This study investigated the antiviral, chemoprotective and proliferative effects of strawberry anthocyanins on herpes simplex virus type-1, cancerous cell lines HT-29 and AGS, and normal cell lines Hs 738.St/Int and CCD-18Co. Antiviral properties were measured by infecting vero cells from adult grivet (Cercopithecus aethiops) with herpes simplex virus type-1 (HSV-1) and treating with a concentration of 1.25-20 µg/mL of strawberry anthocyanins. Infectivity and replication were quantified for herpes simplex virus type-1 using the direct plaque assay and reporting PFU/mL. Strawberry anthocyanins (>20 µg/mL) inhibited the herpes simplex virus infectivity in vero cells by 100% (p<0.05). Strawberry anthocyanins at concentrations of 5, 10 and 20 μg/mL were reduced to 75.36, 57.98, and 31.46 percent of the control (100%) (p<0.05). Chemoprotective and proliferative effects of strawberry anthocyanins were analyzed for the human cell lines AGS, Hs 738.St/Int, HT-29, and CCD-18Co at a concentration of 25-200 µg/mL and quantified using the sulforhodamine-B assay. Growth inhibition occurred at a level of ≥87% for treatment concentrations 100 and 200 µg/mL for the cancerous AGS and HT-29 cell lines (p<0.0001). Proliferation rates for the normal Hs 738.St/Int and CCD-18Co cell lines increased at all treatment concentrations of 25-200 μg/mL (p<0.0001); suggestingthat the observed proliferative activity may be associated with anthocyanin treatment.Strawberry anthocyanin treatment concentration worked in a dose dependent manner for the HSV-1 and the cancerous AGS and HT-29 cells. The caspase-3 assay was performed to demonstrate potential mechanism of action and confirmed thatanthocyanin treatments play a role in apoptosisby the up regulation of caspase-3.Significantdifferences were seen between the growth characteristics of cancerous cell linescompared to their equivalent normal cell lines (p<0.0001). In summary, the antiviral findings suggest that strawberry anthocyanin extracts could be an effective topical treatment and/or prophylactic agent for oral herpetic infections (HSV-1). Also, the in vitro chemoprotective effect of strawberry anthocyanins found may be relevant to in vivo work in the future because when anthocyanins are consumed in the diet they come in direct contact with the gastrointestinal tract and may provide chemoprotection upon contact with the stomach and gastrointestinal tract’s epithelial cell layer.

Le premier objectif a été de sélectionner des promoteurs de gènes cellulaires actifs spécifiquement dans les HCC à l'aide d'une recherche bibliographique puis en utilisant la base de donnée UniGene. Leur activité a été vérifiée par RT-qPCR et CHIP dans des lignées modèles HCC et dans des hépatocytes. Ces promoteurs ont été clonés en amont de la luciférase dans la région intergénique 20 du génome HSV-1 afin d'étudier leur force d'activité, 2 types de cinétiques et leur activité différentielle en fonction du type cellulaire et dans le contexte d'une infection virale. Le deuxième objectif a été de construire des virus oncolytiques ciblés pour l'expression de la protéine Us3, une protéine virale impliquée dans le contrôle de la réponse apoptotique induite par HSV-1. L'expression de la protéine Us3 est placée sous contrôle d'un promoteur cellulaire spécifique d'HCC. L'hypothèse est qu'en l'absence d'activité du promoteur cellulaire dans les cellules non HCC, la protéine Us3 ne sera pas synthétisée et, par conséquent, l'apoptose qui ne sera pas réprimée, inhibera le cycle de réplication et par conséquent, la production virale dans les cellules saines. Dans les cellules HCC, le promoteur actif permettra la réplication virale aboutissant à la destruction de lamasse tumorale. Un virus HSV-1 Us3- a été construit en utilisant la technique de recombinaison en plasmide BAC (Bacterial artificial chromosome), puis 2 virus oncolytiques en réintroduisant le gène Us3 sous contrôle du promoteur ANGPTL3 ou du promoteur HRE (hypoxia responsive element). Leur comportement oncolytique a été étudié en réalisant des courbes de croissance sur lignées cellulaires d'HCC et cellules hepatocyte-like.

B virus (Cercopithecine herpesvirus 1) is an alphaherpesvirus indigenous to macaque monkeys and is closely related to herpes simplex virus type 1 (HSV-1). Disease caused by B virus, which is often mild or asymptomatic in its natural host, the macaque monkey, is similar in infected macaques to HSV-1 infection in humans. When B virus zoonotically infects foreign hosts, e.g., humans, high morbidity and mortality are evidenced in > 80% of untreated cases. To explore the underlying reasons for differences in pathogenesis between B virus and HSV-1 infection in humans, human microarrays were used to comparatively examine global cellular gene expression patterns engaged as a result of infection of human foreskin fibroblasts (HFFs). Our results demonstrate that these closely related simplexvirus family members have divergent strategies to thwart host cell pathways related to innate defenses. In these studies, B virus did not induce detectable interferon, cytokine or chemokine genes, in sharp contrast to HSV-1, which induced innate immune responsive genes in infected cells. Although no innate immune response genes were found to be up-regulated by B virus infection, B virus induced I£eB£a, which was the only gene found to be involved in the NF-£eB signaling pathway within the innate immunity biological network. Quantification of NF-£eB p50 DNA binding activity in virus-infected nuclear extracts demonstrated that NF-£eB p50 DNA binding activity was lower in B virus-infected cells. Suppression of I£eB£a in B virus infected cells by siRNA restored NF-£eB-induced cytokine and chemokine expressions. Data presented here support the model that I£eB£a inhibits NF-£eB regulated immune responsive genes in B virus-infected HFF cells, and this response differs from that observed in HFF cells infected with HSV-1. The result is that B virus alters the NF-£eB regulated expression of cytokine and chemokine genes of HFF cells differently from HSV-1 early after infection. These differences in cytokine and chemokine expression may be associated with the delayed or reduced host responses observed in B virus infected humans and underlie the failure of adaptive responses in zoonotically infected humans.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia. / Made available in DSpace on 2012-10-22T08:38:12Z (GMT). No. of bitstreams: 1 226339.pdf: 2710123 bytes, checksum: a20d190831a6779c0388646cfa5bd118 (MD5)

HSV-1 and HSV-2, both double-stranded DNA viruses of the Alphaherpesvirinae subfamily, reportedly have sub-clinical prevalence in nearly 67% of the world population. During their intra-nuclear virus replication, four types of capsids (procapsids, A, B, and C capsids) are produced while only C-capsids contain mature DNA. Given their larger size (125 nm) than the nuclear pores (30-50 nm), they exit the nucleus by an unusual route called nuclear egress. On the other hand, our lab previously found that HSV-1 incorporates 49 distinct host proteins, including DDX3X, a DEAD (Asp-Glu-Ala-Asp) box ATP-dependent RNA helicase that modulates gene expression of both DNA and RNA viruses. We also showed that DDX3X is redirected to the inner nuclear membrane late during the infection and interacts with pUL31, a component of the viral nuclear egress complex, to promote the nuclear exit of the viral capsids to the cytoplasm. However, the exact nature of such interactions remains elusive. On the other hand, our lab also reported that PCBP1 is specifically present on the C-capsids, and its depletion causes a reduction in viral titer. PCBP1 is known to bind with poly(c) RNA through K-homology (KH) domains and plays a role in mRNA stabilization, transcriptional control, RNA translation, antiviral immunity, and the modulation of viral propagation. Using confocal microscopy and co-immunoprecipitation studies, the present work reveals that DDX3X interacts with both components of the nuclear egress complex, i.e., pUL31 and pUL34, and that this interaction is independent of their phosphorylation by the pUS3 kinase that normally modulates their localization. Moreover, we also show that DDX3X interacts with PCBP1, which could explain the preferential selection of the C-capsids during the nuclear egress. This study is a step forward to map the complex multiple host protein interactions with viral partners and elucidate their possible role in the enigmatic selective escape of HSV-1 C-capsids. / HSV-1 et HSV-2, tous deux des virus à ADN double brin de la sous-famille des Alphaherpesvirinae, ont une prévalence subclinique chez près de 67 % de la population mondiale. Au cours de leur réplication virale intra-nucléaire, quatre types de capsides (procapsides, capsides A, B et C) sont produites tandis que seules les capsides C contiennent de l'ADN mature. Compte tenu de leur plus grande taille (125 nm) que les pores nucléaires (30 à 50 nm), ils quittent le noyau par une voie inhabituelle appelée sortie nucléaire. Notre laboratoire a précédemment découvert que le HSV-1 incorpore 49 protéines hôtes distinctes, dont DDX3X, une hélicase à ARN de type DEAD (Asp-Glu-Ala-Asp) dépendante de l'ATP qui module l'expression génique des virus à ADN et à ARN. DDX3X est redirigé vers la membrane nucléaire interne à la fin de l'infection et interagit avec pUL31, un composant du complexe de sortie nucléaire viral, pour favoriser la sortie nucléaire des capsides virales vers le cytoplasme. Cependant, la nature exacte de ces interactions reste incertaine. D'autre part, notre laboratoire a également signalé que PCBP1 est spécifiquement présent sur les capsides C et que sa déplétion entraîne une réduction du titre viral. PCBP1 est connu pour se lier à l'ARN poly (c) via les domaines d'homologie K (KH) et joue un rôle dans la stabilisation de l'ARNm, le contrôle transcriptionnel, la traduction de l'ARN, l'immunité antivirale et la modulation de la propagation virale. À l'aide de microscopie confocale et d'études de co-immunoprécipitation, le présent travail révèle que DDX3X interagit avec les deux composants du complexe de sortie nucléaire, à savoir pUL31 et pUL34, et que cette interaction est indépendante de leur phosphorylation par la kinase pUS3 qui module normalement leur localisation. De plus, nous montrons également que DDX3X interagit avec PCBP1, ce qui pourrait expliquer la sélection préférentielle des capsides C lors de la sortie nucléaire. Cette étude constitue un pas en avant dans la cartographie des interactions complexes entre protéines hôtes multiples et partenaires viraux et pour élucider leur rôle possible dans l’évasion sélective énigmatique des capsides C du HSV-1.

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