No abstract

bHepatitis C virus (HCV) nonstructural protein 5A (NS5A) is involved in regulating viral replication through its direct interaction with the HCV RNA-dependent RNA polymerase. NS5A also alters infected cell metabolism through complex interactions with numerous host cell proteins. NS5A has furthermore been suggested to act as a transcriptional activator, although the impact on viral replication is unclear. To study this, HCV NS5A variants were amplified from hepatic tissue from an HCV-infected patient, and their abilities to activate gene transcription were analyzed in a single-hybrid yeast (Saccharomyces cerevisiae) model. Different variants isolated from the same patient displayed different transactivational activities. When these variants were inserted into the HCV subgenomic replicon system, they demonstrated various levels of RNA replication, which correlated with their transactivational activities. We showed that the C-terminal fragment of NS5A was localized to the nucleus and that a functional NS5A nuclear localization signal and cellular caspase activity were required for this process. Furthermore, nuclear localization of NS5A was necessary for viral replication. Finally, we demonstrate that nuclear NS5A binds to host cell promoters of several genes previously identified as important for efficient HCV RNA replication, inducing their transcription. Taken together, these results demonstrate a new mechanism by which HCV modulates its cellular environment, thereby enhancing viral replication. H epatitis C virus (HCV) infection is characterized by a highfrequency of chronicity and is responsible for chronic infection of approximately 130 million carriers-i.e., 2.2% of the worldwide population (1). Chronic HCV infection is responsible for chronic hepatitis, a major risk factor in the development of cirrhosis and hepatocellular carcinoma (HCC) or primary liver cancer, which occurs in 1 to 4% of cirrhotic patients every year (2, 3). Chronic HCV infection has become the principal cause of HCC in Japan, and modeling of the ongoing epidemics predicts a similar trend in Europe (4, 5).HCV is an enveloped, positive-strand RNA virus with a cytoplasmic life cycle. HCV replication is catalyzed by an RNA-dependent RNA polymerase (RdRp). It takes place within a membranous web (replication complex) located close to perinuclear membranes, in close association with the nonstructural HCV proteins and a number of host cell factors that play an important role in this process (6, 7). The precise functions of HCV nonstructural protein 5A (NS5A) remains largely unclear. However, it has been shown to be a pleiotropic serine phosphoprotein, involved in the regulation of RdRp function as well as in a number of interactions with host cell mechanisms (8). Studies have also suggested a role for NS5A in the establishment of chronicity of HCV infection and in procarcinogenic events (9).Among its numerous properties, the HCV NS5A protein has been shown to bear transcriptional activation properties in yeast (Saccharomyces cerevisiae) and mamma...

Volume 87, no. 10, p 5523-5539, 2013. Page 5528, Fig. 3G: We mistakenly introduced an incomplete set of data and plotted these data as percentage of NS5A-v1 (100%) which was inconsistent with Fig. 3E which was plotted as percentage of NS5A-Nim (100%). The data set presented corresponded to only one experiment, while the transactivation levels and replication efficiencies of the constructs presented in the rest of the manuscript corresponded to an average of 3 independent experiments. Therefore, we corrected Fig. 3G by inserting the complete set of data (average of 3 experiments, instead of 1 experiment) and, to be consistent with Fig. 3E, by replotting this data set using a percentage of NS5A-Nim (100%) for the replication efficiency instead of a percentage of NS5A-v1.In addition, the figure legend was clarified by adding the source of all constructs represented in the graph, including the two swapped chimeras. We also clarified the y and x axes and the origin of each data point by using a letter code. This letter code is explained in the corrected legend. Figure 3G and its corrected legend should appear as shown below. (G) Relationship between the transcriptional activation capacities of NS5A variants and the replication efficiencies of the corresponding subgenomic replicons. Data points represent the mean transactivational capacities as a function of replication efficiencies for NS5A variants v1, v2, v3, v4, and v5 (described in the legend for Fig. 3A), labeled g, f, e, b, and a, respectively, and AR2 swapped variants v1-AR2/v5 and v5-AR2/v1 (described in the legend to Fig. 5), labeled c and d, respectively. Percentages of replication efficiencies were calculated using NS5A-Nim (g) ϭ 100%.