The current emergency due to the worldwide spread of the COVID-19 caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a great concern for global public health. Already in the past, the outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle Eastern respiratory syndrome (MERS) in 2012 demonstrates the potential of coronaviruses to cross-species borders and further underlines the importance of identifying new-targeted drugs. An ideal antiviral agent should target essential proteins involved in the lifecycle of SARS-CoV. Currently, some HIV protease inhibitors (i.e., Lopinavir) are proposed for the treatment of COVID-19, although their effectiveness has not yet been assessed. The main protease (M pro ) provides a highly validated pharmacological target for the discovery and design of inhibitors. We identified potent M pro inhibitors employing computational techniques that entail the screening of a Marine Natural Product (MNP) library. MNP library was screened by a hyphenated pharmacophore model, and molecular docking approaches. Molecular dynamics and re-docking further confirmed the results obtained by structure-based techniques and allowed this study to highlight some crucial aspects. Seventeen potential SARS-CoV-2 M pro inhibitors have been identified among the natural substances of marine origin. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds could be bioactive is excellent.

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-B was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-B␣, indicating that NF-B activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-B-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.Interest in the understanding of mechanisms by which viruses belonging to a variety of families regulate cell apoptosis has grown rapidly in recent years (1-3). Herpesviruses, due to the relatively large quantity of information contained in their genomes, seem particularly well equipped to exert a fine control over cell apoptosis (4). This occurs through various interactions among viral and cell products acting at different levels (5).Among herpesviruses, herpes simplex viruses have been shown to regulate apoptosis of infected cells both positively and negatively, according to the presence or absence of specific genes, experimental conditions, or specificity of target cells (6 -21).Glycoprotein D (gD) 1 is a main component of the external structure of HSV-1, and its function is essential for HSV-1 spread. Interaction between gD and cell receptors allows virion entry into cells to be infected (22)(23)(24)(25). At least one of the cell receptors for gD, namely herpesvirus entry mediator A (HveA; also known as HVEM, TNFRSF14), belongs to the family of tumor necrosis factor receptors, which play a central role in mediating signal transduction leading to death receptor-associated apoptosis (26 -28). Recent results have shown that gD delivered in trans blocks the apoptotic cascade triggered by HSV-1 mutants lacking the gene encoding gD in SK-N-SH cells (29,30). Cellular signals involved in the antiapoptotic action exerted by HSV-1-gD remain to be elucidated. Interestingly, overexpression of the gD receptor HveA has been shown to cause activation of the transcription factor, NF-B (28). Furthermore, it has been reported that engagement with HveA receptor of its natural ligand, LIGHT, can stimulate the activation of NF-B in different cellular systems (31,32). This suggests the possibility that also engagement of gD with HveA could lead to NF-B activation. The transcription factor NF-B consists of a homodimeric or heterodimeric complex of two subunits belonging to the highly conserved family of Rel-related proteins (33). The most important complex is that formed by two proteins with molecular masses of 50 kDa (p50) and 65 kDa (p65), respectively. This heterodimer is pre...

In recent years, both cellular and viral RNAs have been reported to be packaged in virions of human cytomegalovirus (CMV) (1, 4, 7). The observations are of particular interest for several reasons. Foremost, human CMVs are among the largest viruses infecting cells of higher organisms. In addition, these viruses, members of the Herpesviridae family, incorporate into their virions numerous proteins with multiple functions that effectively assist in the creation of an effective intracellular environment for rapid takeover and redirection of cellular functions to the benefit of the virus. In comparison with other members of Herpesviridae family, the functions encoded in their genomes and the ready-made proteins brought into cells during infection should be more than sufficient to render the infected cell a very pliant client. The presence of the RNAs in virions is therefore an unexpected, novel, intriguing facet of herpesvirus biology.Following the basic premise that viruses do not perform gratuitous functions, we decided to determine whether herpes simplex virus type 1 (HSV-1) virions also contain RNAs and to determine their function. The advantage of HSV-1 is twofold. First, HSV-1 contains fewer open reading frames (ORFs) and the pattern of transcription of the viral genome has been extensively studied. Second, the major functions of HSV-1 gene products are at least in part understood. Hence, if RNAs were packaged in virions, we would have a basis on which to evaluate the significance of the packaged mRNAs.The purification of HSV-1 virions has been extensively studied and characterized (5, 10). In this report, RNAs extracted from either intracellular or extracellular purified virions after RNase digestion were reverse transcribed, amplified by PCR, and subjected to two kinds of analyses. We report that a fraction of the HSV RNAs are represented in RNAs extracted from purified preparations and that the RNA transcripts of nine ORFs were detected in all purified preparations tested. We also report the presence of cellular RNAs in purified virions. In this instance, the selectivity of the packaged RNAs is less well apparent. MATERIALS AND METHODS Cells and viruses.Vero and HEp-2 cell lines (American Type Culture Collection) and a rabbit skin cell line (originally obtained from J. McLaren) were propagated in Dulbecco's modified Eagle's medium supplemented with 5% newborn calf serum. HSV-1(F) is the prototype HSV-1 strain used in this laboratory (2). Isolation of the mutant virus R7023 was described elsewhere (6). R7023 lacks the genes U S 8 through U S 12. Titers of the stocks of HSV-1(F) and R7023 on Vero cells were determined.Purification of virions. HSV-1 and R7023 virions were purified as described by Spear and Roizman (10). Briefly, Vero or HEp-2 cells grown in roller bottles or in 150-cm 2 flasks were exposed to 5 PFU of virus per cell. The cells were harvested 22 to 24 h after infection and centrifuged at 2,000 rpm for 10 min in an Allegra 6R centrifuge equipped with a GH-3.8 rotor (Beckman Coulter, Inc., Fuller...

Nuclear phosphoprotein HMGA1a, high mobility group A1a, (previously HMGI) has been investigated during apoptosis. A change in the degree of phosphorylation of HMGA1a has been observed during apoptosis induced in four leukemic cell lines (HL60, K562, NB4, and U937) by drugs (etoposide, camptothecin) or herpes simplex virus type-1. Both hyper-phosphorylation and de-phosphorylation of HMGA1a have been ascertained by liquid chromatography-mass spectrometry. Hyperphosphorylation (at least five phosphate groups/ HMGA1a molecule) occurs at the early apoptotic stages and is probably related to HMGA1a displacement from DNA and chromatin release from the nuclear scaffold. De-phosphorylation (one phosphate or no phosphate groups/HMGA1a molecule) accompanies the later formation of highly condensed chromatin in the apoptotic bodies. We report for the first time a direct link between the degree of phosphorylation of HMGA1a protein and apoptosis according to a process that involves the entire amount of HMGA1a present in the cells and, consequently, whole chromatin. At the same time we report that variously phosphorylated forms of HMGA1a protein are also mono-methylated.Among nonhistone nuclear proteins of mammalian cells, a family of three proteins called HMGA1a, HMGA1b, and HMGA2 (previously termed HMGI, HMGY, and HMGI-C, respectively) 1 have aroused great interest in many laboratories over the last few years, due to the variety of biological processes in which they are involved (Refs. 1-4 and references therein). HMGA1a and HMGA1b are very similar, differing by only 11 amino acid residues, because they are the splicing products of the same gene (5), whereas HMGA2 is the product of another gene (6). These proteins are composed of about a hundred residues, and all contain three characteristic short basic regions, called AT-hooks, that interact with AT-rich stretches of DNA in the minor groove (7,8).A property that is characteristic not only of the three proteins under discussion, but also of other HMG proteins, is a C-terminal domain having a very high content of acidic residues (1, 9). In HMGA1a, HMGA1b, and HMGA2 the acidic C-terminal domain is constitutively phosphorylated in vivo by CK2 (10 -12), but additional sites for cell-cycle-dependent phosphorylation by other kinases have also been reported (13-15).High levels of HMGA1a, HMGA1b, and HMGA2 proteins have been found in embryonic cell lines or tissues as well as in neoplastic cell lines and tumors, but they are absent or expressed at very low levels in normal cells (1, 12, 16 -23). These findings support the hypothesis that the three proteins could be linked to growth, differentiation, and neoplastic transformation.Given the importance of HMGA1a, HMGA1b, and HMGA2 proteins in chromatin dynamics, we addressed the question of chromatin assembly/disassembly by studying their post-translational modifications during apoptosis, a process where profound changes in chromatin structure occur. Apoptosis has been induced in four leukemic cell lines (HL60, K562, NB4, and U937), and...

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The topic dealt with in this paper concerns an experimental study, performed by means of ultrasonic techniques, on aqueous solutions of α,α-trehalose, an effective bioprotector against dehydration. The ultrasonic velocity and density data show that, in these aqueous solutions, the mixing process is not ideal. The behavior of the excess compressibility as a function of concentration can be interpreted by supposing that α,α-trehalose molecules form intramolecular hydrogen bonds by folding over at high concentration. When water molecules are added, they spread out again, thereby releasing hydrogen bond sites for water interactions and increasing the water−sugar interaction strength. Furthermore, the temperature dependence of the hydration number has been evaluated, and its value at room temperature is very close to that given in the literature.