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Herpes simplex virus 1 (HSV-1) can infect a wide range of cell types, including cells of the adaptive and innate immunity but, normally, it completes a fully-permissive replication cycle only in epithelial or neural cells. Complex mechanisms controlling this delicate balance in immune cells and consequent restriction of HSV-1 infection in these cells have not been completely elucidated. We have recently demonstrated that the transcription factor nuclear factor kappa B (NF-&kappa, B) can act as a main permissiveness regulator of HSV-1 infection in monocytic cells, however, mediators involved in this regulation have not been identified. To better define mechanisms involved in this phenomenon and, particularly, the possible involvement of ROS, wild type U937 cells or U937 cells stably transfected with a dominant-negative (DN) I&kappa, B-mutant and selenium-containing compounds, as anti-oxidants, were utilized. The main results can be summarized as follows. HSV-1 infection induces an immediate ROS production in U937 monocytic cells that can efficiently activate NF-&kappa, B but not in DN-I&kappa, B-mutant cells. Treatment with selenium-containing antioxidants efficiently inhibited HSV-1-induced ROS generation while producing increased levels of HSV-1 replication and a reduction of HSV-1-induced NF-&kappa, B activation in U937 monocytic cells. Our results suggest a scenario in which an efficient NF-&kappa, B-dependent ROS production in response to infection could contribute in limiting HSV-1 replication in monocytes/macrophages, thus avoiding possible irreparable damage to the innate immune system of the host during HSV-1 infection.

Assessing the actual efficacy of compounds to directly inhibit HIV reverse transcriptase (RT) activity is a main goal in preclinical antiretroviral studies. Our previous studies demonstrated that the effects of inhibitor compounds towards HIV-RT could be efficiently assessed through a simple cell-free assay based on conventional reverse transcription PCR. In the present study, we describe a modified variant of our assay, termed RT real-time quantitative PCR inhibitory assay (RT-qPCR-IA), in which the ability of compounds to restrict the complementary DNA (cDNA) generation by HIV-RT using a specific RNA template is performed by the real-time technique, in order to improve both accuracy and sensitivity of the method. As specific RNA template, RNA extracted from stable transfectants ectopically expressing the herpes simplex virus 1 glycoprotein D gene was utilized. HIV-RT, of both commercial or house-made viral lysate origin, was employed for the assay. To assess the reliability of RT-qPCR-IA, we performed a comparative, quantitative analysis of the dose-dependent effect exerted by known nucleotide and non-nucleotide reverse-transcriptase inhibitors, using the SYBR Green dye chemistry as detection system. The results obtained with RT-qPCR-IA were compared to that obtained using a one-step PicoGreen technology-based commercial kit. The outcome of our study indicates that the development of the novel RT-qPCR-IA will provide rapid and accurate evaluation of the inhibitory efficacy of compounds towards HIV-RT activity. This evaluation could be very useful for large-scale screening of potential new anti-HIV drugs.

Herpes simplex virus type 1 (HSV-1) is responsible of common and widespread viral infections in humans through the world, and of rare, but extremely severe, clinical syndromes in the central nervous system. The emergence of resistant strains to drugs actually in use encourages the searching for novel antiviral compounds, including those of natural origin. In this study, the recently described poly-γ-glutamic acid (γ-PGA-APA), produced by the marine thermotolerant Bacillus horneckiae strain APA, and previously shown to possess biological and antiviral activity, was evaluated for its anti-HSV-1 and immunomodulatory properties. The biopolymer hindered the HSV-1 infection in the very early phase of virus replication. In addition, the γ-PGA-APA was shown to exert low cytotoxicity and noticeable immunomodulatory activities towards TNF-α and IL-1β gene expression. Moreover, the capacity to positively modulate the transcriptional activity of the cytokine genes was paired with increased level of activation of the transcription factor NF-kB by γ-PGA-APA. Overall, as non-cytotoxic biopolymer able to contribute in the antiviral defense against HSV-1, γ-PGA-APA could lead to the development of novel natural drugs for alternative therapies.