F HCV NS5A protein was not altered in these cells (from 0.84 to 1.28, Fig. 8B). Thus, HCV replication was not affected by cdN protein.DiscussionIn this study, cellular cdN protein was found to interact with NCV 1317923 NS3 protein in the yeast two-hybrid system (Fig. 1) and in cultured cells (Figs. 2 and 3). This interaction results in the partial suppression of cdN activity by NS3 protein (Fig. 5). Moreover, the cdN activity was also partially repressed by HCV in the systems of HCV sub-genomic replicons and infectious HCV virions (Figs. 6?7). DNA replication and repair requires a balanced supply of four deoxyribonucleotides (dNTPs) [24]. The intracellular dNTPs pools in mammalian cells are regulated by substrate cycles. Substrate cycles depend on the interplay between a deoxynucleoside kinase and a nucleotidase [25,26]. Nucleoside monophosphate phosphohydrases or 59-nucleotidases dephosphorylate non-cyclic nucleoside monophosphate to nucleosides and inorganic phosphates. At least seven 59-nucleotidases with different subcellular localization have been cloned [19]. Some 59-nucleotidases are ubiquitous (eN,cN-II, cdN, and mdN) while others display tissue-specific expression (cN-I and cN-III). All 59-nucleotidases have relatively broad substrate specificities. Detection of individual nucleotidases by enzymatic assays in cell lysates is problematic because different nucleotidases are co-expressed in the same tissue or cell type. cdN is first purified to homogeneity from human placenta [6]. Our results (Fig. 4) showed that cdN contributed more than mdN to the total cellular 59(39)-deoxyribonucleotidase activity, which is consistent to previous reports demonstrating that cdN is responsible for the major 59(39)-deoxyribonucleotidase activity in cultured human cells [15,27]. Our results showed that HCV NS3 protein caused a 30 reduction of the cellular 59(39)-deoxyribonucleotidase activity in both transiently and stably expressed systems but it did not repress the expression of the cdN protein (Fig. 5). The apparent lowered 59(39)-deoxyribonucleotidase activity should be due to a reduction of cdN but not mdN activity for the following reasons: (i) NS3 protein binds cdN (Figs 1-3); (ii) mdN does not co-localize with NS3 protein although it is highly homologous to cdN (52 amino acid identity); (iii) mdN activity is lower in HuH7 cells (Fig. 4C). The presence of at least seven genes for 59-nucleotidases in human genome suggests that these enzymes perform importantHCV NS3 Interacts with cdN ProteinFigure 6. The cdN activity but not its protein Epigenetics amount was increased after interferon treatment in HCV replicon cells. (A) HuH7 cells (lanes 1 and 2) or HCV replicon cells (lanes 3-6) were mocktreated (lanes 1, 3 and 5) or treated with interferon-a (104 U/ml in lanes 2 and 6; 103 U/ml in lane 4). At 72 hrs after treatment, proteins derived from these cells were Epigenetics analyzed using antibodies against NS5A to reflect HCV replication (upper panel), against cdN protein (middle panel) or against Erk-2 as a loading control (bottom panel). (B) The 59(39)deoxyribonucleotidase activity was analyzed using cell lysates derived from (A). doi:10.1371/journal.pone.0068736.gmetabolic functions [19]. HCV NS3 protein is involved in cell transformation. The serine protease domain of NS3 protein is responsible for the cell transformation [4,5]. HCV NS3 proteinderived from certain genotypes has been demonstrated to generate an internally cleaved product containing the protease domain [2.F HCV NS5A protein was not altered in these cells (from 0.84 to 1.28, Fig. 8B). Thus, HCV replication was not affected by cdN protein.DiscussionIn this study, cellular cdN protein was found to interact with NCV 1317923 NS3 protein in the yeast two-hybrid system (Fig. 1) and in cultured cells (Figs. 2 and 3). This interaction results in the partial suppression of cdN activity by NS3 protein (Fig. 5). Moreover, the cdN activity was also partially repressed by HCV in the systems of HCV sub-genomic replicons and infectious HCV virions (Figs. 6?7). DNA replication and repair requires a balanced supply of four deoxyribonucleotides (dNTPs) [24]. The intracellular dNTPs pools in mammalian cells are regulated by substrate cycles. Substrate cycles depend on the interplay between a deoxynucleoside kinase and a nucleotidase [25,26]. Nucleoside monophosphate phosphohydrases or 59-nucleotidases dephosphorylate non-cyclic nucleoside monophosphate to nucleosides and inorganic phosphates. At least seven 59-nucleotidases with different subcellular localization have been cloned [19]. Some 59-nucleotidases are ubiquitous (eN,cN-II, cdN, and mdN) while others display tissue-specific expression (cN-I and cN-III). All 59-nucleotidases have relatively broad substrate specificities. Detection of individual nucleotidases by enzymatic assays in cell lysates is problematic because different nucleotidases are co-expressed in the same tissue or cell type. cdN is first purified to homogeneity from human placenta [6]. Our results (Fig. 4) showed that cdN contributed more than mdN to the total cellular 59(39)-deoxyribonucleotidase activity, which is consistent to previous reports demonstrating that cdN is responsible for the major 59(39)-deoxyribonucleotidase activity in cultured human cells [15,27]. Our results showed that HCV NS3 protein caused a 30 reduction of the cellular 59(39)-deoxyribonucleotidase activity in both transiently and stably expressed systems but it did not repress the expression of the cdN protein (Fig. 5). The apparent lowered 59(39)-deoxyribonucleotidase activity should be due to a reduction of cdN but not mdN activity for the following reasons: (i) NS3 protein binds cdN (Figs 1-3); (ii) mdN does not co-localize with NS3 protein although it is highly homologous to cdN (52 amino acid identity); (iii) mdN activity is lower in HuH7 cells (Fig. 4C). The presence of at least seven genes for 59-nucleotidases in human genome suggests that these enzymes perform importantHCV NS3 Interacts with cdN ProteinFigure 6. The cdN activity but not its protein amount was increased after interferon treatment in HCV replicon cells. (A) HuH7 cells (lanes 1 and 2) or HCV replicon cells (lanes 3-6) were mocktreated (lanes 1, 3 and 5) or treated with interferon-a (104 U/ml in lanes 2 and 6; 103 U/ml in lane 4). At 72 hrs after treatment, proteins derived from these cells were analyzed using antibodies against NS5A to reflect HCV replication (upper panel), against cdN protein (middle panel) or against Erk-2 as a loading control (bottom panel). (B) The 59(39)deoxyribonucleotidase activity was analyzed using cell lysates derived from (A). doi:10.1371/journal.pone.0068736.gmetabolic functions [19]. HCV NS3 protein is involved in cell transformation. The serine protease domain of NS3 protein is responsible for the cell transformation [4,5]. HCV NS3 proteinderived from certain genotypes has been demonstrated to generate an internally cleaved product containing the protease domain [2.
