Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae)

Microsporogenesis and pollen development were analyzed in a tetraploid (2n = 4x = 36) accession of the forage grass Brachiaria jubata (BRA 007820) from the Embrapa Beef Cattle Brachiaria collection that showed partial male sterility. Microsporocytes and pollen grains were prepared by squashing and staining with 0.5% propionic carmine. The meiotic process was typical of polyploids, with precocious chromosome migration to the poles and laggards in both meiosis I and II, resulting in tetrads with micronuclei in some microspores. After callose dissolution, microspores were released into the anther locule and appeared to be normal. Although each microspore initiated its differentiation into a pollen grain, in 11.1% of them nucleus polarization was not observed, i.e., pollen mitosis I was symmetric and the typical hemispherical cell plate was not detected. After a central cytokinesis, two equal-sized cells showing equal chromatin condensation and the same nuclear shape and size were formed. Generative cells and vegetative cells could not be distinguished. These cells did not undergo the second pollen mitosis and after completion of pollen wall synthesis each gave rise to a sterile and uninucleate pollen grain. The frequency of abnormal pollen mitosis varied among flowers and also among inflorescences. All plants were equally affected. The absence of fertile sperm cells in a considerable amount of pollen grains in this accession of B. jubata may compromise its use in breeding and could explain, at least in part, why seed production is low when compared with the amount of flowers per raceme.

Human T-cell lymphotropic virus type I (HTLV-I) proviral DNA viral load among asymptomatic patients and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis

To evaluate the human T-cell lymphotropic virus type I (HTLV-I) proviral DNA load among asymptomatic HTLV-I-infected carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), real time PCR using TaqMan probes for the pol gene was performed in two million peripheral blood mononuclear cells (PBMC). The albumin gene was the internal genomic control and MT2 cells were used as positive control. The results are reported as copies/10,000 PBMC, and the detection limit was 10 copies. A total of 89 subjects (44 HAM/TSP and 45 healthy HTLV-I-infected carriers) followed up at the Institute of Infectious Diseases "Emilio Ribas" and in the Neurology Division of Hospital of Clínicas were studied. The asymptomatic HTLV-I-infected carriers had a median number of 271 copies (ranging from 5 to 4756 copies), whereas the HAM/TSP cases presented a median of 679 copies (5-5360 copies) in 10,000 PBMC. Thus, HAM/TSP patients presented a significantly higher HTLV-I proviral DNA load than healthy HTLV-I carriers (P = 0.005, one-way Mann-Whitney test). As observed in other persistent infections, proviral DNA load quantification may be an important tool for monotoring HTLV-I-infected subjects. However, long-term follow-up is necessary to validate this assay in the clinical setting.

Dexamethasone-induced reactivation of bovine herpesvirus type 5 latent infection in experimentally infected rabbits results in a broader distribution of latent viral DNA in the brain

Bovine herpesvirus type 5 (BHV-5) is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8) or submitted to dexamethasone treatment (2.6 mg kg-1 day-1, im, for 5 days) and euthanized 60 days later (group B, N = 7) for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3) submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8), frequently in cerebellum (5/8), anterior cerebral cortex and pons-medulla (3/8) and occasionally in dorsolateral (2/8), ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8). Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7) and posterior cerebral cortices (5/7), pons-medulla (6/7), thalamus (4/7), and midbrain (3/7). In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbit's brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.

A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98%) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

Participation of the cholinergic system in the ethanol-induced suppression of paradoxical sleep in rats

Sleep disturbance is among the many consequences of ethanol abuse in both humans and rodents. Ethanol consumption can reduce REM or paradoxical sleep (PS) in humans and rats, respectively. The first aim of this study was to develop an animal model of ethanol-induced PS suppression. This model administered intragastrically (by gavage) to male Wistar rats (3 months old, 200-250 g) 0.5 to 3.5 g/kg ethanol. The 3.5 g/kg dose of ethanol suppressed the PS stage compared with the vehicle group (distilled water) during the first 2-h interval (0-2 h; 1.3 vs 10.2; P < 0.001). The second aim of this study was to investigate the mechanisms by which ethanol suppresses PS. We examined the effects of cholinergic drug pretreatment. The cholinergic system was chosen because of the involvement of cholinergic neurotransmitters in regulating the sleep-wake cycle. A second set of animals was pretreated with 2.5, 5.0, and 10 mg/kg pilocarpine (cholinergic agonist) or atropine (cholinergic antagonist). These drugs were administered 1 h prior to ethanol (3.5 g/kg) or vehicle. Treatment with atropine prior to vehicle or ethanol produced a statistically significant decrease in PS, whereas pilocarpine had no effect on minutes of PS. Although the mechanism by which ethanol induces PS suppression is not fully understood, these data suggest that the cholinergic system is not the only system involved in this interaction.

Differential gene expression profiles of hepatocellular carcinomas associated or not with viral infection

Chronic hepatitis B (HBV) and C (HCV) virus infections are the most important factors associated with hepatocellular carcinoma (HCC), but tumor prognosis remains poor due to the lack of diagnostic biomarkers. In order to identify novel diagnostic markers and therapeutic targets, the gene expression profile associated with viral and non-viral HCC was assessed in 9 tumor samples by oligo-microarrays. The differentially expressed genes were examined using a z-score and KEGG pathway for the search of ontological biological processes. We selected a non-redundant set of 15 genes with the lowest P value for clustering samples into three groups using the non-supervised algorithm k-means. Fisher’s linear discriminant analysis was then applied in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Different transcriptional levels of genes were identified in HCC of different etiologies and from different HCC samples. When comparing HBV-HCC vs HCV-HCC, HBV-HCC/HCV-HCC vs non-viral (NV)-HCC, HBC-HCC vs NV-HCC, and HCV-HCC vs NV-HCC of the 58 non-redundant differentially expressed genes, only 6 genes (IKBKβ, CREBBP, WNT10B, PRDX6, ITGAV, and IFNAR1) were found to be associated with hepatic carcinogenesis. By combining trios, classifiers could be generated, which correctly classified 100% of the samples. This expression profiling may provide a useful tool for research into the pathophysiology of HCC. A detailed understanding of how these distinct genes are involved in molecular pathways is of fundamental importance to the development of effective HCC chemoprevention and treatment.

Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

Digital content and making it viral in social media

Tutkimuksen tavoite on selvittää digitaalisen sisällön ominaisuuksia, jotka vaikuttavat ryhtyvätkö kuluttajat jakamaan, tykkäämään ja kommentoimaan sitä sosiaalisessa mediassa. Tällä pyritään auttamaan yrityksiä ymmärtämään paremmin viraalisuutta, jotta he pystyisivät tuottamaan ja julkaisemaan nettisivuillaan ja sosiaalisessa mediassa parempaa sisältöä, jota kuluttajat jakaisivat enemmän. Tutkimus toteutetaan muodostamalla hypoteeseja mahdollisista ominaisuuksista kirjallisuuden perusteella ja testaamalla niitä regressioanalyyseillä empiirisessä osiossa. Tulokset paljastavat yhdeksän piirrettä, jotka lisäävät viraalisuutta: kiinnostavuus, neutraalisuus, yllättävyys, viihdyttävyys, epäkäytännöllisyys, artikkelin ja Facebook julkaisun pituus, eri sisältö taktiikoiden käyttö (erityisesti blogit ja kuvat lisäävät viraalisuutta) sekä kun mielipidevaikuttaja tai kuuluisuus jakaa sisällön.

Lettre à un ami sur la suppression de l'habit ecclésiastique, & sur le danger que court le clergé de France, de pertes plus réelles ([Reprod.])

Collection : Les archives de la Révolution française ; 8.262

Edit du Roy, portant suppression des offices de contrôlleurs des bans de mariage qui avoient esté créez par l'Edit du mois de septembre 1697 . Et tarif des droits que Sa Majesté veut estre payez à l'avenir pour le contrôlle & l'enregistrement de chacune publication de ban de mariage conformement audit Edit. Donné à Versailles au mois de mars 1702. Registré en Parlement le 26 avril 1702.

[Acte royal. 1702-03-00. Versailles]

Arrêt du conseil d'Etat qui commet J.-B. Dumont pour l'exécution de l'Edit de janvier 1717, portant suppression des offices de contrôleurs des greffiers garde-minutes des chancelleries près les cours et sièges présidiaux

[Acte. 1717-02-20. Paris]

Édit... portant suppression des offices de conseillers du Roy notaires et secrétaires dans les cours de Parlement, Chambres des Comptes et Cours des Aydes, Grand Conseil, Requestes de l'Hostel et du Palais, Conseils supérieurs et provinciaux, Cours des monnoies et Bureaux des Finances ; ordonne que les greffiers en chef des cours seront tous tenus de se pourvoir de l'un des offices de secrétaires, créez dans les chancelleries près les Cours par édit... de juin 1715 ; que la vente des offices de receveurs et payeurs des gages des officiers desdites chancelleries créez par édits... de novembre 1707 et décembre 1708 sera continuée ; et restablit les gardes des sceaux des chancelleries près les Cours dans leurs offices, en payant un supplément de finance... [Enregistré au Parlement le 13 juin 1716.]

[Acte royal. 1716-05-00. Paris]