Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers. We performed exome sequencing on 113 tumor-normal pairs, yielding a mean of 82 non-silent mutations per tumor, and 8 cell lines. The mutational profile of ESCC closely resembles those of squamous cell carcinomas of other tissues but differs from that of esophageal adenocarcinoma. Genes involved in cell cycle and apoptosis regulation were mutated in 99% of cases by somatic alterations of TP53 (93%), CCND1 (33%), CDKN2A (20%), NFE2L2 (10%) and RB1 (9%). Histone modifier genes were frequently mutated, including KMT2D (also called MLL2; 19%), KMT2C (MLL3; 6%), KDM6A (7%), EP300 (10%) and CREBBP (6%). EP300 mutations were associated with poor survival. The Hippo and Notch pathways were dysregulated by mutations in FAT1, FAT2, FAT3 or FAT4 (27%) or AJUBA (JUB; 7%) and NOTCH1, NOTCH2 or NOTCH3 (22%) or FBXW7 (5%), respectively. These results define the mutational landscape of ESCC and highlight mutations in epigenetic modulators with prognostic and potentially therapeutic implications.
Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-␣ genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA.
The mechanisms of hepatitis C virus (HCV) replication remain poorly understood, and the cellular factors required for HCV replication are yet to be completely defined. CD81 is known to mediate HCV entry. Our study uncovered an unexpected novel function of CD81 in the HCV life cycle that is important for HCV RNA replication. HCV replication occurred efficiently in infected cells with high levels of CD81 expression. In HCV-infected or RNA-transfected cells with low levels of CD81 expression, initial viral protein synthesis occurred normally, but efficient replication failed to proceed. The aborted replication could be restored by the transient transfection of a CD81 expression plasmid. CD81-dependent replication was demonstrated with both an HCV infectious cell culture and HCV replicon cells of genotypes 1b and 2a. We also showed that CD81 expression is positively correlated with the kinetics of HCV RNA synthesis but inversely related to the kinetics of viral protein production, suggesting that CD81 may control viral replication by directing viral RNA template function to RNA replication. Thus, CD81 may be necessary for the efficient replication of the HCV genome in addition to its role in viral entry.Hepatitis C virus (HCV) infection affects about 170 million people worldwide. Chronic HCV infection is an important cause of liver diseases, leading to cirrhosis and hepatocellular carcinoma (2, 18). The therapy for chronic HCV infection to date is suboptimal and associated with many side effects (12, 13). The mechanisms of HCV replication and persistent infection remain poorly understood (3, 31).HCV carries a positive-and single-stranded RNA genome consisting of approximately 9,600 nucleotides (nt) (36). HCV encodes 10 proteins and exploits cellular factors for replication (24,32,35,41). However, many crucial host factors required for HCV RNA replication remain undefined. The HCV RNA genome, like other positive-stranded RNA viruses, serves as templates for both viral protein translation and RNA replication (4, 15, 28), which are expected to be asynchronous in vivo, as the template pool is constantly replenished from ongoing HCV infection and replication (4). However, a coordinated translation/transcription process would be predicted if the use of HCV RNA as a template is subjected to cellular factor control that directs HCV RNA for specific template functions and synchronizes the translation/transcription process. CD81 has diverse functions in various biological processes (23,39,48) and is known to mediate HCV entry (10,30,34,49). CD81 was recently suggested to play a role in postentry events (8). In this study we identified CD81 as a key cellular factor required for efficient HCV RNA replication, and inefficient RNA replication occurs in HCV-infected or RNA-transfected cells with low levels of CD81. Our data also showed that the utilization of HCV RNA as templates for viral protein synthesis and RNA synthesis is mutually exclusive and suggested that HCV RNA template function for RNA replication may be subjected to ...
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