A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins.
Bottom Line: To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures.Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains.Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile.
Affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, USA.Show MeSH
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Mentions: To find new nonstructural-protein functions not related to traditional roles in genome replication, we applied the aforementioned criteria (along with a minimum of 20 for input P0 reads) and generated a candidate list of 13 mutants with insertions in nonstructural proteins (Table 2). Because insertions in NS4B dominated this list (8 out of 13 mutants), we focused our validation efforts on NS4B. Individually cloned NS4B mutants with insertions at positions 5571, 5584, 5597, 5607, and 5615 showed a defect in spreading in cell culture, as evidenced by immunofluorescence studies examining the number of HCV-positive cells after transfection of viral genome RNA (Fig. 4A). Moreover, the insertions reduced infectivity to 1 to 6% of the levels seen for cells transfected with a wild-type genome at 3 days following transfection (Fig. 4B). We observed similar defects in infectious virus production at 1 and 5 days (Fig. 4B). Both our immunofluorescence (Fig. 4B) and Western blotting (data not shown) suggested that the insertions in NS4B reduced NS5A protein levels only slightly, which indicated that viral RNA genomes still replicated robustly. Thus, we conclude that the decreases in infectious virus particle production may be due to additional defects in steps after genome replication, such as viral assembly or egress. In summary, we identified a new region in the NS4B protein that may to be essential for steps following genome replication. Importantly, we showed that our data set could serve as a resource to screen for insertion mutants that reveal protein regions with previously unrecognized biological functions.
Affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, USA.