Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.

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 ...

). In this study we further investigated early events of viral infection in both 3D and 3W ducks. We present evidence that a lower level of virus replication in the hepatocytes of 3W birds is an additional factor that probably favors transient infection. We suggest that lower virus replication is due to a less rapid covalently closed circular DNA amplification, leading to lower viremias and a slower spread of infection in the liver, and that the slower spread of infection in 3W ducks makes the infection more sensitive to interruption by the host immune responses.

Elodea nuttallii is well-known for its successful invasion to Europe and Asia. In addition to its high growth rate and nutrient storage capacity, chemical defense was recently found to play an important role in excluding other aquatic organisms. To reveal the chemical nature of its allelopathic effects on cyanobacterial development, extraction of plant exudates with different solvents and eluted procedures followed by 72h bioassays with Microcystis aeruginosa was conducted. The results showed that the fraction eluted with methanol exhibited the strongest inhibitory effects, which indicated that the anti-cyanobacterial active allelochemicals released by E.nuttallii was hydrophilic. Then ten phenolic acids were identified in the purified methanol fraction of Elodea exudates by GC-MS analysis. Quantification of the four phenolic acids therein showed the release contents were lower than 10μg/L when E.nuttallii was cultivated at 10g FW/L, which increased with the increasing culture density. When the culture density was 100g FW/L, the detected contents of vanillic acid, protocatechuic acid, ferulic acid and caffeic acid in E.nuttallii culture water were 23.41μg/L, 17.56 μg/L, 12.31 μg/L and 18.93μg/L, respectively. These results were helpful to understand the allelopathy mechanisms of E.nuttallii in aquatic ecosystems and useful in control of harmful cyanobacterial blooms.