Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and poliovirus

Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and poliovirus

Herpes simplex virus 1 (HSV-1) glycoprotein K (gK) is expressed on virions and functions in entry, inasmuch as HSV-1(KOS) virions devoid of gK enter cells substantially slower than is the case for the parental KOS virus (T. VP16 activates transcription from IE promoters by indirectly binding to specific sequence elements (TAATGARAT) found in the promoter-regulatory regions of all IE genes (19, 33). HSV-2 infection did not increase cellular p53 expression, but led to phosphorylation of this protein at Ser20. Because HSV replicates independently of the cell cycle, we were able to establish conditions that would permit the study of rates of HSV DNA synthesized in logarithmically growing cells in the virtual absence of cellular DNA synthesis. (ii) Pulse chase studies with [35S]methionine and 32P indicate that whereas most polypeptides are stable, the bound phosphate with few exceptions cycles on and off. The SPLCf inhibited virus adsorption and steps after penetration, and inhibited the synthesis of viral protein. Hsieh, R.

In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. In comparison with other native endogenous promoters, which usually contain many other transcription binding sites, pC8-6CC-Luc amplicon vectors should confer better regulated and consistent transgene expression and may be considered a gene delivery vector of choice to target actively proliferating tumor cells. Is one of the most regular human pathogens, being a public health problem, and causal agent of several diseases, estimated to occur in 40–80% of world population and approximately 90% of people between 20 and 40 years have antibodies against HSV-1 [2] and [3]. The UL20 membrane protein and gK were readily detected on double-gradient-purified virion preparations. In addition, the Oct-1 protein is phosphorylated in a cell cycle-dependent manner (23, 27). In Brazil, the HSV-1 is the most common causative agent of sporadic viral encephalitis in adults [7]. Immuno compromised individuals and the recipients of organ transplantations are at high risk for increased severity of HSV-1 infection.

The most of the treatments for HSV are based on nucleoside analogs of guanine, for example, acyclovir (ACV). However, widespread use of ACV has shown HSV develops resistance through mutations in genes coding for thymidine kinase or for DNA polymerase [8]. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Poliovirus type 1 (PV-1), a member of the Picornaviridae family and Enterovirus genus, is a positive single-stranded RNA non-enveloped virus. It is an enteric virus and about 1% of infected individuals develop paralytic poliomyelitis due to viral invasion of the central nervous system and destruction of motor neurons [9]. Beyond its medical and epidemiological importance, poliovirus is one of the best understood models of non-enveloped RNA virus, including for the development of new drugs. 82:6310–6323, 2008).

At 48 h posttransfection, cultures were infected with the equivalent of 10 PFU of UV-inactivated HSV-1 KOS per cell in the presence and absence of 100 μM Roscovitine. There is not a specific drug for the treatment of polio, but, there are compounds capable of blocking viral uncoating and/or viral adsorption on cell receptors, such as disoxaril and pleconaril [11]. Nevertheless, the development of naturals anti-poliovirus drugs that combine low toxicity and selectivity are required. In recent years, compounds originated from natural sources have been investigated and many of them have shown promising as antivirals [12]. These substances have been shown to interact with several viral targets, ranging from the adsorption of virus to the host cell until the release of new viral particles, which may result in complementary mechanisms of action to the existing antiviral drugs [13]. Using the yeast two-hybrid system, coimmunoprecipitation, and in vitro binding assays, Wadd et al. Among these anti-viral substances, anionic polysaccharides from natural sources and of synthetic origin are noteworthy [14] and [15].
Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and poliovirus

It was suggested that these negatively charged molecules, exert their inhibitory effect by interacting with the positive charges on the virus or on the cell surface, thereby, preventing the entry of the virus into the host cells [14]. The Caesalpinia ferrea is a native arboreal species of Brazil, popularly known as pau-ferro or jucá. An alternative pathway involves receptor-mediated endocytosis and fusion of the viral envelope with endocytic membranes facilitated by the low-pH environment of endosomes (30). Since Roscovitine inhibits HSV replication even when added to infected cells at 6 h p.i. ferrea fruits has been demonstrated effective against oral pathogens Streptococcus sp. and Candida albicans [16]. Moreover, ethanol and methanol extracts, and triterpenoids from fruits and seeds of different species of Caesalpinia presented similar effect against several Gram-positive and -negative bacteria [17] and [18].

Although the antimicrobial effect of C. Interestingly, changes in the phosphorylation of RNA polymerase II correlate with enhanced transcription of the HSV-1 genome (17). In this work, we related the activity of an aqueous solution of a sulfated derivative polysaccharide from C. ferrea against HSV and PV, in vitro. The polysaccharide of C. We reported that gK is a structural component of virions as a Golgi complex-dependent glycosylated species and functions in virus entry, inasmuch as virions lacking gK enter susceptible cells in cell culture substantially slower (15). The level of CAT activity when Roscovitine was added at these times was similar to the basal levels in mock-infected samples.

After the reaction, the mixture was cooled to 4 °C and chlorosulfonic acid (4 ml) was slowly added to the mixture with stirring over 24 h at 4 °C. The resulting solution was neutralized with a saturated NaHCO3 solution, dialyzed (molecular weight cutoff 8–12 kDa) for 120 h against distilled water and then centrifuged for 25 min. Three consecutive sulfations were carried out and the sulfated derivative was collected after lyophilization. The degree of sulfation (DS = 8.7%) was determined by elemental analysis. J. ferrea sulfated polysaccharide (SPLCf) stock solution was prepared by dissolving in ultra pure water. SPLCf was characterized by 13C, 1H NMR and FT-IR.

13C and 1H NMR spectra of 2.5% (w/v) solution in D2O were recorded at 353 K on a Fourier transform Brucker Avance-DRX 500 spectrometer with an inverse multinuclear gradient probe-head equipped with z-shielded gradient coils, and with Silicon Graphics. Bioinformatics analysis of predicted protease sites within HSV-1 proteins.The ExPASy PeptideCutter software tool (16), available at the ExPASy Bioinformatics Resource portal (http://web.expasy.org/peptide_cutter/), was utilized to predict potential tobacco etch virus (TEV) cleavage sites within each predicted amino acid sequence of the HSV-1(F) genome (GenBank accession number GU734771). A well-characterized cell cycle inhibitor, Lovastatin, and a broad-spectrum serine-threonine kinase inhibitor, K252a, were tested for their ability to inhibit virion-induced CAT expression (10). The spectrum was obtained from 20 scans. The sample was analyzed as KBr pellet. HEp-2 cells (human larynx epithelial cells carcinoma, ATCC CCL-23), used throughout, were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (Invitrogen – Gibco, USA), 100 IU/ml penicillin (Novafarma Indústria Farmacêutica, BR), 100 μg/ml streptomycin (Gibco BRL, USA) and 2.5 μg/ml of amphotericin B (Meizler Biopharma S/A, BR). The HSV-1 isolated from clinical specimens and was provided by the Departamento de Virologia (IMPPG/UFRJ, BR) and the PV-1 (VR-58) is an ATCC strain.

Preimmune serum or the polyclonal antibody 54 was used for immunoprecipitation of hnRNP K (52). The cytotoxicity of the compound in HEp-2 cells was evaluated by MTT (dimethylthiazolyldiphenyltetrazolium bromide) kit assay (Sigma Chem. Co., USA), according to manufacturer’s specifications. Briefly, cell cultures grown at approximately 70% confluence in 96-well microplates (TPP, Switzerland) were treated with varying concentrations of the compound and incubated for 72 h at 37 °C. The gDΔTEV virus was subsequently used to generate the gK-V5-TEV recombinant virus, which expressed gK containing the V5 epitope and the TEV protease recognition site ENLYFQG inserted in frame immediately after amino acid 68 of gK.

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