect of NPs on DNA replication in vitro was investigated with the polymerase chain reaction (PCR) technique. The results showed that NPs with a high affinity for DNA strongly inhibited DNA replication, whereas NPs with low affinity had no or minimal effects on DNA replication. The methodology here is expected to benefit the genotoxicological testing of NPs as well as the design of safe NPs.

  15. Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

    PubMed Central

    Wu, Hao; Mitra, Mithun; Naufer, M. Nabuan; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity. PMID:24293648

  16. A prospective on drug abuse-associated epigenetics and HIV-1 replication.

    PubMed

    Pandhare, Jui; Dash, Chandravanu

    2011-05-23

    Drugs of abuse serve as cofactors to susceptibility to HIV infection and disease progression. Although clinical reports indicate association between HIV/AIDS and drug use, the molecular mechanism of infection susceptibility and disease progression remains unclear. Drugs such as cocaine exert their addictive effects in part by epigenetic mechanisms. Given that epigenetic modifications play an important role in HIV-1 life cycle, it is essential to unravel whether drug abuse-associated epigenetic changes may contribute to HIV/AIDS. In this article we will provide a prospective on the impact of epigenetic mechanisms on HIV-1 life cycle.

  17. Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment

    PubMed Central

    Zhong, Ting; Zhang, Li-ying; Wang, Zeng-yan; Wang, Yue; Song, Feng-mei; Zhang, Ya-hong; Yu, Jing-hua

    2017-01-01

    Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication

  18. Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment.

    PubMed

    Zhong, Ting; Zhang, Li-Ying; Wang, Zeng-Yan; Wang, Yue; Song, Feng-Mei; Zhang, Ya-Hong; Yu, Jing-Hua

    2017-03-01

    Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication

  19. Bovine Lactoferrampin, Human Lactoferricin, and Lactoferrin 1-11 Inhibit Nuclear Translocation of HIV Integrase.

    PubMed

    Wang, Winston Yan; Wong, Jack Ho; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Cheung, Randy Chifai; Ng, Tzi Bun

    2016-08-01

    This study aimed to investigate fragments derived from human and bovine lactoferrins for ability to inhibit nuclear translocation of HIV-1 integrase. It was shown that human lactoferricin, human lactoferrin 1-11, and bovine lactoferrampin reduced nuclear distribution of HIV-1 integrase. Bovine lactoferrampin could inhibit both the activity and nuclear translocation of HIV-1 integrase. Human lactoferrampin, bovine lactoferricin, and bovine lactoferrin 1-11 had no effect on HIV-1 integrase nuclear translocation. Human lactoferrampin which inhibited the activity of integrase did not prevent its nuclear translocation. Human lactoferricin and lactoferrin 1-11 did not inhibit HIV-1 integrase nuclear translocation despite their ability to attenuate the enzyme activity. The discrepancy between the findings on reduction of HIV-1 activity and inhibition of nuclear translocation of HIV-1 integrase was due to the different mechanisms involved. A similar reasoning can also be applied to the different inhibitory potencies of the milk peptides on different HIV enzymes, i.e., nuclear translocation.

  20. Valganciclovir Inhibits Human Adenovirus Replication and Pathology in Permissive Immunosuppressed Female and Male Syrian Hamsters

    PubMed Central

    Toth, Karoly; Ying, Baoling; Tollefson, Ann E.; Spencer, Jacqueline F.; Balakrishnan, Lata; Sagartz, John E.; Buller, Robert Mark L.; Wold, William S. M.

    2015-01-01

    Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate) is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5) infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients. PMID:25807051

  1. Valganciclovir inhibits human adenovirus replication and pathology in permissive immunosuppressed female and male Syrian hamsters.

    PubMed

    Toth, Karoly; Ying, Baoling; Tollefson, Ann E; Spencer, Jacqueline F; Balakrishnan, Lata; Sagartz, John E; Buller, Robert Mark L; Wold, William S M

    2015-03-23

    Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate) is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5) infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients.

  2. Inhibition of Japanese encephalitis virus (JEV) replication by specific RNA aptamer against JEV methyltransferase.

    PubMed

    Han, Seung Ryul; Lee, Seong-Wook

    2017-01-29

    Japanese encephalitis virus (JEV) is the most common etiological agent of epidemic viral encephalitis. JEV encodes a single methyltransferase (MTase) domain located at the N-terminal region of the viral nonstructural protein NS5. JEV MTase is essential for viral replication and specifically catalyzes methylation of the viral RNA cap, which occurs exclusively in the cytoplasm. Therefore, JEV MTase is a potential target for antiviral therapy. Here, we identified specific and avid RNA aptamer (Kd ∼ 12 nM) with modified 2'-O-methyl pyrimidines against JEV MTase. The RNA aptamer efficiently inhibited viral cap methylation activity of MTase and interfered with JEV production in cells. Moreover, we generated a 24-mer truncated aptamer that could specifically bind to JEV MTase with high affinity (Kd ∼16 nM). The 24-mer aptamer efficiently inhibited JEV production and replication in cells. Therefore, MTase-specific RNA aptamer might be useful as an anti-JEV agent.

  3. Ginseng Protects Against Respiratory Syncytial Virus by Modulating Multiple Immune Cells and Inhibiting Viral Replication

    PubMed Central

    Lee, Jong Seok; Lee, Yu-Na; Lee, Young-Tae; Hwang, Hye Suk; Kim, Ki-Hye; Ko, Eun-Ju; Kim, Min-Chul; Kang, Sang-Moo

    2015-01-01

    Ginseng has been used in humans for thousands of years but its effects on viral infection have not been well understood. We investigated the effects of red ginseng extract (RGE) on respiratory syncytial virus (RSV) infection using in vitro cell culture and in vivo mouse models. RGE partially protected human epithelial (HEp2) cells from RSV-induced cell death and viral replication. In addition, RGE significantly inhibited the production of RSV-induced pro-inflammatory cytokine (TNF-α) in murine dendritic and macrophage-like cells. More importantly, RGE intranasal pre-treatment prevented loss of mouse body weight after RSV infection. RGE treatment improved lung viral clearance and enhanced the production of interferon (IFN-γ) in bronchoalveolar lavage cells upon RSV infection of mice. Analysis of cellular phenotypes in bronchoalveolar lavage fluids showed that RGE treatment increased the populations of CD8+ T cells and CD11c+ dendritic cells upon RSV infection of mice. Taken together, these results provide evidence that ginseng has protective effects against RSV infection through multiple mechanisms, which include improving cell survival, partial inhibition of viral replication and modulation of cytokine production and types of immune cells migrating into the lung. PMID:25658239

  4. MicroRNA-23b inhibits enterovirus 71 replication through downregulation of EV71 VPl protein.

    PubMed

    Wen, Bai-ping; Dai, Hong-jian; Yang, Yue-huang; Zhuang, Yu; Sheng, Ru

    2013-01-01

    Enterovirus 71 (EV71) is one of the causative pathogens of hand-foot-and-mouth disease and effective antiviral agents and vaccines against this virus have, to date, not been available. MicroRNAs (miRNAs) are a recently discovered class of RNAs with the function of post-transcriptional gene expression regulation. It has been demonstrated that miRNAs play important roles in the complicated interaction network between virus and host, while few studies have explored the role of miRNAs in EV71 infection. A recent study showed that hsa-miR-23b was downregulated significantly in cell-infected viruses. To address this issue, biological software miRanda was first used to predict possible target sites of miR-23b at EV71 gene sequence, then to confirm it by luciferase assay. miR-23b mimics were transfected to verify its effects on infection of EV71. These results suggest that miR-23b and upregulation of miR-23b inhibited the replication of EV71 by targeting at EV71 3'UTR conserved sequence. Taken together, miR-23b could inhibit EV71 replication through downregulation of EV71 VPl protein. These results may enhance our understanding on the prevention and treatment of hand-foot-and-mouth disease caused by EV71 infection.

  5. Ferritin protect shrimp Litopenaeus vannamei from WSSV infection by inhibiting virus replication.

    PubMed

    Ye, Ting; Wu, Xiaoting; Wu, Wenlin; Dai, Congjie; Yuan, Jianjun

    2015-01-01

    Iron is considered as an essential element for all living organisms. Therefore, limiting iron availability may be key part of the host's innate immune response to various pathogens. Ferritin is a major iron storage protein in living cells and plays an important role in iron homeostasis. One way the host can transiently reduce iron bioavailability is by ferritin over expression. In invertebrates, ferritin was found to be up-regulated after pathogens challenge and is considered to be an important element in the innate immune system. This study was designed to investigate the involvement of ferritin in shrimp Litopenaeus vannamei defense against WSSV. We discovered that the viral load of shrimp injected with recombinant ferritin protein was lower than that of control group. The suppression of ferritin by dsRNA increased susceptibility to WSSV with 3-fold high viral copies. The present study documented that ferritin protected shrimp L. vannamei from WSSV by inhibiting virus replication. We presume that ferritin reduce iron availability, leading to inhibit the activity of ribonucleotide reductase and delay the replication of virus genome. This study provided new insights into the understanding of molecular responses and defense mechanisms in shrimp against WSSV.

  6. Potent inhibition of scrapie prion replication in cultured cells by bis-acridines

    PubMed Central

    May, Barnaby C. H.; Fafarman, Aaron T.; Hong, Septima B.; Rogers, Michael; Deady, Leslie W.; Prusiner, Stanley B.; Cohen, Fred E.

    2003-01-01

    Prion diseases are characterized by an accumulation of PrPSc, a misfolded isoform of the normal cellular prion protein, PrPC. We previously reported the bioactivity of acridine-based compounds against PrPSc replication in scrapie-infected neuroblastoma cells and now report the improved potency of bis-acridine compounds. Bis-acridines are characterized by a dimeric motif, comprising two acridine heterocycles tethered by a linker. A library of bis-(6-chloro-2-methoxy-acridin-9-yl) and bis-(7-chloro-2-methoxy-benzo[b][1,5]naphthyridin-10-yl) analogs was synthesized to explore the effect of structurally diverse linkers on PrPSc replication in scrapie-infected neuroblastoma cells. Structure–activity analysis revealed that linker length and structure are important determinants for inhibition of prion replication in cultured scrapied cells. Three bis-acridine analogs, (6-chloro-2-methoxy-acridin-9-yl)-(3-{4-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-piperazin-1-yl}-propyl)-amine, N,N′-bis-(6-chloro-2-methoxy-acridin-9-yl)-1,8-diamino-3,6-dioxaoctane, and (1-{[4-(6-chloro-2-methoxy-acridin-9-ylamino)-butyl]-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-carbamoyl}-ethyl)-carbamic acid tert-butyl ester, showed half-maximal inhibition of PrPSc formation at 40, 25, and 30 nM, respectively, and were not cytotoxic to uninfected neuroblastoma cells at concentrations of 500 nM. Our data suggest that bis-acridine analogs may provide a potent alternative to the acridine-based compound quinacrine, which is currently under clinical evaluation for the treatment of prion disease. PMID:12626750

  7. Cytomegalovirus Replication in Semen Is Associated with Higher Levels of Proviral HIV DNA and CD4+ T Cell Activation during Antiretroviral Treatment

    PubMed Central

    Massanella, Marta; Richman, Douglas D.; Little, Susan J.; Spina, Celsa A.; Vargas, Milenka V.; Lada, Steven M.; Daar, Eric S.; Dube, Michael P.; Haubrich, Richard H.; Morris, Sheldon R.; Smith, Davey M.

    2014-01-01

    ABSTRACT Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4+ T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4+ T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4+ (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations. IMPORTANCE Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in

  8. Roles for biological membranes in regulating human immunodeficiency virus replication and progress in the development of HIV therapeutics that target lipid metabolism.

    PubMed

    Haughey, Norman J; Tovar-y-Romo, Luis B; Bandaru, Veera Venkata Ratnam

    2011-06-01

    Infection by the human immunodeficiency virus (HIV) involves a number of important interactions with lipid components in host membranes that regulate binding, fusion, internalization, and viral assembly. Available data suggests that HIV actively modifies the sphingolipid content of cellular membranes to create focal environments that are favorable for infection. In this review, we summarize the roles that membrane lipids play in HIV infection and discuss the current status of therapeutics that attempt to modify biological membranes to inhibit HIV.

  9. RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates.

    PubMed

    Kerr, S G; Anderson, K S

    1997-11-18

    The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.

  10. Experimental infection with Haemophilus ducreyi in persons who are infected with HIV does not cause local or augment systemic viral replication.

    PubMed

    Janowicz, Diane M; Tenner-Racz, Klara; Racz, Paul; Humphreys, Tricia L; Schnizlein-Bick, Carol; Fortney, Kate R; Zwickl, Beth; Katz, Barry P; Campbell, James J; Ho, David D; Spinola, Stanley M

    2007-05-15

    We infected 11 HIV-seropositive volunteers whose CD4(+) cell counts were >350 cells/ microL (7 of whom were receiving antiretrovirals) with Haemophilus ducreyi. The papule and pustule formation rates were similar to those observed in HIV-seronegative historical control subjects. No subject experienced a sustained change in CD4(+) cell count or HIV RNA level. The cellular infiltrate in biopsy samples obtained from the HIV-seropositive and HIV-seronegative subjects did not differ with respect to the percentage of leukocytes, neutrophils, macrophages, or T cells. The CD4(+):CD8(+) cell ratio in biopsy samples from the HIV-seropositive subjects was 1:3, the inverse of the ratio seen in the HIV-seronegative subjects (P<.0001). Although CD4(+) cells proliferated in lesions, in situ hybridization and reverse-transcription polymerase chain reaction for HIV RNA was negative. We conclude that experimental infection in HIV-seropositive persons is clinically similar to infection in HIV-seronegative persons and does not cause local or augment systemic viral replication. Thus, prompt treatment of chancroid may abrogate increases in viral replication associated with natural disease.

  11. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    PubMed

    Zhang, Ling; Yin, Sen; Tan, Wanlong; Xiao, Dong; Weng, Yunceng; Wang, Wenjing; Li, Tingting; Shi, Junwen; Shuai, Lifang; Li, Hongwei; Zhou, Jianhua; Allain, Jean-Pierre; Li, Chengyao

    2012-01-01

    Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  12. Recombinant Interferon-γ Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo

    PubMed Central

    Zhang, Ling; Yin, Sen; Tan, Wanlong; Xiao, Dong; Weng, Yunceng; Wang, Wenjing; Li, Tingting; Shi, Junwen; Shuai, Lifang; Li, Hongwei; Zhou, Jianhua; Allain, Jean-Pierre; Li, Chengyao

    2012-01-01

    Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P = 0.005–0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P = 0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases. PMID:22916129

  13. Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies

    NASA Astrophysics Data System (ADS)

    Sánchez-Luque, Francisco J.; Stich, Michael; Manrubia, Susanna; Briones, Carlos; Berzal-Herranz, Alfredo

    2014-09-01

    The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5'-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

  14. Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

    PubMed Central

    Lorenzi, Julio C. C.; Cohen, Yehuda Z.; Cohn, Lillian B.; Kreider, Edward F.; Oliveira, Thiago; Lavine, Christy L.; Horwitz, Joshua A.; Settler, Allison; Jankovic, Mila; Seaman, Michael S.; Chakraborty, Arup K.; Hahn, Beatrice H.; Caskey, Marina; Nussenzweig, Michel C.

    2016-01-01

    HIV-1–infected individuals harbor a latent reservoir of infected CD4+ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones. PMID:27872306

  15. Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA.

    PubMed

    Lorenzi, Julio C C; Cohen, Yehuda Z; Cohn, Lillian B; Kreider, Edward F; Barton, John P; Learn, Gerald H; Oliveira, Thiago; Lavine, Christy L; Horwitz, Joshua A; Settler, Allison; Jankovic, Mila; Seaman, Michael S; Chakraborty, Arup K; Hahn, Beatrice H; Caskey, Marina; Nussenzweig, Michel C

    2016-12-06

    HIV-1-infected individuals harbor a latent reservoir of infected CD4(+) T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.

  16. Transcriptional control of HIV replication by multiple modulators and their implication for a novel antiviral therapy.

    PubMed

    Victoriano, Ann Florence B; Okamoto, Takashi

    2012-02-01

    Transcriptional regulation is critical for the human immunodeficiency virus 1 (HIV-1) life cycle and is the only step at which the virus amplifies the content of its genetic information. Numerous known and still unknown transcriptional factors, both host and viral, regulate HIV-1 gene expression and latency. This article is a comprehensive review of transcription factors involved in HIV-1 gene expression and presents the significant implications of nuclear factor kappa B (NF-κB) and the HIV-1 transactivator of transcription (Tat) protein. We include recent findings on chromatin remodeling toward HIV transcription and its therapeutic implication is also discussed. The current status of small-molecular-weight compounds that affect HIV transcription is also described.

  17. Behçet's disease diagnosed after acute HIV infection: viral replication activating underlying autoimmunity?

    PubMed

    Roscoe, Clay; Kinney, Rebecca; Gilles, Ryan; Blue, Sky

    2015-05-01

    Behçet's disease is an autoimmune systemic vasculitis that can occur after exposure to infectious agents. Behçet's disease also has been associated with HIV infection, including de novo development of this condition during chronic HIV infection and resolution of Behçet's disease symptoms following initiation of antiretroviral therapy. We describe a patient who presented with systemic vasculitis with skin and mucous membrane ulcerations in the setting of acute HIV infection, who was eventually diagnosed with Behçet's disease, demonstrating a possible link between acute HIV infection, immune activation and development of autoimmunity.

  18. Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

    PubMed Central

    Saito, Akatsuki; Henning, Matthew S.; Serrao, Erik; Dubose, Brittany N.; Teng, Samantha; Huang, Jing; Li, Xiangming; Saito, Namiko; Roy, Saumendra Prasad; Siddiqui, Mohammad Adnan; Ahn, Jinwoo; Tsuji, Moriya; Hatziioannou, Theodora; Engelman, Alan N.

    2016-01-01

    ABSTRACT Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4+ T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains. IMPORTANCE CPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for

  19. "Fusion and binding inhibition" key target for HIV-1 treatment and pre-exposure prophylaxis: targets, drug delivery and nanotechnology approaches.

    PubMed

    Malik, Tanushree; Chauhan, Gaurav; Rath, Goutam; Murthy, R S R; Goyal, Amit K

    2017-11-01

    More than 35 million people are living with HIV worldwide with approximately 2.3 million new infections per year. Cascade of events (cell entry, virus replication, assembly and release of newly formed virions) is involved in the HIV-1 transmission process. Every single step offers a potential therapeutic strategy to halt this progression and HIV fusion into the human host cell is one such stage. Controlling the initial event of HIV-1 transmission is the best way to control its dissemination especially when prophylaxis is concerned. Action is required either on the HIV's or host's cell surface which is logically more rational when compared with other intracellular acting moieties. Aim of this manuscript is to detail the significance and current strategies to halt this initial step, thus blocking the entry of HIV-1 for further infection. Both HIV-1 and the possible host cell's receptors/co-receptors are under focus while specifying the targets available for inhibiting this fusion. Current and under investigation moieties are categorized based on their versatile mechanisms. Advanced drug delivery and nanotechnology approaches present a key tool to exploit the therapeutic potential in a boosted way. Current drug delivery and the impact of nanotechnology in potentiating this strategy are detailed.

  20. Efficacy of an antiviral compound to inhibit replication of multiple pestivirus species.

    PubMed

    Newcomer, Benjamin W; Marley, M Shonda; Ridpath, Julia F; Neill, John D; Boykin, David W; Kumar, Arvind; Givens, M Daniel

    2012-11-01

    Pestiviruses are economically important pathogens of livestock. An aromatic cationic compound (DB772) has previously been shown to inhibit bovine viral diarrhea virus (BVDV) type 1 in vitro at concentrations lacking cytotoxic side effects. The aim of this study was to determine the scope of antiviral activity of DB772 among diverse pestiviruses. Isolates of BVDV 2, border disease virus (BDV), HoBi virus, pronghorn virus and Bungowannah virus were tested for in vitro susceptibility to DB772 by incubating infected cells in medium containing 0, 0.006, 0.01, 0.02, 0.05, 0.1, 0.2, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 or 25μM DB772. The samples were assayed for the presence of virus by virus isolation and titration (BDV and BVDV 2) or PCR (HoBi, pronghorn and Bungowannah viruses). Cytotoxicity of the compound was assayed for each cell type. Complete inhibition of BVDV 2, BDV, and Pronghorn virus was detected when DB772 was included in the culture media at concentrations of 0.20μM and higher. In two of three tests, a concentration of 0.05μM DB772 was sufficient to completely inhibit HoBi virus replication. Bungowannah virus was completely inhibited at a concentration of 0.01μM DB772. Thus, DB772 effectively inhibits all pestiviruses studied at concentrations >0.20μM. As cytotoxicity is not evident at these concentrations, this antiviral compound potentially represents an effective preventative or therapeutic for diverse pestiviruses.

  1. Discovery of gramine derivatives that inhibit the early stage of EV71 replication in vitro.

    PubMed

    Wei, Yanhong; Shi, Liqiao; Wang, Kaimei; Liu, Manli; Yang, Qingyu; Yang, Ziwen; Ke, Shaoyong

    2014-06-27

    Enterovirus 71 (EV71) is a notable causative agent of hand, foot, and mouth disease in children, which is associated with an increased incidence of severe neurological disease and death, yet there is no specific treatment or vaccine for EV71 infections. In this study, the antiviral activity of gramine and 21 gramine derivatives against EV71 was investigated in cell-based assays. Eighteen derivatives displayed some degree of inhibitory effects against EV71, in that they could effectively inhibit virus-induced cytopathic effects (CPEs), but the anti-EV71 activity of the lead compound gramine was not observed. Studies on the preliminary modes of action showed that these compounds functioned by targeting the early stage of the EV71 lifecycle after viral entry, rather than inactivating the virus directly, inhibiting virus adsorption or affecting viral release from the cells. Among these derivatives, one (compound 4s) containing pyridine and benzothiazole units showed the most potency against EV71. Further studies demonstrated that derivative 4s could profoundly inhibit viral RNA replication, protein synthesis, and virus-induced apoptosis in RD cells. These results indicate that derivative 4s might be a feasible therapeutic agent against EV71 infection and that these gramine derivatives may provide promising lead scaffolds for the further design and synthesis of potential antiviral agents.

  2. Antisense Oligonucleotides Targeting Influenza A Segment 8 Genomic RNA Inhibit Viral Replication

    PubMed Central

    Lenartowicz, Elzbieta; Nogales, Aitor; Kierzek, Elzbieta; Kierzek, Ryszard; Martínez-Sobrido, Luis

    2016-01-01

    Influenza A virus (IAV) affects 5%–10% of the world's population every year. Through genome changes, many IAV strains develop resistance to currently available anti-influenza therapeutics. Therefore, there is an urgent need to find new targets for therapeutics against this important human respiratory pathogen. In this study, 2′-O-methyl and locked nucleic acid antisense oligonucleotides (ASOs) were designed to target internal regions of influenza A/California/04/2009 (H1N1) genomic viral RNA segment 8 (vRNA8) based on a base-pairing model of vRNA8. Ten of 14 tested ASOs showed inhibition of viral replication in Madin-Darby canine kidney cells. The best five ASOs were 11–15 nucleotides long and showed inhibition ranging from 5- to 25-fold. In a cell viability assay they showed no cytotoxicity. The same five ASOs also showed no inhibition of influenza B/Brisbane/60/2008 (Victoria lineage), indicating that they are sequence specific for IAV. Moreover, combinations of ASOs slightly improved anti-influenza activity. These studies establish the accessibility of IAV vRNA for ASOs in regions other than the panhandle formed between the 5′ and 3′ ends. Thus, these regions can provide targets for the development of novel IAV antiviral approaches. PMID:27463680

  3. Mutation of Host Δ9 Fatty Acid Desaturase Inhibits Brome Mosaic Virus RNA Replication between Template Recognition and RNA Synthesis

    PubMed Central

    Lee, Wai-Ming; Ishikawa, Masayuki; Ahlquist, Paul

    2001-01-01

    All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae. BMV encodes RNA replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain. Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA replication complexes on the perinuclear ER. We show that BMV RNA replication is severely inhibited by a mutation in OLE1, an essential yeast chromosomal gene encoding Δ9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis. OLE1 deletion and medium supplementation show that BMV RNA replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels. In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally. However, RNA replication is blocked prior to initiation of negative-strand RNA synthesis. The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA replication. The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy. PMID:11160714

  4. Activation of COX-2/PGE2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production

    PubMed Central

    Alfajaro, Mia Madel; Choi, Jong-Soon; Kim, Deok-Song; Seo, Ja-Young; Kim, Ji-Yun; Park, Jun-Gyu; Soliman, Mahmoud; Baek, Yeong-Bin; Cho, Eun-Hyo; Kwon, Joseph; Kwon, Hyung-Jun; Park, Su-Jin; Lee, Woo Song; Kang, Mun-Il; Hosmillo, Myra

    2016-01-01

    ABSTRACT Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product, prostaglandin E2 (PGE2), are known to play important roles in the modulation of both the host response to infection and the replicative cycles of several viruses. However, the precise mechanism(s) by which the COX/PGE2 pathway regulates sapovirus replication remains largely unknown. In this study, infection with porcine sapovirus (PSaV) strain Cowden, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 h postinfection (hpi), with only a transient increase in COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors, such as nonsteroidal anti-inflammatory drugs or small interfering RNAs (siRNAs) against COX-1 and COX-2, significantly reduced PGE2 production, as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-l-methyl-arginine ester. This study identified a pivotal role for the COX/PGE2 pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of this poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection. IMPORTANCE Sapoviruses are among the major etiological agents of acute gastroenteritis in both humans and animals, but little is known about sapovirus host factor requirements. Here, using only cultivable porcine sapovirus (PSaV) strain Cowden, we demonstrate that PSaV induced the vitalization of the cyclooxygenase (COX) and prostaglandin E2 (PGE2

  5. NIRF, a Novel Ubiquitin Ligase, Inhibits Hepatitis B Virus Replication Through Effect on HBV Core Protein and H3 Histones.

    PubMed

    Qian, Guanhua; Hu, Bin; Zhou, Danlin; Xuan, Yanyan; Bai, Lu; Duan, Changzhu

    2015-05-01

    Np95/ICBP90-like RING finger protein (NIRF), a novel E3 ubiquitin ligase, has been shown to interact with HBc and promote its degradation. This study investigated the effects of NIRF on replication of hepatitis B virus (HBV) and the mechanisms. We have shown that NIRF inhibits replication of HBV DNA and secretion of HBsAg and HBeAg in HepG2 cells transfected with pAAV-HBV1.3. NIRF also inhibits the replication and secretion of HBV in a mouse model that expressed HBV. NIRF reduces acetylation of HBV cccDNA-bound H3 histones. These results showed that NIRF is involved in the HBV replication cycle not only through direct interaction with HBc but also reduces acetylation of HBV cccDNA-bound H3 histones.

  6. Toll-interacting protein inhibits HIV-1 infection and regulates viral latency.

    PubMed

    Li, Chuan; Kuang, Wen-Dong; Qu, Di; Wang, Jian-Hua

    2016-06-24

    HIV-1 latency is mainly characterized by a reversible silencing of long-terminal repeat (LTR)-driven transcription of provirus. The existing of repressive factors has been described to contribute to transcription silencing of HIV-1. Toll-interacting protein (Tollip) has been identified as a repressor of Toll like receptors (TLR)-mediated signaling. Our previous study has found that Tollip inhibited NF-κB-dependent HIV-1 promoter LTR-driven transcription, indicating the potential role of Tollip in governing viral latency. In this study, by using HIV-1 latently infected Jurkat T-cell and central memory CD4(+) T-cells, we demonstrate the role of Tollip in regulating HIV-1 latency, as the knock-down of Tollip promoted HIV-1 reactivation from both HIV-1 latently infected Jurkat CD4(+) T cells and primary central memory T cells (TCM). Moreover, we found that the activities of LTRs derived from multiple HIV-1 subtypes could be repressed by Tollip; Knock-down of Tollip promoted HIV-1 transcription and infection in CD4(+) T cells. Our data indicate a key role of Tollip in suppressing HIV-1 infection and regulating viral latency, which provides a potential host target for combating HIV-1 infection and latency.

  7. Inhibition of Rift Valley Fever Virus Replication and Perturbation of Nucleocapsid-RNA Interactions by Suramin

    PubMed Central

    Ellenbecker, Mary; Lanchy, Jean-Marc

    2014-01-01

    Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. There are currently no proven safe and effective treatment options for RVFV infection. Inhibition of RNA binding to RVFV nucleocapsid protein (N) represents an attractive antiviral therapeutic strategy because several essential steps in the RVFV replication cycle involve N binding to viral RNA. In this study, we demonstrate the therapeutic potential of the drug suramin by showing that it functions well as an inhibitor of RVFV replication at multiple stages in human cell culture. Suramin has been used previously to treat trypanosomiasis in Africa. We characterize the dynamic and cooperative nature of N-RNA binding interactions and the dissociation of high-molecular-mass ribonucleoprotein complexes using suramin, which we previously identified as an N-RNA binding inhibitor in a high-throughput screen. Finally, we elucidate the molecular mechanism used by suramin in vitro to disrupt both specific and nonspecific binding events important for ribonucleoprotein formation. PMID:25267680

  8. Inhibition of human coronavirus NL63 infection at early stages of the replication cycle.

    PubMed

    Pyrc, Krzysztof; Bosch, Berend Jan; Berkhout, Ben; Jebbink, Maarten F; Dijkman, Ronald; Rottier, Peter; van der Hoek, Lia

    2006-06-01

    Human coronavirus NL63 (HCoV-NL63), a recently discovered member of the Coronaviridae family, has spread worldwide and is associated with acute respiratory illness in young children and elderly and immunocompromised persons. Further analysis of HCoV-NL63 pathogenicity seems warranted, in particular because the virus uses the same cellular receptor as severe acute respiratory syndrome-associated coronavirus. As there is currently no HCoV-NL63-specific and effective vaccine or drug therapy available, we evaluated several existing antiviral drugs and new synthetic compounds as inhibitors of HCoV-NL63, targeting multiple stages of the replication cycle. Of the 28 compounds that we tested, 6 potently inhibited HCoV-NL63 at early steps of the replication cycle. Intravenous immunoglobulins, heptad repeat 2 peptide, small interfering RNA1 (siRNA1), siRNA2, beta-D-N(4)-hydroxycytidine, and 6-azauridine showed 50% inhibitory concentrations of 125 microg/ml, 2 microM, 5 nM, 3 nM, 400 nM, and 32 nM, respectively, and low 50% cytotoxicity concentrations (>10 mg/ml, >40 microM, >200 nM, >200 nM, >100 microM, and 80 microM, respectively). These agents may be investigated further for the treatment of coronavirus infections.

  9. Ganciclovir Inhibits Human Adenovirus Replication and Pathogenicity in Permissive Immunosuppressed Syrian Hamsters

    PubMed Central

    Ying, Baoling; Tollefson, Ann E.; Spencer, Jacqueline F.; Balakrishnan, Lata; Dewhurst, Stephen; Capella, Cristina; Buller, R. Mark L.; Wold, William S. M.

    2014-01-01

    Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment. PMID:25224011

  10. Ganciclovir inhibits human adenovirus replication and pathogenicity in permissive immunosuppressed Syrian hamsters.

    PubMed

    Ying, Baoling; Tollefson, Ann E; Spencer, Jacqueline F; Balakrishnan, Lata; Dewhurst, Stephen; Capella, Cristina; Buller, R Mark L; Toth, Karoly; Wold, William S M

    2014-12-01

    Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.

  11. Inhibition of hepatitis C virus replication in vitro by xanthohumol, a natural product present in hops.

    PubMed

    Lou, Sai; Zheng, Yi-Min; Liu, Shan-Lu; Qiu, Jianming; Han, Qunying; Li, Na; Zhu, Qianqian; Zhang, Pingping; Yang, Cuiling; Liu, Zhengwen

    2014-02-01

    Hepatitis C virus is a major cause of chronic liver disease worldwide. Xanthohumol, a prenylated flavonoid from hops, has various biological activities including an antiviral effect. It was previously characterized as a compound that inhibits bovine viral diarrhea virus, a surrogate model of hepatitis C virus. In the present work, xanthohumol was examined for its ability to inhibit hepatitis C virus replication in a cell culture system carrying replicating hepatitis C virus RNA replicon. 0.2 % DMSO and 500 units/mL interferon-alpha treatments were set as a negative and positive control, respectively. The inhibitory effect by xanthohumol was determined by the luciferase activity of the infected Huh7.5 cell lysates and the hepatitis C virus RNA levels in the culture. Xanthohumol at 3.53 µM significantly decreased the luciferase activity compared to the negative control (p < 0.01). Xanthohumol at 7.05 µM further decreased the luciferase activity compared to xanthohumol at 3.53 µM (p = 0.015). Xanthohumol at 7.05 µM or 14.11 µM achieved an inhibitory effect similar to that of interferon-alpha 2b (p > 0.05). Xanthohumol at 3.53 µM significantly reduced the hepatitis C virus RNA level compared to the negative control (p = 0.001). Although the results of xanthohumol at 7.05 µM had a higher variation, xanthohumol at the 7.05 µM and 14.11 µM decreased the hepatitis C virus RNA level to that achieved by interferon-alpha (p > 0.05). In conclusion, xanthohumol displays anti-hepatitis C virus activity in a cell culture system and may be potentially used as an alternative or complementary treatment against the hepatitis C virus.

  12. Chloroquine inhibits Rhodococcus equi replication in murine and foal alveolar macrophages by iron-starvation.

    PubMed

    Gressler, Leticia T; Bordin, Angela I; McQueen, Cole M; Cohen, Noah D; de Vargas, Agueda Castagna

    2016-05-30

    Rhodococcus equi preferentially infects macrophages causing pyogranulomatous pneumonia in young foals. Both the vapA and rhbC genes are up-regulated in an iron (Fe)-deprived environment, such as that found within macrophages. Chloroquine (CQ) is a drug widely used against malaria that suppresses the intracellular availability of Fe in eukaryotic cells. The main objective of this study was to evaluate the ability of CQ to inhibit replication of virulent R. equi within murine (J774A.1) and foal alveolar macrophages (AMs) and to verify whether the mechanism of inhibition could be Fe-deprivation-dependent. CQ effect on R. equi extracellular survival and toxicity to J774A.1 were evaluated. R. equi survival within J774A.1 and foal AMs was evaluated under CQ (10 and 20μM), bovine saturated transferrin (bHTF), and bovine unsaturated transferrin (bATF) exposure. To explore the action mechanism of CQ, the superoxide anion production, the lysozyme activity, as well as the relative mRNA expression of vapA and rhbC were examined. CQ at≤20μM had no effect on R. equi extracellular multiplication and J774A.1 viability. Exposure to CQ significantly and markedly reduced survival of R. equi within J774A.1 and foal AMs. Treatment with bHTF did not reverse CQ effect on R. equi. Exposure to CQ did not affected superoxide anion production or lysozyme activity, however vapA and rhbC expression was significantly increased. Our results reinforce the hypothesis that intracellular availability of Fe is required for R. equi survival, and our initial hypothesis that CQ can limit replication of R. equi in J774A.1 and foal AMs, most likely by Fe starvation.

  13. Bleomycin-induced over-replication involves sustained inhibition of mitotic entry through the ATM/ATR pathway.

    PubMed

    Nakayama, Yuji; Igarashi, Asae; Kikuchi, Ikue; Obata, Yuuki; Fukumoto, Yasunori; Yamaguchi, Naoto

    2009-09-10

    Polyploid cells result in aneuploidy through aberrant chromosome segregation, possibly leading to tumorigenesis. Although polyploid cells are induced through over-replication by a variety of agents, including DNA-damaging drugs, the mechanisms that induce polyploidy have been hitherto unknown. Here, we show that treatment with bleomycin, a glycopeptide anticancer drug, induces over-replication at low cytotoxic doses. During bleomycin-induced over-replication, mitotic entry is inhibited through tyrosine phosphorylation of CDK1 along the ATM/ATR pathway in the early phase of treatment. Bleomycin-induced over-replication is inhibited by the inhibitors of the ATM/ATR pathway through abrogation of bleomycin-induced G2 arrest, and the ATM/ATR inhibitors promote cell death instead of over-replication. Following the phosphorylation of CDK1, the level of cyclin B1 is decreased in the late phase of treatment. Time-lapse imaging of clone cells that express a live cell marker of endogenous cyclin B1 revealed that cyclin B1 is degraded in G2-arrested cells upon bleomycin treatment. Our findings lead to a model of how the ATM/ATR pathway acts as a molecular switch for regulating cell fates, flipping between cell death via progress into mitosis, and over-replication via sustained G2 arrest upon DNA damage, where cyclin B1 degradation is an important factor for inducing over-replication.

  14. A short sequence motif in the 5' leader of the HIV-1 genome modulates extended RNA dimer formation and virus replication.

    PubMed

    van Bel, Nikki; Das, Atze T; Cornelissen, Marion; Abbink, Truus E M; Berkhout, Ben

    2014-12-19

    The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.

  15. HCV dsRNA-Activated Macrophages Inhibit HCV Replication in Hepatocytes

    PubMed Central

    Wang, Yizhong; Li, Jieliang; Wang, Xu; Zhou, Yu; Zhang, Ting; Ho, Wenzhe

    2015-01-01

    Background: Macrophages play critical roles in innate immune response in the liver. Whether macrophages participate in liver innate immunity against HCV replication is poorly understood Objectives: The aim of this study was to investigate the role of macrophages in liver innate immunity against HCV replication. Materials and Methods: Freshly isolated monocytes were purified from peripheral blood of healthy adult donors. Macrophages refer to 7-day-cultured monocytes in vitro. A hepatoma cell line (Huh7) was infected with HCV JFH-1 to generate in vitro HCV infectious system. RT-PCR was used to determine HCV RNA and mRNA levels of genes expression. ELISA was used to measure the protein level of interferon-α (IFN-α) and western blot was used to determine protein expression level of Toll-like receptor 3 (TLR3). Results: HCV dsRNA induced the expression of type I IFN (IFN-α/β) in monocyte-derived macrophages. HCV dsRNA also induced the expression of TLR3 and IFN regulatory factor-7 (IRF-7), the key regulators of the IFN signaling pathway. When HCV JFH-1-infected Huh7 cells were co-cultured with macrophages activated with HCV dsRNA or incubated in media conditioned with supernatant (SN) from HCV dsRNA-activated macrophages, HCV replication was significantly suppressed. This macrophage SN action on HCV inhibition was mediated through type I IFN, which was evidenced by the observation that antibody to type I IFN receptor could neutralize the macrophages-mediated anti-HCV effect. The role of type I IFN in macrophages-mediated anti-HCV activity is further supported by the observation that HCV dsRNA-activated macrophages SN treatment induced the expression of several IFN-stimulated genes (ISGs), ISG15, ISG56, OAS-1, OAS-2, MxA and Viperin in HCV-infected Huh7 cells. Conclusions: Macrophages may play an important role in liver innate immunity against HCV replication through a type I IFN-dependent mechanism. PMID:26322111

  16. Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells

    PubMed Central

    Mouser, Emily E. I. M.; Pollakis, Georgios; Yazdanbakhsh, Maria; Harnett, William

    2016-01-01

    One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs. PMID:26808476

  17. Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

    SciTech Connect

    Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C.

    2016-01-15

    Cardiovirus Leader proteins (L{sub X}) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus L{sub M} structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus L{sub E}, and also larger complexes with L{sub E}:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant L{sub S} and L{sub T} from Theiloviruses. When mutations were introduced to alter the L{sub E} zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on L{sub E} must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by L{sub E}.

  18. Bis-biguanide dihydrochloride inhibits intracellular replication of M. tuberculosis and controls infection in mice

    PubMed Central

    Shen, Hongbo; Wang, Feifei; Zeng, Gucheng; Shen, Ling; Cheng, Han; Huang, Dan; Wang, Richard; Rong, Lijun; Chen, Zheng W.

    2016-01-01

    While there is an urgent need to develop new and effective drugs for treatment of tuberculosis (TB) and multi-drug resistant TB (MDR-TB), repurposing FDA (U.S. Food and Drug Administration) -approved drugs for development of anti-TB agents may decrease time and effort from bench to bedside. Here, we employed host cell-based high throughput screening (HTS) assay to screen and characterize FDA-approved, off-patent library drugs for anti-Mycobacterium tuberculosis (MTB) activities. The cell-based HTS allowed us to identify an anti-cancer drug of bis-biguanide dihydrochloride (BBD) as potent anti-mycobacteria agent. Further characterization showed that BBD could inhibit intracellular and extracellular growth of M. smegmatis and slow-growing M. bovis BCG. BBD also potently inhibited replication of clinically-isolated MTB and MDR-TB strains. The proof-of-concept study showed that BBD treatment of MTB-infected mice could significantly decrease CFU counts in the lung and spleen. Notably, comparative evaluation showed that MTB CFU counts in BBD-treated mice were lower than those in rifampicin-treated mice. No apparent BBD side effects were found in BBD-treated mice. Thus, our findings support further studies to develop BBD as a new and effective drug against TB and MDR-TB. PMID:27601302

  19. Aureonitol, a Fungi-Derived Tetrahydrofuran, Inhibits Influenza Replication by Targeting Its Surface Glycoprotein Hemagglutinin

    PubMed Central

    Sacramento, Carolina Q.; Marttorelli, Andressa; Fintelman-Rodrigues, Natalia; de Freitas, Caroline S.; de Melo, Gabrielle R.; Rocha, Marco E. N.; Kaiser, Carlos R.; Rodrigues, Katia F.; da Costa, Gisela L.; Alves, Cristiane M.; Santos-Filho, Osvaldo; Barbosa, Jussara P.; Souza, Thiago Moreno L.

    2015-01-01

    The influenza virus causes acute respiratory infections, leading to high morbidity and mortality in groups of patients at higher risk. Antiviral drugs represent the first line of defense against influenza, both for seasonal infections and pandemic outbreaks. Two main classes of drugs against influenza are in clinical use: M2-channel blockers and neuraminidase inhibitors. Nevertheless, because influenza strains that are resistant to these antivirals have been described, the search for novel compounds with different mechanisms of action is necessary. Here, we investigated the anti-influenza activity of a fungi-derived natural product, aureonitol. This compound inhibited influenza A and B virus replication. This compound was more effective against influenza A(H3N2), with an EC50 of 100 nM. Aureonitol cytoxicity was also very low, with a CC50 value of 1426 μM. Aureonitol inhibited influenza hemagglutination and, consequently, significantly impaired virus adsorption. Molecular modeling studies revealed that aureonitol docked in the sialic acid binding site of hemagglutinin, forming hydrogen bonds with highly conserved residues. Altogether, our results indicate that the chemical structure of aureonitol is promising for future anti-influenza drug design. PMID:26462111

  20. Aureonitol, a Fungi-Derived Tetrahydrofuran, Inhibits Influenza Replication by Targeting Its Surface Glycoprotein Hemagglutinin.

    PubMed

    Sacramento, Carolina Q; Marttorelli, Andressa; Fintelman-Rodrigues, Natalia; de Freitas, Caroline S; de Melo, Gabrielle R; Rocha, Marco E N; Kaiser, Carlos R; Rodrigues, Katia F; da Costa, Gisela L; Alves, Cristiane M; Santos-Filho, Osvaldo; Barbosa, Jussara P; Souza, Thiago Moreno L

    2015-01-01

    The influenza virus causes acute respiratory infections, leading to high morbidity and mortality in groups of patients at higher risk. Antiviral drugs represent the first line of defense against influenza, both for seasonal infections and pandemic outbreaks. Two main classes of drugs against influenza are in clinical use: M2-channel blockers and neuraminidase inhibitors. Nevertheless, because influenza strains that are resistant to these antivirals have been described, the search for novel compounds with different mechanisms of action is necessary. Here, we investigated the anti-influenza activity of a fungi-derived natural product, aureonitol. This compound inhibited influenza A and B virus replication. This compound was more effective against influenza A(H3N2), with an EC50 of 100 nM. Aureonitol cytoxicity was also very low, with a CC50 value of 1426 μM. Aureonitol inhibited influenza hemagglutination and, consequently, significantly impaired virus adsorption. Molecular modeling studies revealed that aureonitol docked in the sialic acid binding site of hemagglutinin, forming hydrogen bonds with highly conserved residues. Altogether, our results indicate that the chemical structure of aureonitol is promising for future anti-influenza drug design.

  1. Resveratrol induces cell death and inhibits human herpesvirus 8 replication in primary effusion lymphoma cells.

    PubMed

    Tang, Feng-Yi; Chen, Chang-Yu; Shyu, Huey-Wen; Hong, Shin; Chen, Hung-Ming; Chiou, Yee-Hsuan; Lin, Kuan-Hua; Chou, Miao-Chen; Wang, Lin-Yu; Wang, Yi-Fen

    2015-12-05

    Resveratrol (3,4',5-trihydroxy-trans-stilbene) has been reported to inhibit proliferation of various cancer cells. However, the effects of resveratrol on the human herpesvirus 8 (HHV8) harboring primary effusion lymphoma (PEL) cells remains unclear. The anti-proliferation effects and possible mechanisms of resveratrol in the HHV8 harboring PEL cells were examined in this study. Results showed that resveratrol induced caspase-3 activation and the formation of acidic vacuoles in the HHV8 harboring PEL cells, indicating resveratrol treatment could cause apoptosis and autophagy in PEL cells. In addition, resveratrol treatment increased ROS generation but did not lead to HHV8 reactivation. ROS scavenger (N-acetyl cysteine, NAC) could attenuate both the resveratrol induced caspase-3 activity and the formation of acidic vacuoles, but failed to attenuate resveratrol induced PEL cell death. Caspase inhibitor, autophagy inhibitors and necroptosis inhibitor could not block resveratrol induced PEL cell death. Moreover, resveratrol disrupted HHV8 latent infection, inhibited HHV8 lytic gene expression and decreased virus progeny production. Overexpression of HHV8-encoded viral FLICE inhibitory protein (vFLIP) could partially block resveratrol induced cell death in PEL cells. These data suggest that resveratrol-induced cell death in PEL cells may be mediated by disruption of HHV8 replication. Resveratrol may be a potential anti-HHV8 drug and an effective treatment for HHV8-related tumors.

  2. Inhibition of porcine transmissible gastroenteritis virus (TGEV) replication in mini-pigs by shRNA.

    PubMed

    Zhou, Junfang; Huang, Fen; Hua, Xiuguo; Cui, Li; Zhang, Wen; Shen, Yan; Yan, Yijia; Chen, Piren; Ding, Dezhong; Mou, Jing; Chen, Qi; Lan, Daoliang; Yang, Zhibiao

    2010-04-01

    Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis (TGE), characterized by high mortality and severely retarded growth in piglets that dramatically affects the porcine industry. Previously, we have identified two shRNA-expressing plasmids pEGFP-U6/P1 and pEGFP-U6/P2 that target RNA-dependent RNA polymerase (RdRP) gene of TGEV with more than 95% of virus inhibition in vitro. In this study, inhibition of the TGEV replication by pEGFP-U6/P1 and pEGFP-U6/P2 was tested in mini-pigs. SPF mini-pigs at 25 days old were injected with the shRNA-expressing plasmids and then infected with TGEV. The results from the analyses of clinical signs, histopathology, indirect immunofluorescence (IIF) and RT-PCR show that the two shRNA-expressing plasmids could significantly decrease the quantity of TGEV in different organs and protect mini-pigs from TGEV infection. These findings illustrate the prospect for TGEV-specific shRNAs to be new anti-TGEV agents.

  3. Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition.

    PubMed

    Ciomperlik, Jessica J; Basta, Holly A; Palmenberg, Ann C

    2016-01-01

    Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE.

  4. Cardiovirus Leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition

    PubMed Central

    Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C.

    2015-01-01

    Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE. PMID:26492198

  5. Peptide-Derivatized SB105-A10 Dendrimer Inhibits the Infectivity of R5 and X4 HIV-1 Strains in Primary PBMCs and Cervicovaginal Histocultures

    PubMed Central

    Bon, Isabella; Lembo, David; Rusnati, Marco; Clò, Alberto; Morini, Silvia; Miserocchi, Anna; Bugatti, Antonella; Grigolon, Sonia; Musumeci, Giuseppina; Landolfo, Santo; Re, Maria Carla; Gibellini, Davide

    2013-01-01

    Peptide dendrimers are a class of molecules that exhibit a large array of biological effects including antiviral activity. In this report, we analyzed the antiviral activity of the peptide-derivatized SB105-A10 dendrimer, which is a tetra-branched dendrimer synthetized on a lysine core, in activated peripheral blood mononuclear cells (PBMCs) that were challenged with reference and wild-type human immunodeficiency virus type 1 (HIV-1) strains. SB105-A10 inhibited infections by HIV-1 X4 and R5 strains, interfering with the early phases of the viral replication cycle. SB105-A10 targets heparan sulfate proteoglycans (HSPGs) and, importantly, the surface plasmon resonance (SPR) assay revealed that SB105-A10 strongly binds gp41 and gp120, most likely preventing HIV-1 attachment/entry through multiple mechanisms. Interestingly, the antiviral activity of SB105-A10 was also detectable in an organ-like structure of human cervicovaginal tissue, in which SB105-A10 inhibited the HIV-1ada R5 strain infection without altering the tissue viability. These results demonstrated the strong antiviral activity of SB105-A10 and suggest a potential microbicide use of this dendrimer to prevent the heterosexual transmission of HIV-1. PMID:24116111

  6. Systems Biology Reveals NS4B-Cyclophilin A Interaction: A New Target to Inhibit YFV Replication.

    PubMed

    Vidotto, Alessandra; Morais, Ana T S; Ribeiro, Milene R; Pacca, Carolina C; Terzian, Ana C B; Gil, Laura H V G; Mohana-Borges, Ronaldo; Gallay, Philippe; Nogueira, Mauricio L

    2017-04-07

    Yellow fever virus (YFV) replication is highly dependent on host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1-DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein that has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. 59 proteins were found to be significantly increased when compared with a negative control, and CypA exhibited the greatest difference, with a 22-fold change. Fisher's exact test was significant for 58 proteins, and the p value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified 16 pathways, and this analysis indicated sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using sirolimus and cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction.

  7. The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas.

    PubMed

    Borysov, Sergiy I; Nepon-Sixt, Brook S; Alexandrow, Mark G

    2015-12-28

    The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G1-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.

  8. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

    PubMed Central

    Duval, Mark; Posner, Marshall R.

    2016-01-01

    Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells. PMID:27419146

  9. miR-370 mimic inhibits replication of Japanese encephalitis virus in glioblastoma cells

    PubMed Central

    Li, Wenjuan; Cheng, Peng; Nie, Shangdan; Cui, Wen

    2016-01-01

    Japanese encephalitis (JE) is one of the most severe viral infections of the central nervous system. No effective treatment for JE currently exists, because its pathogenesis remains largely unknown. The present study was designed to screen the potential microRNAs (miRNAs) involved in JE. Glioblastoma cells were collected, after being infected with the Japanese encephalitis virus (JEV). Total miRNAs were extracted and analyzed using an miRNA chip. One of the most severely affected miRNAs was selected, and the role of miR-370 in JEV infection was investigated. Cell viability and apoptosis of the host cells were evaluated. JEV replication was detected via analysis of gene E expression. Real-time polymerase chain reaction was used to determine the levels of endogenous miR-370 and expression of innate immunity-related genes. Following JEV infection, 114 miRNAs were affected, as evidenced by the miRNA chip. Among them, 30 miRNAs were upregulated and 84 were downregulated. The changes observed in five miRNAs were confirmed by real-time polymerase chain reaction. One of the significantly downregulated miRNAs was miR-370. Therefore, miR-370 mimic was transfected into the cells, following which the levels of endogenous miR-370 were significantly elevated. Concurrently, JEV replication was significantly reduced 24 hours after transfection of miR-370 mimic. Functionally, miR-370 mimic mitigated both JEV-induced apoptosis and the inhibition of host cell proliferation. Following JEV infection, interferon-β and nuclear factor-kappa B were upregulated, whereas miR-370 mimic prevented the upregulation of the genes induced by JEV infection. The present study demonstrated that miR-370 expression in host cells is downregulated following JEV infection, which further mediates innate immunity-related gene expression. Taken together, miR-370 mimic might be useful to prevent viral replication and infection-induced host cell injury. PMID:27703358

  10. A restriction enzyme based cloning method to assess the in vitro replication capacity of HIV-1 subtype C Gag-MJ4 chimeric viruses.

    PubMed

    Claiborne, Daniel T; Prince, Jessica L; Hunter, Eric

    2014-08-31

    The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

  11. Genistein interferes with SDF-1- and HIV-mediated actin dynamics and inhibits HIV infection of resting CD4 T cells

    PubMed Central

    2013-01-01

    Background Binding of HIV to the chemokine coreceptor CXCR4 mediates viral fusion and signal transduction that promotes actin dynamics critical for HIV infection of blood resting CD4 T cells. It has been suggested that this gp120-mediated actin activity resembles the chemotactic actin dynamics mediated by chemokines such as SDF-1. To determine whether inhibiting SDF-1-mediated chemotactic activity can also inhibit HIV infection, we screened several inhibitors known to reduce SDF-1-mediated chemotaxis of T cells. Results We found that a tyrosine kinase inhibitor, genistein, inhibited both SDF-1-mediated chemotaxis and HIV infection of resting CD4 T cells. Genistein was also found to interfere with SDF-1- and HIV-mediated actin dynamics in CD4 T cells. This reduction in actin activity correlates with genistein-mediated inhibition of viral DNA accumulation in resting CD4 T cells. In addition, we also tested two other tyrosine kinase inhibitors, sunitinib and AG1478. Sunitinib, but not AG1478, inhibited HIV infection of resting CD4 T cells. We further tested the safety of genistein in 3 Chinese rhesus macaques (Macaca mulatta), and each animal was given a monotherapy of genistein at 10 mg/kg orally for 12 weeks. No adverse drug effects were observed in these animals. Conclusions Our results suggest that novel therapeutic strategies can be developed based on targeting cellular proteins involved in HIV-dependent signaling. This approach can interfere with HIV-mediated actin dynamics and inhibit HIV infection. PMID:23782904

  12. Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells

    PubMed Central

    Lin, C.; Agnes, J. T.; Behrens, N.; Tagawa, Y.; Gershwin, L. J.; Corbeil, L. B.

    2016-01-01

    Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2—RSAD2) and ISG15 (IFN-stimulated gene 15—ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo. PMID:26859677

  13. Replication Study: BET bromodomain inhibition as a therapeutic strategy to target c-Myc

    PubMed Central

    Aird, Fraser; Kandela, Irawati; Mantis, Christine

    2017-01-01

    In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Kandela et al., 2015) that described how we intended to replicate selected experiments from the paper "BET bromodomain inhibition as a therapeutic strategy to target c-Myc" (Delmore et al., 2011). Here we report the results of those experiments. We found that treatment of human multiple myeloma (MM) cells with the small-molecular inhibitor of BET bromodomains, (+)-JQ1, selectively downregulated MYC transcription, which is similar to what was reported in the original study (Figure 3B; Delmore et al., 2011). Efficacy of (+)-JQ1 was evaluated in an orthotopically xenografted model of MM. Overall survival was increased in (+)-JQ1 treated mice compared to vehicle control, similar to the original study (Figure 7E; Delmore et al., 2011). Tumor burden, as determined by bioluminescence, was decreased in (+)-JQ1 treated mice compared to vehicle control; however, while the effect was in the same direction as the original study (Figure 7C-D; Delmore et al., 2011), it was not statistically significant. The opportunity to detect a statistically significant difference was limited though, due to the higher rate of early death in the control group, and increased overall survival in (+)-JQ1 treated mice before the pre-specified tumor burden analysis endpoint. Additionally, we evaluated the (−)-JQ1 enantiomer that is structurally incapable of inhibiting BET bromodomains, which resulted in a minimal impact on MYC transcription, but did not result in a statistically significant difference in tumor burden or survival distributions compared to treatment with (+)-JQ1. Finally, we report meta-analyses for each result. DOI: http://dx.doi.org/10.7554/eLife.21253.001 PMID:28100400

  14. How HIV Takes Advantage of the Cytoskeleton in Entry and Replication

    PubMed Central

    Stolp, Bettina; Fackler, Oliver T.

    2011-01-01

    The host cell cytoskeleton plays a key role in the life cycle of viral pathogens whose propagation depends on mandatory intracellular steps. Accordingly, also the human immunodeficiency virus type 1 (HIV-1) has evolved strategies to exploit and modulate in particular the actin cytoskeleton for its purposes. This review will recapitulate recent findings on how HIV-1 hijacks the cytoskeleton to facilitate entry into, transport within and egress from host cells as well as to commandeer communication of infected with uninfected bystander cells. PMID:21994733

  15. Tomatidine inhibits replication of Staphylococcus aureus small-colony variants in cystic fibrosis airway epithelial cells.

    PubMed

    Mitchell, Gabriel; Gattuso, Mariza; Grondin, Gilles; Marsault, Éric; Bouarab, Kamal; Malouin, François

    2011-05-01

    Small-colony variants (SCVs) often are associated with chronic Staphylococcus aureus infections, such as those encountered by cystic fibrosis (CF) patients. We report here that tomatidine, the aglycon form of the plant secondary metabolite tomatine, has a potent growth inhibitory activity against SCVs (MIC of 0.12 μg/ml), whereas the growth of normal S. aureus strains was not significantly altered by tomatidine (MIC, >16 μg/ml). The specific action of tomatidine was bacteriostatic for SCVs and was clearly associated with their dysfunctional electron transport system, as the presence of the electron transport inhibitor 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) caused normal S. aureus strains to become susceptible to tomatidine. Inversely, the complementation of SCVs' respiratory deficiency conferred resistance to tomatidine. Tomatidine provoked a general reduction of macromolecular biosynthesis but more specifically affected the incorporation of radiolabeled leucine in proteins of HQNO-treated S. aureus at a concentration corresponding to the MIC against SCVs. Furthermore, tomatidine inhibited the intracellular replication of a clinical SCV in polarized CF-like epithelial cells. Our results suggest that tomatidine eventually will find some use in combination therapy with other traditional antibiotics to eliminate persistent forms of S. aureus.

  16. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    SciTech Connect

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S.; Arbuthnot, Patrick

    2009-11-20

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  17. Inhibition of HCV replication by humanized-single domain transbodies to NS4B.

    PubMed

    Glab-Ampai, Kittirat; Malik, Aijaz Ahmad; Chulanetra, Monrat; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Srimanote, Potjanee; Tongtawe, Pongsri; Chaicumpa, Wanpen

    2016-08-05

    NS4B of hepatitis C virus (HCV) initiates membrane web formation, binds RNA and other HCV proteins for viral replication complex (RC) formation, hydrolyses NTP, and inhibits innate anti-viral immunity. Thus, NS4B is an attractive target of a novel anti-HCV agent. In this study, humanized-nanobodies (VHs/VHHs) that bound to recombinant NS4B were produced by means of phage display technology. The nanobodies were linked molecularly to a cell penetrating peptide, penetratin (PEN), for making them cell penetrable (become transbodies). Human hepatic (Huh7) cells transfected with HCV JFH1-RNA that were treated with transbodies from four Escherichia coli clones (PEN-VHH7, PEN-VHH9, PEN-VH33, and PEN-VH43) had significant reduction of HCV RNA amounts in their culture fluids and intracellularly when compared to the transfected cells treated with control transbody and medium alone. The results were supported by the HCV foci assay. The transbody treated-transfected cells also had upregulation of the studied innate cytokine genes, IRF3, IFNβ and IL-28b. The transbodies have high potential for testing further as a novel anti-HCV agent, either alone, adjunct of existing anti-HCV agents/remedies, or in combination with their cognates specific to other HCV enzymes/proteins.

  18. HIVsirDB: A Database of HIV Inhibiting siRNAs

    PubMed Central

    Thakur, Nishant; Sharma, Arun; Raghava, Gajendra P. S.; Kumar, Manoj

    2011-01-01

    Background Human immunodeficiency virus (HIV) is responsible for millions of deaths every year. The current treatment involves the use of multiple antiretroviral agents that may harm patients due to their toxic nature. RNA interference (RNAi) is a potent candidate for the future treatment of HIV, uses short interfering RNA (siRNA/shRNA) for silencing HIV genes. In this study, attempts have been made to create a database HIVsirDB of siRNAs responsible for silencing HIV genes. Descriptions HIVsirDB is a manually curated database of HIV inhibiting siRNAs that provides comprehensive information about each siRNA or shRNA. Information was collected and compiled from literature and public resources. This database contains around 750 siRNAs that includes 75 partially complementary siRNAs differing by one or more bases with the target sites and over 100 escape mutant sequences. HIVsirDB structure contains sixteen fields including siRNA sequence, HIV strain, targeted genome region, efficacy and conservation of target sequences. In order to facilitate user, many tools have been integrated in this database that includes; i) siRNAmap for mapping siRNAs on target sequence, ii) HIVsirblast for BLAST search against database, iii) siRNAalign for aligning siRNAs. Conclusion HIVsirDB is a freely accessible database of siRNAs which can silence or degrade HIV genes. It covers 26 types of HIV strains and 28 cell types. This database will be very useful for developing models for predicting efficacy of HIV inhibiting siRNAs. In summary this is a useful resource for researchers working in the field of siRNA based HIV therapy. HIVsirDB database is accessible at http://crdd.osdd.net/raghava/hivsir/. PMID:22022467

  19. Biased small-molecule ligands for selective inhibition of HIV-1 cell entry via CCR5.

    PubMed

    Berg, Christian; Spiess, Katja; Lüttichau, Hans R; Rosenkilde, Mette M

    2016-12-01

    Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small-molecule receptor antagonists for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small-molecule CCR5 agonists as HIV-1 fusion inhibitors. A virus-free cell-based fusion reporter assay, based on mixing "effector cells" (expressing HIV Env and luciferase activator) with "target cells" (expressing CD4, CCR5 wild type or a selection of well-described mutations, and luciferase reporter), was used as fusion readout. Receptor expression was evaluated by ELISA and fluorescence microscopy. On CCR5 WT, Maraviroc and Aplaviroc inhibited fusion with high potencies (EC 50 values of 91 and 501 nM, respectively), whereas removal of key residues for both antagonists (Glu283Ala) or Maraviroc alone (Tyr251Ala) prevented fusion inhibition, establishing this assay as suitable for screening of HIV entry inhibitors. Both ligands inhibited HIV fusion on signaling-deficient CCR5 mutations (Tyr244Ala and Trp248Ala). Moreover, the steric hindrance CCR5 mutation (Gly286Phe) impaired fusion, presumably by a direct hindrance of gp120 interaction. Finally, the efficacy switch mutation (Leu203Phe) - converting small-molecule antagonists/inverse agonists to full agonists biased toward G-protein activation - uncovered that also small-molecule agonists can function as direct HIV-1 cell entry inhibitors. Importantly, no agonist-induced receptor internalization was observed for this mutation. Our studies of the pharmacodynamic requirements for HIV-1 fusion inhibitors highlight the possibility of future development of biased ligands with selective targeting of the HIV-CCR5 interaction without interfering with the normal functionality of CCR5.

  20. Similar Replicative Fitness Is Shared by the Subtype B and Unique BF Recombinant HIV-1 Isolates that Dominate the Epidemic in Argentina

    PubMed Central

    Rubio, Andrea E.; Abraha, Awet; Carpenter, Crystal A.; Troyer, Ryan M.; Reyes-Rodríguez, Ángel L.; Salomon, Horacio; Arts, Eric J.; Tebit, Denis M.

    2014-01-01

    The HIV-1 epidemic in South America is dominated by pure subtypes (mostly B and C) and more than 7 BF and BC recombinant forms. In Argentina, circulating recombinant forms (CRFs) comprised of subtypes B and F make up more than 50% of HIV infections. For this study, 28 HIV-1 primary isolates were obtained from patients in Buenos Aires, Argentina and initially classified into subtype B (n = 9, 32.1%), C (n = 1, 3.6%), and CRFs (n = 18, 64.3%) using partial pol and vpu-env sequences, which proved to be inconsistent and inaccurate for these phylogenetic analyses. Near full length genome sequences of these primary HIV-1 isolates revealed that nearly all intersubtype BF recombination sites were unique and countered previous “CRF” B/F classifications. The majority of these Argentinean HIV-1 isolates were CCR5-using but 4 had a dual/mixed tropism as predicted by both phenotypic and genotypic assays. Comparison of the replicative fitness of these BF primary HIV-1 isolates to circulating B, F, and C HIV-1 using pairwise competitions in peripheral blood mononuclear cells (PBMCs) indicated a similarity in fitness of these BF recombinants to subtypes B and F HIV-1 (of the same co-receptor usage) whereas subtype C HIV-1 was significantly less fit than all as previously reported. These results suggest that the multitude of BF HIV-1 strains present within the Argentinean population do not appear to have gained replicative fitness following recent B and F recombination events. PMID:24727861

  1. In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

    PubMed

    Wang, Gaili; He, Wenqi; Song, Deguang; Li, Jida; Bao, Yingfu; Lu, Rongguang; Bi, Jingying; Zhao, Kui; Gao, Feng

    2014-05-01

    Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications.

  2. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

    SciTech Connect

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika; E-mail: hiokada@med.nagoya-cu.ac.jp

    2007-02-23

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1{sub IIIB} infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.

  3. CPSF6 Defines a Conserved Capsid Interface that Modulates HIV-1 Replication

    PubMed Central

    Schaller, Torsten; Elliott, Tom; Lee, KyeongEun; KewalRamani, Vineet N.; Chin, Jason W.; Towers, Greg J.; James, Leo C.

    2012-01-01

    The HIV-1 genome enters cells inside a shell comprised of capsid (CA) protein. Variation in CA sequence alters HIV-1 infectivity and escape from host restriction factors. However, apart from the Cyclophilin A-binding loop, CA has no known interfaces with which to interact with cellular cofactors. Here we describe a novel protein-protein interface in the N-terminal domain of HIV-1 CA, determined by X-ray crystallography, which mediates both viral restriction and host cofactor dependence. The interface is highly conserved across lentiviruses and is accessible in the context of a hexameric lattice. Mutation of the interface prevents binding to and restriction by CPSF6-358, a truncated cytosolic form of the RNA processing factor, cleavage and polyadenylation specific factor 6 (CPSF6). Furthermore, mutations that prevent CPSF6 binding also relieve dependence on nuclear entry cofactors TNPO3 and RanBP2. These results suggest that the HIV-1 capsid mediates direct host cofactor interactions to facilitate viral infection. PMID:22956906

  4. Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates.

    PubMed

    van der Vliet, P C; Kwant, M M

    1981-04-28

    In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.

  5. A species-specific amino acid difference in the macaque CD4 receptor restricts replication by global circulating HIV-1 variants representing viruses from recent infection.

    PubMed

    Humes, Daryl; Emery, Sandra; Laws, Elizabeth; Overbaugh, Julie

    2012-12-01

    HIV-1 replicates poorly in macaque cells, and this had hindered the advancement of relevant nonhuman primate model systems for HIV-1 infection and pathogenesis. Several host restriction factors have been identified that contribute to this species-specific restriction to HIV-1 replication, but these do not fully explain the poor replication of most strains of HIV-1 in macaque cells. Only select HIV-1 envelope variants, typically those derived from viruses that have been adapted in cell culture, result in infectious chimeric SIVs encoding HIV-1 envelope (SHIVs). Here we demonstrate that most circulating HIV-1 variants obtained directly from infected individuals soon after virus acquisition do not efficiently mediate entry using the macaque CD4 receptor. The infectivity of these viruses is ca. 20- to 50-fold lower with the rhesus and pig-tailed macaque versus the human CD4 receptor. In contrast, culture-derived HIV-1 envelope variants that facilitate efficient replication in macaques showed similar infectivity with macaque and human CD4 receptors (within ∼2-fold). The ability of an envelope to mediate entry using macaque CD4 correlated with its ability to mediate entry of cells expressing low levels of the human CD4 receptor and with soluble CD4 sensitivity. Species-specific differences in the functional capacity of the CD4 receptor to mediate entry mapped to a single amino acid difference at position 39 that is under strong positive selection, suggesting that the evolution of CD4 may have been influenced by its function as a viral receptor. These results also suggest that N39 in human CD4 may be a critical residue for interaction of transmitted HIV-1 variants. These studies provide important insights into virus-host cell interactions that have hindered the development of relevant nonhuman primate models for HIV-1 infection and provide possible markers, such as sCD4 sensitivity, to identify potential HIV-1 variants that could be exploited for development of better

  6. Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication.

    PubMed

    Dreer, Marcel; Fertey, Jasmin; van de Poel, Saskia; Straub, Elke; Madlung, Johannes; Macek, Boris; Iftner, Thomas; Stubenrauch, Frank

    2016-04-01

    Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins.

  7. Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication

    PubMed Central

    Dreer, Marcel; Fertey, Jasmin; van de Poel, Saskia; Straub, Elke; Madlung, Johannes; Macek, Boris; Iftner, Thomas; Stubenrauch, Frank

    2016-01-01

    Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins

  8. Vaccine-delivered HIV envelope inhibits CD4+ T-cell activation, a mechanism for poor HIV vaccine responses

    PubMed Central

    Fernando, Kathy; Hu, Haitao; Ni, Houping; Hoxie, James A.

    2007-01-01

    The human immunodeficiency virus (HIV) causes impairment of the immune system in part by targeting CD4+ T cells for infection and dysfunction. HIV envelope (Env) present on free virions and infected cells causes dysfunction of uninfected bystander CD4+ T cells via interaction with both CD4 and coreceptors. Env is commonly used as part of a cocktail of HIV antigens in current vaccines. In DNA and viral vector vaccine approaches, antigen-presenting cells (APCs) and non-APCs in the vicinity of the vaccine delivery site and draining lymph node express vaccine-derived antigens. The studies here demonstrate that cell-surface expression of Env on APCs and non-APCs as part of the vaccine action causes an inhibition of antigen-induced CD4+ T-cell activation and proliferation mediated by CD4 binding and suggests a potential mechanism for reduced activity of Env-containing HIV vaccines. Similar studies using a functional Env lacking CD4 binding circumvented suppression, suggesting an alternative and potentially superior approach to HIV vaccine design. PMID:17158230

  9. Cleavage of the HIV replication primer tRNALys,3 in human cells expressing bacterial anticodon nuclease.

    PubMed Central

    Shterman, N; Elroy-Stein, O; Morad, I; Amitsur, M; Kaufmann, G

    1995-01-01

    Anticodon nuclease is a bacterial restriction enzyme directed against tRNA(Lys). We report that anticodon nuclease also cleaves mammalian tRNA(Lys) molecules, with preference and site specificity shown towards the natural substrate. Expression of the anticodon nuclease core polypeptide PrrC in HeLa cells from a recombinant vaccinia virus elicited cleavage of intracellular tRNA(Lys),3. The data justify an inquiry into the possible application of anticodon nuclease as an inhibitor of tRNA(Lys),3-primed HIV replication. They also indicate that the anticodon region of tRNA(Lys) is a substrate recognition site and suggest that PrrC harbors the enzymatic activity. Images PMID:7784179

  10. The stress-activated protein kinases p38α/β and JNK1/2 cooperate with Chk1 to inhibit mitotic entry upon DNA replication arrest.

    PubMed

    Llopis, Alba; Salvador, Noelia; Ercilla, Amaia; Guaita-Esteruelas, Sandra; Barrantes, Ivan del Barco; Gupta, Jalaj; Gaestel, Matthias; Davis, Roger J; Nebreda, Angel R; Agell, Neus

    2012-10-01

    Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G 2/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.

  11. Inhibition of Hepatitis C Virus Replication and Viral Helicase by Ethyl Acetate Extract of the Marine Feather Star Alloeocomatella polycladia

    PubMed Central

    Yamashita, Atsuya; Salam, Kazi Abdus; Furuta, Atsushi; Matsuda, Yasuyoshi; Fujita, Osamu; Tani, Hidenori; Fujita, Yoshihisa; Fujimoto, Yuusuke; Ikeda, Masanori; Kato, Nobuyuki; Sakamoto, Naoya; Maekawa, Shinya; Enomoto, Nobuyuki; Nakakoshi, Masamichi; Tsubuki, Masayoshi; Sekiguchi, Yuji; Tsuneda, Satoshi; Akimitsu, Nobuyoshi; Noda, Naohiro; Tanaka, Junichi; Moriishi, Kohji

    2012-01-01

    Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC50 of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC50 value of 23 to 44 µg/mL, which is similar to the IC50 value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C. PMID:22690141

  12. Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

    PubMed Central

    Levy, D N; Refaeli, Y; Weiner, D B

    1995-01-01

    The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics. PMID:7815499

  13. Antibody Against Integrin Lymphocyte Function-Associated Antigen 1 Inhibits HIV Type 1 Infection in Primary Cells Through Caspase-8-Mediated Apoptosis

    PubMed Central

    Walker, Tiffany N.; Cimakasky, Lisa M.; Coleman, Ebony M.; Madison, M. Nia

    2013-01-01

    Abstract HIV-1 infection induces formation of a virological synapse wherein CD4, chemokine receptors, and cell-adhesion molecules such as lymphocyte function-associated antigen 1 (LFA-1) form localized domains on the cell surface. Studies show that LFA-1 on the surface of HIV-1 particles retains its adhesion function and enhances virus attachment to susceptible cells by binding its counterreceptor intercellular adhesion molecule 1 (ICAM-1). This virus–cell interaction augments virus infectivity by facilitating binding and entry events. In this study, we demonstrate that inhibition of the LFA-1/ICAM-1 interaction by a monoclonal antibody leads to decreased virus production and spread in association with increased apoptosis of HIV-infected primary T cells. The data indicate that the LFA-1/ICAM-1 interaction may limit apoptosis in HIV-1-infected T cells. This phenomenon appears similar to anoikis wherein epithelial cells are protected from apoptosis conferred by ligand-bound integrins. These results have implications for further understanding HIV pathogenesis and replication in peripheral compartments and lymphoid organs. PMID:22697794

  14. Inhibition of hepatitis C virus replication by chalepin and pseudane IX isolated from Ruta angustifolia leaves.

    PubMed

    Wahyuni, Tutik Sri; Widyawaruyanti, Aty; Lusida, Maria Inge; Fuad, Achmad; Soetjipto; Fuchino, Hiroyuki; Kawahara, Nobuo; Hayashi, Yoshitake; Aoki, Chie; Hotta, Hak

    2014-12-01

    Hepatitis C virus (HCV) infection is highly prevalent among global populations, with an estimated number of infected patients being 170 million. Approximately 70-80% of patients acutely infected with HCV will progress to chronic liver disease, such as liver cirrhosis and hepatocellular carcinoma, which is a substantial cause of morbidity and mortality worldwide. New therapies for HCV infection have been developed, however, the therapeutic efficacies still need to be improved. Medicinal plants are promising sources for antivirals against HCV. A variety of plants have been tested and proven to be beneficial as antiviral drug candidates against HCV. In this study, we examined extracts, their subfractions and isolated compounds of Ruta angustifolia leaves for antiviral activities against HCV in cell culture. We isolated six compounds, chalepin, scopoletin, γ-fagarine, arborinine, kokusaginine and pseudane IX. Among them, chalepin and pseudane IX showed strong anti-HCV activities with 50% inhibitory concentration (IC₅₀) of 1.7 ± 0.5 and 1.4 ± 0.2 μg/ml, respectively, without apparent cytotoxicity. Their anti-HCV activities were stronger than that of ribavirin (2.8 ± 0.4 μg/ml), which has been widely used for the treatment of HCV infection. Mode-of-action analyses revealed that chalepin and pseudane IX inhibited HCV at the post-entry step and decreased the levels of HCV RNA replication and viral protein synthesis. We also observed that arborinine, kokusaginine and γ-fagarine possessed moderate levels of anti-HCV activities with IC₅₀ values being 6.4 ± 0.7, 6.4 ± 1.6 and 20.4 ± 0.4 μg/ml, respectively, whereas scopoletin did not exert significant anti-HCV activities at 30 μg/ml.

  15. A Scorpion Defensin BmKDfsin4 Inhibits Hepatitis B Virus Replication in Vitro

    PubMed Central

    Zeng, Zhengyang; Zhang, Qian; Hong, Wei; Xie, Yingqiu; Liu, Yun; Li, Wenxin; Wu, Yingliang; Cao, Zhijian

    2016-01-01

    Hepatitis B virus (HBV) infection is a major worldwide health problem which can cause acute and chronic hepatitis and can significantly increase the risk of liver cirrhosis and primary hepatocellular carcinoma (HCC). Nowadays, clinical therapies of HBV infection still mainly rely on nucleotide analogs and interferons, the usage of which is limited by drug-resistant mutation or side effects. Defensins had been reported to effectively inhibit the proliferation of bacteria, fungi, parasites and viruses. Here, we screened the anti-HBV activity of 25 scorpion-derived peptides most recently characterized by our group. Through evaluating anti-HBV activity and cytotoxicity, we found that BmKDfsin4, a scorpion defensin with antibacterial and Kv1.3-blocking activities, has a comparable high inhibitory rate of both HBeAg and HBsAg in HepG2.2.15 culture medium and low cytotoxicity to HepG2.2.15. Then, our experimental results further showed that BmKDfsin4 can dose-dependently decrease the production of HBV DNA and HBV viral proteins in both culture medium and cell lysate. Interestingly, BmKDfsin4 exerted high serum stability. Together, this study indicates that the scorpion defensin BmKDfsin4 also has inhibitory activity against HBV replication along with its antibacterial and potassium ion channel Kv1.3-blocking activities, which shows that BmKDfsin4 is a uniquely multifunctional defensin molecule. Our work also provides a good molecule material which will be used to investigate the link or relationship of its antiviral, antibacterial and ion channel–modulating activities in the future. PMID:27128943

  16. Combination fluconazole/paroxetine treatment is neuroprotective despite ongoing neuroinflammation and viral replication in an SIV model of HIV neurological disease.

    PubMed

    Meulendyke, Kelly A; Queen, Suzanne E; Engle, Elizabeth L; Shirk, Erin N; Liu, Jiayang; Steiner, Joseph P; Nath, Avindra; Tarwater, Patrick M; Graham, David R; Mankowski, Joseph L; Zink, M Christine

    2014-12-01

    Effective combined antiretroviral therapy (cART) in HIV-infected patients has made HIV a treatable infection; however, debilitating HIV-associated neurocognitive disorders (HAND) can still affect approximately 50% of HIV-infected individuals even under cART. While cART has greatly reduced the prevalence of the most severe form of HAND, milder forms have increased in prevalence, leaving the total proportion of HIV-infected individuals suffering from HAND relatively unchanged. In this study, an in vitro drug screen identified fluconazole and paroxetine as protective compounds against HIV gp120 and Tat neurotoxicity. Using an accelerated, consistent SIV/macaque model of HIV-associated CNS disease, we tested the in vivo neuroprotective capabilities of combination fluconazole/paroxetine (FluPar) treatment. FluPar treatment protected macaques from SIV-induced neurodegeneration, as measured by neurofilament light chain in the CSF, APP accumulation in axons, and CaMKIIα in the frontal cortex, but did not significantly reduce markers of neuroinflammation or plasma or CNS viral loads. Since HIV and SIV neurodegeneration is often attributed to accompanying neuroinflammation, this study provides proof of concept that neuroprotection can be achieved even in the face of ongoing neuroinflammation and viral replication.

  17. miR-28-3p is a cellular restriction factor that inhibits human T cell leukemia virus, type 1 (HTLV-1) replication and virus infection.

    PubMed

    Bai, Xue Tao; Nicot, Christophe

    2015-02-27

    Human T cell leukemia virus, type 1 (HTLV-1) replication and spread are controlled by different viral and cellular factors. Although several anti-HIV cellular microRNAs have been described, such a regulation for HTLV-1 has not been reported. In this study, we found that miR-28-3p inhibits HTLV-1 virus expression and its replication by targeting a specific site within the genomic gag/pol viral mRNA. Because miR-28-3p is highly expressed in resting T cells, which are resistant to HTLV-1 infection, we investigated a potential protective role of miR-28-3p against de novo HTLV-1 infection. To this end, we developed a new sensitive and quantitative assay on the basis of the detection of products of reverse transcription. We demonstrate that miR-28-3p does not prevent virus receptor interaction or virus entry but, instead, induces a post-entry block at the reverse transcription level. In addition, we found that HTLV-1, subtype 1A isolates corresponding to the Japanese strain ATK-1 present a natural, single-nucleotide polymorphism within the miR-28-3p target site. As a result of this polymorphism, the ATK-1 virus sequence was not inhibited by miR-28. Interestingly, genetic studies on the transmission of the virus has shown that the ATK-1 strain, which carries a Thr-to-Cys transition mutation, is transmitted efficiently between spouses, suggesting that miR-28 may play an important role in HTLV-1 transmission.

  18. The Effect of Root, Shoot and Seed Extracts of The Iranian Thymus L. (Family: Lamiaceae) Species on HIV-1 Replication and CD4 Expression

    PubMed Central

    Soleimani Farsani, Maryam; Behbahani, Mandana; Isfahani, Hamid Zarkesh

    2016-01-01

    Objective The genus Thymus L. is a cushion plant that was previously used for the treatment of bronchitis and rheumatism. The present investigation was carried out to study the effects of root, shoot, leaf and seed extracts of five Thymus species and subspecies on peripheral blood mononuclear cells (PBMCs) toxicity and HIV-1 replication. Materials and Methods In this experimental study, the activity of the Thymus extracts on HIV-1 replication and lymphocytes population were examined respectively using HIV-1 p24 Antigen kit and flow-cytometer. The Thymus species effect was investigated, including Thymus kotschyanus, Thymus vulgaris, Thymus carmanicus, Thymus daenensis subspecies lancifolius and Thymus daenensis subspecies daenensis. Results The effect of root methanol extracts of all species on PBMCs proliferation was significantly higher than the other extracts. The intensity of CD4, CD3 and CD45 were decreased in the presence of all root extracts. Although the average median fluorescence intensity (MFI) values of CD19 were increased in the cells treated with these extracts. All methanol extracts showed anti-HIV-1 activity at high concentrations (200 and 500 µg/ml). Anti-HIV-1 activity of Thymus daenensis subspecies daenensis was significantly more than the other species. Conclusion These results demonstrated that root extracts of Thymus species might be a good candidate to investigate anti-HIV infection in vivo. PMID:27540531

  19. High-dose vitamin D3 reduces deficiency caused by low UVB exposure and limits HIV-1 replication in urban Southern Africans

    NASA Astrophysics Data System (ADS)

    Coussens, Anna K.; Naude, Celeste E.; Goliath, Rene; Chaplin, George; Wilkinson, Robert J.; Jablonski, Nina G.

    2015-06-01

    Cape Town, South Africa, has a seasonal pattern of UVB radiation and a predominantly dark-skinned urban population who suffer high HIV-1 prevalence. This coexistent environmental and phenotypic scenario puts residents at risk for vitamin D deficiency, which may potentiate HIV-1 disease progression. We conducted a longitudinal study in two ethnically distinct groups of healthy young adults in Cape Town, supplemented with vitamin D3 in winter, to determine whether vitamin D status modifies the response to HIV-1 infection and to identify the major determinants of vitamin D status (UVB exposure, diet, pigmentation, and genetics). Vitamin D deficiency was observed in the majority of subjects in winter and in a proportion of individuals in summer, was highly correlated with UVB exposure, and was associated with greater HIV-1 replication in peripheral blood cells. High-dosage oral vitamin D3 supplementation attenuated HIV-1 replication, increased circulating leukocytes, and reversed winter-associated anemia. Vitamin D3 therefore presents as a low-cost supplementation to improve HIV-associated immunity.

  20. High-dose vitamin D3 reduces deficiency caused by low UVB exposure and limits HIV-1 replication in urban Southern Africans

    PubMed Central

    Coussens, Anna K.; Naude, Celeste E.; Goliath, Rene; Chaplin, George; Wilkinson, Robert J.; Jablonski, Nina G.

    2015-01-01

    Cape Town, South Africa, has a seasonal pattern of UVB radiation and a predominantly dark-skinned urban population who suffer high HIV-1 prevalence. This coexistent environmental and phenotypic scenario puts residents at risk for vitamin D deficiency, which may potentiate HIV-1 disease progression. We conducted a longitudinal study in two ethnically distinct groups of healthy young adults in Cape Town, supplemented with vitamin D3 in winter, to determine whether vitamin D status modifies the response to HIV-1 infection and to identify the major determinants of vitamin D status (UVB exposure, diet, pigmentation, and genetics). Vitamin D deficiency was observed in the majority of subjects in winter and in a proportion of individuals in summer, was highly correlated with UVB exposure, and was associated with greater HIV-1 replication in peripheral blood cells. High-dosage oral vitamin D3 supplementation attenuated HIV-1 replication, increased circulating leukocytes, and reversed winter-associated anemia. Vitamin D3 therefore presents as a low-cost supplementation to improve HIV-associated immunity. PMID:26080414

  1. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner

    SciTech Connect

    Miller, S.D.; Farmer, G.; Prives, C.

    1995-12-01

    This report discusses new findings that the tumor supressor gene product p53 may play a role as a DNA-binding-dependent regulator of DNA replication. The results were obtained using polyomavirus in replication assays. Details regarding effects on cell growth arrest and transcriptional activation are discussed. 61 refs., 7 figs.

  2. Inhibition of HIV-1 infection by aqueous extracts of Prunella vulgaris L.

    PubMed Central

    2011-01-01

    Background The mint family (Lamiaceae) produces a wide variety of constituents with medicinal properties. Several family members have been reported to have antiviral activity, including lemon balm (Melissa officinalis L.), sage (Salvia spp.), peppermint (Mentha × piperita L.), hyssop (Hyssopus officinalis L.), basil (Ocimum spp.) and self-heal (Prunella vulgaris L.). To further characterize the anti-lentiviral activities of Prunella vulgaris, water and ethanol extracts were tested for their ability to inhibit HIV-1 infection. Results Aqueous extracts contained more anti-viral activity than did ethanol extracts, displaying potent antiviral activity against HIV-1 at sub μg/mL concentrations with little to no cellular cytotoxicity at concentrations more than 100-fold higher. Time-of-addition studies demonstrated that aqueous extracts were effective when added during the first five hours following initiation of infection, suggesting that the botanical constituents were targeting entry events. Further analysis revealed that extracts inhibited both virus/cell interactions and post-binding events. While only 40% inhibition was maximally achieved in our virus/cell interaction studies, extract effectively blocked post-binding events at concentrations similar to those that blocked infection, suggesting that it was targeting of these latter steps that was most important for mediating inhibition of virus infectivity. Conclusions We demonstrate that aqueous P. vulgaris extracts inhibited HIV-1 infectivity. Our studies suggest that inhibition occurs primarily by interference of early, post-virion binding events. The ability of aqueous extracts to inhibit early events within the HIV life cycle suggests that these extracts, or purified constituents responsible for the antiviral activity, are promising microbicides and/or antivirals against HIV-1. PMID:21513560

  3. Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System

    PubMed Central

    Mahdi, Mohamed; Szojka, Zsófia; Mótyán, János András; Tőzsér, József

    2015-01-01

    Retroviral protease inhibitors (PIs) are fundamental pillars in the treatment of HIV infection and acquired immunodeficiency syndrome (AIDS). Currently used PIs are designed against HIV-1, and their effect on HIV-2 is understudied. Using a modular HIV-2 protease cassette system, inhibition profiling assays were carried out for protease inhibitors both in enzymatic and cell culture assays. Moreover, the treatment-associated resistance mutations (I54M, L90M) were introduced into the modular system, and comparative inhibition assays were performed to determine their effect on the susceptibility of the protease. Our results indicate that darunavir, saquinavir, indinavir and lopinavir were very effective HIV-2 protease inhibitors, while tipranavir, nelfinavir and amprenavir showed a decreased efficacy. I54M, L90M double mutation resulted in a significant reduction in the susceptibility to most of the inhibitors with the exception of tipranavir. To our knowledge, this modular system constitutes a novel approach in the field of HIV-2 protease characterization and susceptibility testing. PMID:26633459

  4. Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types.

    PubMed

    Collman, R; Hassan, N F; Walker, R; Godfrey, B; Cutilli, J; Hastings, J C; Friedman, H; Douglas, S D; Nathanson, N

    1989-10-01

    To characterize the host range of different strains of HIV-1, we have used four types of cells, primary monocyte-derived macrophages (MDM), primary PBL, a promonocyte cell line (U937), and a CD4+ T cell line (SUP-T1). These cells were infected with three prototype strains of HIV-1, a putative lymphocyte-tropic strain (IIIB), and two putative monocyte-tropic strains (SF162 and DV). Infections were monitored by assays for infectious virus, for cell-free and cell-associated viral antigen (p24), and for the proportion of cells infected by immunohistochemical staining. It was concluded that: (a) the use of four different cell types provides a useful biological matrix for distinguishing the tropism of different strains of HIV-1; this matrix yields more information than the infection of any single cell type. (b) A monocyte-tropic strain of HIV-1, such as strain SF162, shows a reciprocal host range when compared with a lymphocyte-tropic strain such as IIIB; strain SF162 replicates well in primary MDM but not in U937 or SUP-T1 cells, while strain IIIB replicates well in both U937 and SUP-T1 cells but not in MDM. (c) Both lymphocyte-tropic and monocyte-tropic strains of HIV-1 replicate well in PBL. (d) The promonocyte cell line, U937, and the T cell line, SUP-T1, differ markedly from primary cells, such as MDM and PBL, in their ability to support the replication of different strains of HIV-1; these cell lines cannot be used as surrogates for primary cells in host range studies of HIV-1 strains.

  5. MCMV-mediated Inhibition of the Pro-apoptotic Bak Protein Is Required for Optimal In Vivo Replication

    PubMed Central

    Fleming, Peter; Kvansakul, Marc; Voigt, Valentina; Kile, Benjamin T.; Kluck, Ruth M.; Huang, David C. S.; Degli-Esposti, Mariapia A.; Andoniou, Christopher E.

    2013-01-01

    Successful replication and transmission of large DNA viruses such as the cytomegaloviruses (CMV) family of viruses depends on the ability to interfere with multiple aspects of the host immune response. Apoptosis functions as a host innate defence mechanism against viral infection, and the capacity to interfere with this process is essential for the replication of many viruses. The Bcl-2 family of proteins are the principle regulators of apoptosis, with two pro-apoptotic members, Bax and Bak, essential for apoptosis to proceed. The m38.5 protein encoded by murine CMV (MCMV) has been identified as Bax-specific inhibitor of apoptosis. Recently, m41.1, a protein product encoded by the m41 open reading frame (ORF) of MCMV, has been shown to inhibit Bak activity in vitro. Here we show that m41.1 is critical for optimal MCMV replication in vivo. Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in Bak−/− mice. Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during in vivo infection. The results show that Bax and Bak mediate non-redundant functions during MCMV infection and that the virus produces distinct inhibitors for each protein to counter the activity of these proteins. PMID:23468630

  6. Porcine circovirus type 2 replication is impaired by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

    SciTech Connect

    Wei Li; Liu Jue

    2009-03-30

    Postweaning multisystemic wasting syndrome, which is primarily caused by porcine circovirus type 2 (PCV2), is an emerging and important swine disease. We have recently shown that PCV2 induces nuclear factor kappa B activation and its activation is required for active replication, but the other cellular factors involved in PCV2 replication are not well defined. The extracellular signal-regulated kinase (ERK) which served as an important component of cellular signal transduction pathways has been shown to regulate many viral infections. In this report, we show that PCV2 activates ERK1/2 in PCV2-infected PK15 cells dependent on viral replication. The PCV2-induced ERK1/2 leads to phosphorylation of the ternary complex factor Elk-1, which kinetically paralleled ERK1/2 activation. Inhibition of ERK activation with U0126, a specific MEK1/2 inhibitor, significantly reduced viral progeny release. Investigations into the mechanism of ERK1/2 regulation revealed that inhibition of ERK activation leads to decreased viral transcription and lower virus protein expression. These data indicate that the ERK signaling pathway is involved in PCV2 infection and beneficial to PCV2 replication in the cultured cells.

  7. Inhibition of Bim enhances replication of varicella-zoster virus and delays plaque formation in virus-infected cells.

    PubMed

    Liu, Xueqiao; Cohen, Jeffrey I

    2014-01-01

    Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication.

  8. Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication.

    PubMed

    Muylaert, Isabella; Elias, Per

    2007-04-13

    Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.

  9. A novel candidate HIV vaccine vector based on the replication deficient Capripoxvirus, Lumpy skin disease virus (LSDV)

    PubMed Central

    2011-01-01

    Background The Capripoxvirus, Lumpy skin disease virus (LSDV) has a restricted host-range and is being investigated as a novel HIV-1 vaccine vector. LSDV does not complete its replication cycle in non-ruminant hosts. Methods The safety of LSDV was tested at doses of 104 and 106 plaque forming units in two strains of immunocompromised mice, namely RAG mice and CD4 T cell knockout mice. LSDV expressing HIV-1 subtype C Gag, reverse transcriptase (RT), Tat and Nef as a polyprotein (Grttn), (rLSDV-grttn), was constructed. The immunogenicity of rLSDV-grttn was tested in homologous prime-boost regimens as well as heterologous prime-boost regimes in combination with a DNA vaccine (pVRC-grttn) or modified vaccinia Ankara vaccine (rMVA-grttn) both expressing Grttn. Results Safety was demonstrated in two strains of immunocompromised mice. In the immunogenicity experiments mice developed high magnitudes of HIV-specific cells producing IFN-gamma and IL-2. A comparison of rLSDV-grttn and rMVA-grttn to boost a DNA vaccine (pVRC-grttn) indicated a DNA prime and rLSDV-grttn boost induced a 2 fold (p < 0.01) lower cumulative frequency of Gag- and RT-specific IFN-γ CD8 and CD4 cells than a boost with rMVA-grttn. However, the HIV-specific cells induced by the DNA vaccine prime rLSDV-grttn boost produced greater than 3 fold (p < 0.01) more IFN- gamma than the HIV-specific cells induced by the DNA vaccine prime rMVA-grttn boost. A boost of HIV-specific CD4 cells producing IL-2 was only achieved with the DNA vaccine prime and rLSDV-grttn boost. Heterologous prime-boost combinations of rLSDV-grttn and rMVA-grttn induced similar cumulative frequencies of IFN- gamma producing Gag- and RT-specific CD8 and CD4 cells. A significant difference (p < 0.01) between the regimens was the higher capacity (2.1 fold) of Gag-and RT-specific CD4 cells to produce IFN-γ with a rMVA-grttn prime - rLSDV-grttn boost. This regimen also induced a 1.5 fold higher (p < 0.05) frequency of Gag- and RT-specific CD

  10. Pyrazinoic acid and its n-propyl ester inhibit fatty acid synthase type I in replicating tubercle bacilli.

    PubMed

    Zimhony, Oren; Vilchèze, Catherine; Arai, Masayoshi; Welch, John T; Jacobs, William R

    2007-02-01

    The activity of different analogs of pyrazinamide on Mycobacterium tuberculosis fatty acid synthase type I (FASI) in replicating bacilli was studied. Palmitic acid biosynthesis was diminished by 96% in bacilli treated with n-propyl pyrazinoate, 94% in bacilli treated with 5-chloro-pyrazinamide, and 97% in bacilli treated with pyrazinoic acid, the pharmacologically active agent of pyrazinamide. We conclude that the minimal structure of pyrazine ring with an acyl group is sufficient for FASI inhibition and antimycobacterial activity.

  11. Inhibition of simian immunodeficiency virus (SIV) replication by CD8+ cells of SIV-infected rhesus macaques: implications for immunopathogenesis.

    PubMed

    Blackbourn, D J; Chuang, L F; Killam, K F; Chuang, R Y

    1994-08-01

    The ability of the CD8+ cells from simian immunodeficiency virus (SIV)-infected rhesus macaques to inhibit SIV replication was investigated. Inhibition was produced by a heat-stable soluble factor of molecular size greater than 10kDa. CD8+ supernatants from some macaques were found not only to suppress SIV growth but also to be cytolytic toward both infected and uninfected CD4+ cells. Such indiscriminate CD8+ cell-mediated cell killing may therefore account for DC4+ cell depletion in certain SIV-infected macaques.

  12. HLA-B*57 Elite Suppressor and Chronic Progressor HIV-1 Isolates Replicate Vigorously and Cause CD4+ T Cell Depletion in Humanized BLT Mice

    PubMed Central

    Salgado, Maria; Swanson, Michael D.; Pohlmeyer, Christopher W.; Buckheit, Robert W.; Wu, Jin; Archin, Nancie M.; Williams, Thomas M.; Margolis, David M.; Siliciano, Robert F.

    2014-01-01

    ABSTRACT Elite controllers or suppressors (ES) are HIV-1-infected patients who maintain undetectable viral loads without antiretroviral therapy. The mechanism of control remains unclear, but the HLA-B*57 allele is overrepresented in cohorts of these patients. However, many HLA-B*57 patients develop progressive disease, and some studies have suggested that infection with defective viruses may be the cause of the lack of high levels of virus replication and disease progression in ES. We therefore performed a comprehensive comparative in vivo and in vitro characterization of viruses isolated from well-defined ES. For this purpose, we first performed full-genome sequence analysis and in vitro fitness assays on replication-competent isolates from HLA-B*57 ES and HLA-B*57 chronic progressors (CPs). Under our experimental conditions, we found that isolates from ES and CPs can replicate in vitro. However, since inherently these assays involve the use of unnaturally in vitro-activated cells, we also investigated the replication competence and pathogenic potential of these HIV isolates in vivo using humanized BLT mice. The results from these analyses demonstrate that virus isolates from ES are fully replication competent in vivo and can induce peripheral and systemic CD4 T cell depletion. These results provide the first direct in vivo evidence that viral fitness does not likely determine clinical outcome in HLA-B*57 patients and that elite suppressors can control replication-competent, fully pathogenic viruses. A better understanding of the immunological bases of viral suppression in ES will serve to inform novel approaches to preventive and therapeutic HIV vaccine design. IMPORTANCE Elite suppressors are HIV-1-infected patients who have undetectable levels of viremia despite not being on antiviral drugs. One of the most fundamental questions about this phenomenon involves the mechanism of control. To address this question, we isolated virus from elite suppressors and from

  13. Genetic Subtype-Independent Inhibition of Human Immunodeficiency Virus Type 1 Replication by CC and CXC Chemokines

    PubMed Central

    Trkola, Alexandra; Paxton, William A.; Monard, Simon P.; Hoxie, James A.; Siani, Michael A.; Thompson, Darren A.; Wu, Lijun; Mackay, Charles R.; Horuk, Richard; Moore, John P.

    1998-01-01

    We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1α (MIP-1α), MIP-1β, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1α against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1β, MIP-1α. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1β, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1β, MIP-1α. PMID:9420238

  14. Cytotoxicity of p-tyrosol and its derivatives may correlate with the inhibition of DNA replication initiation.

    PubMed

    Ahn, Eun-Young; Jiang, Yahong; Zhang, Yanjun; Son, Eun Mi; You, Song; Kang, Shin-Won; Park, Jang-Su; Jung, Jee H; Lee, Burm-Jong; Kim, Dong-Kyoo

    2008-02-01

    p-Tyrosol is a phenolic compound present in different dietary sources that can exert mild antioxidant properties based on in vitro and in vivo studies. In our study, two p-tyrosol derivatives (p-tyrosyl gallate and p-tyrosyl acetate) were synthesized and compared together with p-tyrosol and gallic acid for their cytotoxic activities on human cancer cells. p-Tyrosyl gallate had the most potent cytotoxicity and the major cytotoxic mechanism of its action was studied. We found that in HeLa cells, p-tyrosyl gallate can effectively induce cell cycle arrest during S phase and inhibited in vitro simian virus (SV40 DNA) replication. In addition, p-tyrosyl gallate can inhibit three important functional replication proteins (topoisomerase I, RPA and pol alpha-primase), especially pol alpha-primase. These results suggest that p-tyrosyl gallate-induced cell cycle arrest during S phase correlates with the inhibition of DNA replication. Pol alpha-primase may be the main target molecule. Taken together, we suggest that p-tyrosyl gallate is a strong anticancer drug candidate that warrants further investigation.

  15. Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15

    PubMed Central

    Zhao, Zhong-Fu; Yang, Hui; Han, De-Wu; Zhao, Long-Feng; Zhang, Guo-Ying; Zhang, Yun; Liu, Ming-She

    2006-01-01

    AIM: To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA (siRNA) pGenesil-HBV X targeting HBV X gene region into HepG2.2.15 cells. METHODS: pGenesil-HBV X was constructed and transfected into HepG2.2.15 cells via lipofection. HBV antigen secretion was determined 24, 48, and 72 h after transfection by time-resolved immunofluorometric assays (TRFIA). HBV replication was examined by fluorescence quantitative PCR, and the expression of cytoplasmic viral proteins was determined by immunohistochemistry. RESULTS: The secretion of HBsAg and HBeAg into the supernatant was found to be inhibited by 28.5% and 32.2% (P < 0.01), and by 38.67% (P < 0.05) and 42.86% (P < 0.01) at 48 h and 72 h after pGenesil-HBV X transfection, respectively. Immunohistochemical staining for cytoplasmic HBsAg showed a similar decline in HepG2.2.15 cells 48 h after transfection. The number of HBV genomes within culture supernatants was also significantly decreased 48 h and 72 h post-transfection as quantified by fluorescence PCR (P < 0.05). CONCLUSION: In HepG2.2.15 cells, HBV replication and expression is inhibited by vector-based siRNA pGenesil-HBV X targeting the HBV X coding region. PMID:17009407

  16. Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication

    PubMed Central

    Huang, Hanxia; Falgout, Barry; Takeda, Kazuyo; Yamada, Kenneth M.; Dhawan, Subhash

    2017-01-01

    We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of > 90% in ZIKV replication, with little toxicity to infected cells. Silencing expression of HO-1 or its upstream regulatory gene, nuclear factor erythroid-related factor 2 (Nrf2), attenuated hemin-induced suppression of ZIKV infection, suggesting an important role for induction of these intracellular mediators in retarding ZIKV replication. The inverse correlation between hemin-induced HO-1 levels and ZIKV replication provides a potentially useful therapeutic modality based on stimulation of an innate cellular response against Zika virus infection. PMID:28068513

  17. Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication.

    PubMed

    Huang, Hanxia; Falgout, Barry; Takeda, Kazuyo; Yamada, Kenneth M; Dhawan, Subhash

    2017-03-01

    We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of >90% in ZIKV replication, with little toxicity to infected cells. Silencing expression of HO-1 or its upstream regulatory gene, nuclear factor erythroid-related factor 2 (Nrf2), attenuated hemin-induced suppression of ZIKV infection, suggesting an important role for induction of these intracellular mediators in retarding ZIKV replication. The inverse correlation between hemin-induced HO-1 levels and ZIKV replication provides a potentially useful therapeutic modality based on stimulation of an innate cellular response against Zika virus infection.

  18. Parental LTRs are important in a construct of a stable and efficient replication-competent infectious molecular clone of HIV-1 CRF08_BC.

    PubMed

    Zhang, Qiwei; Zhang, Xiaomin; Wu, Hao; Seto, Donald; Zhang, Hao-Jie; Chen, Zhiwei; Wan, Chengsong; Zheng, Bo-Jian

    2012-01-01

    Circulating recombinant forms (CRFs) of HIV-1 have been identified in southern China in recent years. CRF08_BC is one of the most predominant subtypes circulating in China. In order to study HIV subtype biology and to provide a tool for biotechnological applications, the first full-length replication-competent infectious molecular clone harboring CRF08_BC is reported. The construction of this clone pBRGX indicates that a moderate-copy number vector is required for its amplification in E. coli. In addition, it is shown that the parental CRF08_BC LTRs are important for generating this efficient replication-competent infectious clone. These observations may aid in the construction of infectious clones from other subtypes. Both the pBRGX-derived virus and its parental isolate contain CCR5 tropism. Their full-length genomes were also sequenced, analyzed, compared and deposited in GenBank (JF719819 and JF719818, respectively). The availability of pBRGX as the first replication-competent molecular clone of CRF08_BC provides a useful tool for a wide range of studies of this newly emergent HIV subtype, including the development of HIV vaccine candidates, antiviral drug screening and drug resistance analysis.

  19. Inhibiting the HIV Integration Process: Past, Present, and the Future

    PubMed Central

    2013-01-01

    HIV integrase (IN) catalyzes the insertion into the genome of the infected human cell of viral DNA produced by the retrotranscription process. The discovery of raltegravir validated the existence of the IN, which is a new target in the field of anti-HIV drug research. The mechanism of catalysis of IN is depicted, and the characteristics of the inhibitors of the catalytic site of this viral enzyme are reported. The role played by the resistance is elucidated, as well as the possibility of bypassing this problem. New approaches to block the integration process are depicted as future perspectives, such as development of allosteric IN inhibitors, dual inhibitors targeting both IN and other enzymes, inhibitors of enzymes that activate IN, activators of IN activity, as well as a gene therapy approach. PMID:24025027

  20. Inhibition of HIV-1 gp41 expression with hammerhead ribozymes.

    PubMed

    Fedoruk-Wyszomirska, Agnieszka; Szymański, Maciej; Głodowicz, Paweł; Gabryelska, Marta; Wyszko, Eliza; Estrin, William J; Barciszewski, Jan

    2015-10-01

    Despite great progress in the treatment of AIDS, HIV-1 remains one of the major concerns as a human pathogen. One of the therapeutic strategies against viral infections is the application of catalytic ribonucleic acids (ribozymes) that can significantly reduce expression of a target gene by site-specific hydrolysis of its mRNA. In the present paper, we report a study on the activity of several variants of hammerhead ribozymes targeting a conserved region within mRNA encoding HIV-1 envelope glycoprotein gp41. On the basis of the data from in vitro assays and gene silencing in the cultured cells, we propose a new hammerhead ribozyme targeting the gp41-encoding sequence that can be potentially used as a therapeutic agent in AIDS treatment. Moreover, we demonstrate that the hydrolytic activity of the ribozyme in the intracellular environment cannot be inferred solely from the results of in vitro experiments.

  1. Synthetic α-Hydroxytropolones Inhibit Replication of Wild-Type and Acyclovir-Resistant Herpes Simplex Viruses

    PubMed Central

    Ireland, Peter J.; Tavis, John E.; D'Erasmo, Michael P.; Hirsch, Danielle R.; Murelli, Ryan P.; Cadiz, Mark M.; Patel, Bindi S.; Gupta, Ankit K.; Edwards, Tiffany C.; Korom, Maria; Moran, Eileen A.

    2016-01-01

    Herpes simplex virus 1 (HSV-1) and HSV-2 remain major human pathogens despite the development of anti-HSV therapeutics as some of the first antiviral drugs. Current therapies are incompletely effective and frequently drive the evolution of drug-resistant mutants. We recently determined that certain natural troponoid compounds such as β-thujaplicinol readily suppress HSV-1 and HSV-2 replication. Here, we screened 26 synthetic α-hydroxytropolones with the goals of determining a preliminary structure-activity relationship for the α-hydroxytropolone pharmacophore and providing a starting point for future optimization studies. Twenty-five compounds inhibited HSV-1 and HSV-2 replication at 50 μM, and 10 compounds inhibited HSV-1 and HSV-2 at 5 μM, with similar inhibition patterns and potencies against both viruses being observed. The two most powerful inhibitors shared a common biphenyl side chain, were capable of inhibiting HSV-1 and HSV-2 with a 50% effective concentration (EC50) of 81 to 210 nM, and also strongly inhibited acyclovir-resistant mutants. Moderate to low cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] of 50 to >100 μM). Therapeutic indexes ranged from >170 to >1,200. These data indicate that troponoids and specifically α-hydroxytropolones are a promising lead scaffold for development as anti-HSV drugs provided that toxicity can be further minimized. Troponoid drugs are envisioned to be employed alone or in combination with existing nucleos(t)ide analogs to suppress HSV replication far enough to prevent viral shedding and to limit the development of or treat nucleos(t)ide analog-resistant mutants. PMID:26787704

  2. Replication-Dependent Radiosensitization of Human Glioma Cells by Inhibition of Poly(ADP-Ribose) Polymerase: Mechanisms and Therapeutic Potential

    SciTech Connect

    Dungey, Fiona A.; Loeser, Dana A.; Chalmers, Anthony J.

    2008-11-15

    Purpose: Current treatments for glioblastoma multiforme are inadequate and limited by the radiation sensitivity of normal brain. Because glioblastoma multiforme are rapidly proliferating tumors within nondividing normal tissue, the therapeutic ratio might be enhanced by combining radiotherapy with a replication-specific radiosensitizer. KU-0059436 (AZD2281) is a potent and nontoxic inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) undergoing a Phase II clinical trial as a single agent. Methods and Materials: Based on previous observations that the radiosensitizing effects of PARP inhibition are more pronounced in dividing cells, we investigated the mechanisms underlying radiosensitization of human glioma cells by KU-0059436, evaluating the replication dependence of this effect and its therapeutic potential. Results: KU-0059436 increased the radiosensitivity of four human glioma cell lines (T98G, U373-MG, UVW, and U87-MG). Radiosensitization was enhanced in populations synchronized in S phase and abrogated by concomitant exposure to aphidicolin. Sensitization was further enhanced when the inhibitor was combined with a fractionated radiation schedule. KU-0059436 delayed repair of radiation-induced DNA breaks and was associated with a replication-dependent increase in {gamma}H2AX and Rad51 foci. Conclusion: The results of our study have shown that KU-0059436 increases radiosensitivity in a replication-dependent manner that is enhanced by fractionation. A mechanism is proposed whereby PARP inhibition increases the incidence of collapsed replication forks after ionizing radiation, generating persistent DNA double-strand breaks. These observations indicate that KU-0059436 is likely to enhance the therapeutic ratio achieved by radiotherapy in the treatment of glioblastoma multiforme. A Phase I clinical trial is in development.

  3. Neutralizing antibodies inhibit HIV-1 infection of plasmacytoid dendritic cells by an FcγRIIa independent mechanism and do not diminish cytokines production.

    PubMed

    Lederle, Alexandre; Su, Bin; Holl, Vincent; Penichon, Julien; Schmidt, Sylvie; Decoville, Thomas; Laumond, Géraldine; Moog, Christiane

    2014-08-18

    Plasmacytoid dendritic cells (pDC) expressing FcγRIIa are antigen-presenting cells able to link innate and adaptive immunity and producing various cytokines and chemokines. Although highly restricted, they are able to replicate HIV-1. We determined the activity of anti-HIV-1 neutralizing antibodies (NAb) and non-neutralizing inhibitory antibodies (NNIAb) on the infection of primary pDC by HIV-1 primary isolates and analyzed cytokines and chemokines production. Neutralization assay was performed with primary pDC in the presence of serial antibodies (Ab) concentrations. In parallel, we measured the release of cytokines and chemokines by ELISA and CBA Flex assay. We found that NAb, but not NNIAb, inhibit HIV-1 replication in pDC. This inhibitory activity was lower than that detected for myeloid dendritic cells (mDC) infection and independent of FcγRIIa expressed on pDC. Despite the complete protection, IFN-α production was detected in the supernatant of pDC treated with NAb VRC01, 4E10, PGT121, 10-1074, 10E8, or polyclonal IgG44 but not with NAb b12. Production of MIP-1α, MIP-1β, IL-6, and TNF-α by pDC was also maintained in the presence of 4E10, b12 and VRC01. These findings suggest that pDC can be protected from HIV-1 infection by both NAb and IFN-α release triggered by the innate immune response during infection.

  4. Knockdown of MAP4 and DNAL1 produces a post-fusion and pre-nuclear translocation impairment in HIV-1 replication

    SciTech Connect

    Gallo, Daniel E. Hope, Thomas J.

    2012-01-05

    DNAL1 and MAP4 are both microtubule-associated proteins. These proteins were identified as HIV-1 dependency factors in a screen with wild-type HIV-1. In this study we demonstrate that knockdown using DNAL1 and MAP4 siRNAs and shRNAs inhibits HIV-1 infection regardless of envelope. Using a fusion assay, we show that DNAL1 and MAP4 do not impact fusion. By assaying for late reverse transcripts and 2-LTR circles, we show that DNAL1 and MAP4 inhibit both by approximately 50%. These results demonstrate that DNAL1 and MAP4 impact reverse transcription but not nuclear translocation. DNAL1 and MAP4 knockdown cells do not display cytoskeletal defects. Together these experiments indicate that DNAL1 and MAP4 may exert their functions in the HIV life cycle at reverse transcription, prior to nuclear translocation.

  5. Knockdown of MAP4 and DNAL1 produces a post-fusion and pre-nuclear translocation impairment in HIV-1 replication.

    PubMed

    Gallo, Daniel E; Hope, Thomas J

    2012-01-05

    DNAL1 and MAP4 are both microtubule-associated proteins. These proteins were identified as HIV-1 dependency factors in a screen with wild-type HIV-1. In this study we demonstrate that knockdown using DNAL1 and MAP4 siRNAs and shRNAs inhibits HIV-1 infection regardless of envelope. Using a fusion assay, we show that DNAL1 and MAP4 do not impact fusion. By assaying for late reverse transcripts and 2-LTR circles, we show that DNAL1 and MAP4 inhibit both by approximately 50%. These results demonstrate that DNAL1 and MAP4 impact reverse transcription but not nuclear translocation. DNAL1 and MAP4 knockdown cells do not display cytoskeletal defects. Together these experiments indicate that DNAL1 and MAP4 may exert their functions in the HIV life cycle at reverse transcription, prior to nuclear translocation.

  6. Antiviral effects of autologous CD4 T cells genetically modified with a conditionally replicating lentiviral vector expressing long antisense to HIV.

    PubMed

    Tebas, Pablo; Stein, David; Binder-Scholl, Gwendolyn; Mukherjee, Rithun; Brady, Troy; Rebello, Tessio; Humeau, Laurent; Kalos, Michael; Papasavvas, Emmanouil; Montaner, Luis J; Schullery, Daniel; Shaheen, Farida; Brennan, Andrea L; Zheng, Zhaohui; Cotte, Julio; Slepushkin, Vladimir; Veloso, Elizabeth; Mackley, Adonna; Hwang, Wei-Ting; Aberra, Faten; Zhan, Jenny; Boyer, Jean; Collman, Ronald G; Bushman, Frederic D; Levine, Bruce L; June, Carl H

    2013-02-28

    We report the safety and tolerability of 87 infusions of lentiviral vector–modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engra