An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.

Antiviral host defence peptides.

The on going global mortality and morbidity associated with viral pathogens highlights the need for the continued development of effective, novel antiviral molecules. The antiviral activity of cationic host defence peptides is of significant interest as novel therapeutics for treating viral infection and predominantly due to their broad spectrum antiviral activity. These peptides also display powerful immunomodulatory activity and are key mediators of inflammation. Therefore, they offer a significant opportunity to inform the development of novel therapeutics for treating viral infections by either directly targeting the pathogen or by enhancing the innate immune response. In this chapter, we review the antiviral activity of cathelicidins and defensins, and examine the potential for these peptides to be used as novel antiviral agents.

Oncolytic adenoviruses for treatment of ovarian cancer

Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible.

Molecular mechanism for the specific inhibition of reverse transcriptase of rous sarcoma virus by the copper complexes of isonicotinic acid hydrazide

Isonicotinic acid hydrazide (isoniazid), one of the most potent antitubercular drugs, was recently shown, in our laboratory, to form two different complexes with copper, depending upon the oxidation state of the metal ion. Both the complexes have been shown to possess antiviral activity against Rous sarcoma virus, an RNA tumor virus. The antiviral activity of the complexes has been attributed to their ability to inhibit the endogenous reverse transcriptase activity of RSV. More recent studies in our laboratory indicate that both these complexes inhibit both endogenous and exogenous reactions. As low a final concentration as 50 μM of the cupric and the cuprous complexes inhibits the endogenous reaction to the extent of 93 and 75 per cent respectively. Inhibition of the exogenous reaction varies with the templates. The inhibition can be reversed by either β-mercaptoethanol or ethylene-diamine-tetra-acetic acid. The specificity of this inhibition has been ascertained by using a synthetic primer-template, −(dG)not, vert, similar15−(rCm)n, which is highly specific for reverse transcriptases. The inhibition is found to be template specific. The studies carried out, using various synthetic primer-templates, show the inhibition of both the steps of reverse transcription by the copper complexes of isoniazid.

Makrodomeeni-inhibiittorit antiviraalisina yhdisteinä

Alfavirukset ovat positiivissäkeisiä RNA-viruksia, jotka kuuluvat Togaviridea –heimoon. Alfaviruksia levittävät Aedes –suvun hyttyset ja niitä esiintyy Etelämanteretta lukuunottamatta kaikilla mantereilla. Alfaviruksia on tähän mennessä löydetty 29 lajia ja ne voidaan jakaa uuden ja vanhan maailman viruksiin niiden maantieteellisen esiintyvyyden ja taudinaiheuttamiskyvyn mukaan. Chikunkunyavirus (CHIKV) on yksi vanhan maailman alfaviruksista, jota esiintyy muun muassa Afrikassa ja Aasiassa. Ilmaston lämmettyä se on leviämässä myös eteläiseen Eurooppaan. Ihmisessä se aiheuttaa muun muassa kuumetta, päänsärkyä, ihottumaa ja niveltulehdusta, joka voi kestää useita vuosia ja ne voivat olla hyvinkin kivuliaita. Pienillä lapsilla chikungunya on todettu aiheuttavan myös neurologisia oireita kuten aivotulehdusta. Alfaviruksen genomi koodaa neljää rakenneproteiinia ja neljää replikaatioproteiinia. Replikaatioproteiineista nsP3 sisältää makrodomeeniosan. Makrodomeeniproteiinit ovat eliökunnassa konservoituneita, mutta makrodomeeniproteiinien tarkkaa merkitystä ei vielä tunneta. Makrodomeenien on osoitettu sitovan ADP-riboosia ja sen johdannaisia ja alfaviruksen nsP3-proteiinin on osoitettu olevan tärkeä osa viruksen replikaatiossa. Tutkimuksen tavoitteena oli tutkia makrodomeeniproteiiniin sitoutuvien yhdisteiden käyttöä antiviraalisena yhdisteinä. Tietokonemallinnuksella valittiin antiviraalitutkimuksiin 45 yhdistettä, joiden oletettiin sitoutuvan makrodomeeniproteiiniin. Kilpailevassa sitoutumiskokeessa viisi yhdistettä esti yli 50 % poly-ADP-riboosia (PAR) sitoutumasta MDO1-makrodomeeniproteiiniin, jolla tietokonemallinnus oli tehty. SFV-makrodomeeniproteiinilla tehdyssä kokeessa vain yksi yhdiste esti yli 50 % poly-ADP-riboosin sitoutumisen. SFV-antiviraalikokeessa seitsemällä yhdisteellä inhibitioprosentti oli yli 50 %. Näillä yhdisteillä ei kuitenkaan ollut merkittävää vaikutusta poly-ADP-riboosin sitoutumisen estossa. CHIKV-replikonikokeessa yli 50 % inhibitioprosentti oli viidellä yhdisteellä. Muiden mahdollisia vaikutusmekanismeja tutkittiin selvittämällä estävätkö yhdisteet virusta pääsemästä solun sisään. Tässä kokeessa tutkituista yhdisteistä lähes kaikilla oli vaikutusta viruksen soluun pääsyn estossa. Yleisesti ottaen kyky estää PAR:n sitoutuminen makrodomeeniproteiineihin ja antiviraaliset vaikutukset eivät korreloineet keskenään tutkittavilla yhdisteillä. Vaikka antiviraalista vaikutusta omaavat yhdisteet eivät osoittaneetkaan makrodomeeni-inhibiitiota, työssä löydettiin potentiaalisia antiviraalisia yhdisteitä joiden käyttö viruksen soluun pääsyn estäjinä antaa aihetta jatkotutkimuksille.

Structural characterization and in vitro biomedical activities of sulfated chitosan from Sepia pharaonis

A low molecular weight sulfated chitosan (SP-LMWSC) was isolated from the cuttlebone of Sepia pharaonis. Elemental analysis established the presence of C, H and N. The sulfation of SP-LMWSC was confirmed by the presence of characteristic peaks in FT-IR and FT-Raman spectra. The thermal properties of SP-LMWSC were studied by thermogravimetric analysis and differential scanning calorimetry. Electrolytic conductivity of SP-LMWSC was measured by cyclic voltammetry and the molecular weight was determined by MALDI-TOF/MS. The molecular structure and sulfation sites of SP-LMWSC were unambiguously confirmed using H-1,C-13, 2D COSY and 2D HSQC NMR spectroscopy. SP-LMWSC exhibited increased anticoagulant activity in avian blood by delaying coagulation parameters and displayed cytostatic activity by inhibiting the migration of avian leucocytes. SP-LMWSC demonstrated avian antiviral activity by binding to Newcastle disease virus receptors at a low titer value of 1/64. These findings suggested that SP-LMWSC isolated from an industrial discard holds immense potentials as carbohydrate based pharmaceuticals in future. (C) 2015 Elsevier B.V. All rights reserved.

Trichosanthin suppresses the elevation of p38 MAPK, and Bcl-2 induced by HSV-1 infection in Vero cells

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) effective against HIV-1 and HSV-1 replication. The mechanism of its antiviral activity is not clear. Many believe that it is related to ribosome inactivation. Some RIPs and viral infectio

Anti-HIV Activities of the Compounds Isolated from Polygonum cuspidatum and Polygonum multiflorum

The 70% EtOH extract of Polygonum cuspidatum showed inhibitory action against HIV-1-induced syncytium formation at non-cytotoxic concentrations in vitro with a 50% effective concentration (EC50) of 13.94 +/- 3.41 mu g/mL. Through bioactivity-guided fractionation, 20 phenolic compounds, including eight stilbenoids, were isolated from the roots of Polygonum cuspidatum, and their anti-HIV-1 activities were evaluated. Results showed that compounds 1, 13, 14, and 16 demonstrated fairly strong antiviral activity against HIV-1-induced cytopathic effects in C8166 lymphocytes at non-cytotoxic concentrations, with EC50 values of 4.37 +/- 1.96 mu g/mL, 19.97 +/- 5.09, 14.4 +/- 1.34 mu g/mL, and 11.29 +/- 6.26 mu g/mL and therapeutic index (TI) values of 8.12, > 10.02, > 13.89, and > 17.71, respectively. Other compounds showed either weak or no effects. Compound 6 also showed weak inhibition (153.42 +/- 19.25 mu g/mL); however, it possesses very good water solubility and showed almost no cytotoxicity (> 2000 mu g/mL), therefore achieving a fairly good TI (13.04). The activities of the two compounds (3 and 18) from Polygonum multiflorum were also assayed. The relationship between molecular structures and their bioactivities was also discussed.

The Anti-HIV Actions of 7-and 10-Substituted Camptothecins

Camptothecin (CPT), a traditional anti-tumor drug, has been shown to possess anti-HIV-1 activity. To increase the antiviral potency, the anti-HIV activities of two CPT derivatives, 10-hydroxy-CPT and 7-hydroxymethyl-CPT, were evaluated in vitro. The therapy index (TI) of CPT, 10-hydroxy-CPT and 7-hydroxymethyl-CPT against HIV-1(IIIB) in C8166 were 24.2, 4.2 and 198.1, and against clinical isolated strain HIV-1(KM018) in PBMC were 10.3, 3.5 and 66.0, respectively. While the TI of CPT, 10-hydroxy-CPT and 7-hydroxymethyl-CPT against HIV-2(CBL-20) were 34.5, 10.7 and 317.0, respectively, and the TI of the three compounds against HIV-2(ROD) showed the similar values. However, when the antiviral mechanisms were considered, we found there was no inhibition of 7-hydroxymethyl-CPT on viral cell-to-cell transmission, and was no inhibition on reverse transcriptase, protease or integrase in cell-free systems. 7-Hydroxymethyl-CPT showed no selective killing of chronically infected cells after 3 days of incubation. In conclusion, 7-hydroxymethyl-CPT showed more potent anti-HIV activity, while 10-hydroxy-CPT had less efficient activity, compared with the parent CPT. Though the antiviral mechanisms remain to be further elucidated; the modification of -OH residues at C-7 of CPT could enhance the antiviral activity, while of -OH residues at C-10 of CPT had decreased the antiviral activity, which provides the preliminary modification strategy for anti-viral activities enhancement of this compound.

Enhanced grass carp reovirus resistance of Mx-transgenic rare minnow (Gobiocypris rarus)

In the interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins have direct antiviral activity and inhibit a wide range of viruses by blocking an early stage of the viral genome replication cycle. However, antiviral activity of piscine Mx remains unclear in vivo. In the present study, an Mx-like gene was cloned, characterized and gene-transferred in rare minnow Gobiocypris rarus, and its antiviral activity was confirmed in vivo. The full length of the rare minnow Mx-like cDNA is 2241 bp in length and encodes a polypeptide of 625 amino acids with an estimated molecular mass of 70.928 kDa and a predicted isoelectric point of 7.33. Analysis of the deduced amino acid sequence indicated that the mature peptide contains an amino-terminal tripartite GTP-binding motif, a dynamin family signature sequence, a GTPase effector domain and two carboxy-terminal leucine zipper motifs, and is the most similar to the crucian carp (Carassius auratus) Mx3 sequence with an identity of 89%. Both P0 and F1 generations of Mx-transgenic rare minnow demonstrated very significantly high survival rate to GCRV infection (P < 0.01). The mRNA expression of Mx gene was consistent with survival rate in F1 generation. The virus yield was also concurrent with survival time using electron microscope technology. Rare minnow has Mx gene(s) of its own but introducing more Mx gene improves their resistance to GCRV. Mx-transgenic rare minnow might contribute to control the GCRV diseases. (C) 2008 Published by Elsevier Ltd.

Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

To study the influence of Hypericum perforatum extract (HPE) on piglets infected with porcine respiratory and reproductive syndrome virus (PRRSV), enzyme-labeled immunosorbent assay (ELISA) and cytopathic effect (CPE) were used to determine in vitro whether HPE could induce swine pulmonary alveolar macrophages (PAMs) to secrete IFN-gamma, and whether PRRSV titers in PAMs were affected by the levels of HPE-induced IFN-gamma. HPE (200 mg kg(-1)) was administrated by oral gavage to piglets infected with the PRRSV in vivo to observe whether HPE affected the viremia, lung viral titers, and weight gain of piglets infected with PRRSV. The results showed that HPE was capable of inducing PAMs to produce IFN-gamma in a dose dependent manner and HPE pretreatment was capable of significantly reducing PRRSV viral titers in PAMs (P<0.01). Administration of HPE to the PRRSV-infected animals significantly (P<0.05) reduced viremia over time as compared with the PRRSV-infected animals. But there was not significant decrease in lung viral titers at day 21 post-infection between the HPE-treated animals and the PRRSV-infected control piglets. There were no significant differences in weight gain over time among the HPE-treatment animals, the normal control, and the HPE control animals. The PRRSV-infected animals caused significant (P<0.01) growth retardation as compared with the HPE controls and the normal piglets. It suggested that HPE might be an effective novel therapeutic approach to diminish the PRRSV-induced disease in swine.

Vírus e retrovírus

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.