The effect of the hepatitis C virus (HCV) NS3 protein on the expression of miR-150, miR-199a, miR-335, miR-194 and miR-27a.

Hepatitis C virus (HCV) infection is considered one of the most important causes of chronic liver diseases. Many reports have shown that the proteins of the HCV via interactions with gene expression regulatory networks such as cellular pathways and microRNAs can contribute to the development of chronic liver diseases. The present study aimed to investigate the effects of the HCV NS3 protein on the expression of miR-150 miR-199a, miR-335, miR-194, miR-27a in a cell culture model. Plasmids expressing the full length of the HCV NS3 protein were transfected into the LX-2 cell line, while at the same time a plasmid expressing empty GFP (green fluorescent protein) was used as a negative control group. Subsequently, total RNA was extracted and real-time PCR was performed to measure microRNA expression levels. Additionally, the trypan blue exclusion test was performed to examine the effect of the expressing NS3 protein plasmid on cellular viability. The analysis of microRNA gene expression in LX-2 cells indicated that the NS3 protein, which is endogenous to HCV, can significantly upregulate the expression of miR-27a and downregulate the expression of miR-335 and miR-150 in comparison with the control plasmid expressing GFP and normal cells (p < 0.01). These results suggest that the HCV NS3 protein may play a role in the pathogenesis of chronic hepatic diseases such as liver fibrosis via interaction with cellular microRNAs and modulation of microRNA gene expressions.

Crystal Structure of Human Herpesvirus 6B Tegument Protein U14.

The tegument protein U14 of human herpesvirus 6B (HHV-6B) constitutes the viral virion structure and is essential for viral growth. To define the characteristics and functions of U14, we determined the crystal structure of the N-terminal domain of HHV-6B U14 (U14-NTD) at 1.85 Å resolution. U14-NTD forms an elongated helix-rich fold with a protruding ß hairpin. U14-NTD exists as a dimer exhibiting broad electrostatic interactions and a network of hydrogen bonds. This is first report of the crystal structure and dimerization of HHV-6B U14. The surface of the U14-NTD dimer reveals multiple clusters of negatively- and positively-charged residues that coincide with potential functional sites of U14. Three successive residues, L424, E425 and V426, which relate to viral growth, reside on the ß hairpin close to the dimer's two-fold axis. The hydrophobic side-chains of L424 and V426 that constitute a part of a hydrophobic patch are solvent-exposed, indicating the possibility that the ß hairpin region is a key functional site of HHV-6 U14. Structure-based sequence comparison suggests that U14-NTD corresponds to the core fold conserved among U14 homologs, human herpesvirus 7 U14, and human cytomegalovirus UL25 and UL35, although dimerization appears to be a specific feature of the U14 group.

Construction of an eGFP Expression Plasmid under Control of T7 Promoter and IRES Sequence for Assay of T7 RNA Polymerase Activity in Mammalian Cell Lines.

BACKGROUND: Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site (IRES) sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. METHODS: For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. RESULTS: Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. CONCLUSION: Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics.

HCV NS3 Blocking Effect on IFN Induced ISGs Like Viperin and IL28 With and Without NS4A.

BACKGROUND: Hepatitis C virus (HCV) is able to down-regulate innate immune response. It is important to know the immune pathways that this virus interacts with. HCV non-structural protein 3 (NS3) plays an important role in this viral feature. HCV NS3 protein could affect the expression of antiviral protein such as viperin, and interleukin 28whichare important proteins in antiviral response. OBJECTIVES: HCV has developed different mechanisms to maintain a persistent infection, especially by disrupting type I interferon response and subsequent suppression of expression of Interferon stimulatory genes (ISGs). Viperin, a member of ISGs, is considered as a host antiviral protein, which interferes with viral replication. Since it is a good target for some viruses to evade host responses, it is interesting to study if HCV has evolved a mechanism to interfere with this member of ISGs. MATERIALS AND METHODS: We evaluated the impact of NS3, NS3/4A and a mutated nonfunctional NS3 on ISGs expression such as viperin and IL-28 after the induction of IFN signaling Jak-STAT pathway using IFN-. RESULTS: NS3 protein disrupted the expressions of viperin gene and IL-28, an inducer for the expression of ISGs and viperin itself. By comparing the roles of NS3 and NS3/4A protease activities in suppressing the innate immune responses, we also showed that NS3 (without NS4A) has the ability to down-regulate ISGs expression, similar to that of NS3/4A. CONCLUSIONS: ISGs expression is impeded by NS3 protease activity and its interaction with Jak-STAT pathway proteins. In addition, the NS3/4A substrates spectrum seems to be similar to those of NS3.

Prevalence of GBV-C among Iranian HBV positive patients using PCR-RFLP technique.

AIM: The aim of this study was to investigate the prevalence of GBV-C among Iranian HBV positive patients using PCR-RFLP technique. BACKGROUND: GBV-C was a member of flaviviridae family and recently propose to classify as members of a fourth genus in this family, named Pegivirus and suggest that at least one quarter of the world's population has been infected with this virus. GBV-C can be transmitted via the blood-borne route, although vertical and sexual transmission is very well documented. PATIENTS AND METHODS: 100 serum samples were collected from HBsAg positive patients in 2011-2012. RNA was extracted with Qiagene mini kit. cDNA was synthesized by Reverse Transcriptase method and amplified by Semi- nested PCR method. After designing specific primers, the semi nested PCR was optimized, then sequences of PCR products were analyzed with software such as neb cutter, and sites of restriction enzymes were determined and suitable enzymes were selected for RFLP (Restriction Fragment Length Polymorphism). RESULTS: PCR products were analyzed in 2% agarose gels containing ethidium bromide and were visualized with ultraviolet (UV) light. A 230 bp band was observed in comparison with 100 kb ladder; this band indicates our target gene of GBV-C genome have been isolate from serum samples. CONCLUSION: It seems that Co-infection of GBV-C and HBV are common and This method had acceptable sensitivity for detecting GBV-C and determining its genotype, and more affordable than the other techniques; so the results of this study showed the prevalence of GBV-C were 12 serums of 100 serums HBsAg positive in goal population and one sample from 12 GBV-C positive serums was genotype 3 and the others were genotype 2.

Redistribution of fractions of zinc, cadmium, nickel, copper, and lead in contaminated calcareous soils treated with EDTA.

Effect of ethylene diamine tetraacetic acid (EDTA) on the fractionation of zinc (Zn), cadmium (Cd), nickel (Ni), copper (Cu), and lead (Pb) in contaminated calcareous soils was investigated. Soil samples containing variable levels of contamination, from 105.9 to 5803 mg/kg Zn, from 2.2 to 1361 mg/kg Cd, from 31 to 64.0 mg/kg Ni, from 24 to 84 mg/kg Cu, and from 109 to 24,850 mg/kg Pb, were subjected to EDTA treatment at different dosages of 0, 1.0, and 2.0 g/kg. Metals in the incubated soils were fractionated after 5 months by a sequential extraction procedure, in which the metal fractions were experimentally defined as exchangeable (EXCH), carbonate (CARB), Mn oxide (MNO), Fe oxide (FEO), organic matter (OM), and residual (RES) fractions. In contaminated soils without EDTA addition, Zn, Ni, Cu, and Pb were predominately present in the RES fraction, up to 60.0%, 32.3%, 41.1%, and 36.8%, respectively. In general, with the EDTA addition, the EXCH and CARB fractions of these metals increased dramatically while the OM fraction decreased. The Zn, Ni, Cu, and Pb were distributed mostly in RES, OM, FEO, and CARB fractions in contaminated soils, but Cd was found predominately in the CARB, MNO, and RES fractions. The OM fraction decreased with increasing amounts of EDTA. In the contaminated soils, EDTA removed some Pb, Zn, Cu, and Ni from MNO, FEO, and OM fractions and redistributed them into CARB and EXCH fractions. Based on the relative percent in the EXCH and CARB fractions, the order of solubility was Cd > Pb > Ni > Cu > Zn for contaminated soils, before adding of EDTA, and after adding of EDTA, the order of solubility was Pb > Cd > Zn > Ni > Cu. The risk of groundwater contamination will increase after applying EDTA and it needed to be used very carefully.