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RNAi Screen Uncovers Host Factors Interacting with HCV

NEW YORK (GenomeWeb News) – In a paper scheduled to appear online this week in the Proceedings of the National Academy of Sciences, scientists used an RNA interference screen to find host factors exploited during hepatitis C virus or HIV infection and pathogenicity.

Researchers from the National Institutes of Health, Harvard Medical School, the Massachusetts Institute of Technology, Massachusetts General Hospital, and Brigham and Women's Hospital used a genome-wide small interfering RNA screen in a cell culture system to look for human host factors that interact with HCV. The work implicated both new and previously identified host factors in the way HCV enters and infects cells and is subsequently propagated.

"The identification of host factors participating in the complete HCV lifecycle will both advance our understanding of HCV pathogenesis and illuminate therapeutic targets," senior author Stephen Elledge, a Harvard geneticist, and his co-authors wrote.

Much is already known about the way HCV enters and functions in the cell. For instance, previous work has shown that two HCV envelope proteins interact with receptor proteins on human cells. And once inside the cell, the HCV genome is translated by host ribosomes on the rough endoplasmic reticulum.

The resulting proteins are then processed by both human and viral proteases, with HCV proteins orchestrating the organization of HCV replication complexes in the human cell. These complexes churn out more viruses that, in turn, get packaged and transported to the cell surface before going on to infect other cells.

Even so, Elledge and his team argued that there is still much to learn about HCV-host interactions. They tackled this problem using the HCV cell culture system — an infection competent cell culture that let them recreate not just part of the HCV infection process but the entire viral life cycle. Although siRNA screens focusing on specific targets have been done in such cell systems, the team was keen to take a more genome-wide approach.

"Given the complexity of the virus' relationship with the host cell, and the relatively unexplored mechanisms of HCV replication," they wrote, "we hypothesized that additional host factors might be found by completing an unbiased whole-genome siRNA screen with the [HCV cell culture system].

Elledge and his team infected a liver cell line with an HCV genotype 2a strain called JFH-1. The cell line had been transfected with a whole-genome siRNA library three days earlier. After two days, the researchers stained and imaged the core HCV protein in the cells to find out which were actively infected by the virus.

They also tossed some of the supernatant from this first batch of cells into new liver cells in an effort to find host players involved in later stages of infection. Again, these cells were stained and imaged after two days to determine their infection levels.

After identifying cells in which HCV infection was increased or decreased, and validating their results, the team was left with more than 250 siRNA pools, some affecting genes previously implicated in HCV and others affecting genes with no known role in the viral infection.

In particular, the researchers noted, they found genes involved in processes such as protein metabolism, protein modification, nucleic acid binding and metabolism, and oncogenesis. And more than 30 of the genes found in the screen were implicated in the pathogenesis of HCV or West Nile virus, another flavivirus, in past studies, they noted.

Genes coding for cell surface receptors also showed up, as did a surprising number of nuclear and nucleolar factors. In addition, the researchers pinpointed ten host genes that are apparently involved in both HCV and HIV infection.

Next, the researchers used a meta-analysis to bring together their findings with those from a proteomic and functional study by French researchers called the HCV infection mapping project, or I-MAP. By generating a protein-protein interaction network, the team was able to not only verify some existing HCV interaction networks but also add to these.

"Many interesting connections have emerged from the integration of these efforts that will serve as a resource for future investigations," the authors wrote.

While they cautioned that there are possible limitations to the sort of siRNA screen used in the study — including false positives caused by off-target effects — the team expressed enthusiasm that "[t]he genes identified by these efforts represent a starting point for defining a complete set of HCV-host interactions."

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