A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins

Abstract

Pairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. 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.

Importance: Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. 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. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale.

FIG 1

Functional map of the entire HCV genome. (A) Digital counting of each insertion mutant in sequenced libraries. (Top) Bar graph of raw sequencing reads from all 15-nt insertion mutants at each passage (input [P0], pool 1 [P1], and pool 2 [P2]). Each bar represents a unique mutant, and bar heights indicate corresponding sequencing reads. (Bottom) Cartoon of HCV genome organization. (B) (Top) Closer look at the genetic footprint of insertion sites located in the p7 and NS2 genes. (Bottom) Cartoon of p7 and NS2 protein domain organization. TM1 helix and TM2 helix, transmembrane helix 1 and 2; TMS1, TMS2, and TMS3, transmembrane segments 1, 2, and 3.

FIG 2

Functional annotation of protein structures of HCV transmembrane regions. (A) Heat map of P2/P1 values in the C-terminal region of HCV core protein. Each box provides the phenotype of mutants containing an insertion after the indicated amino acid. Gray shows a lack of the respective insertion mutant in pool 0. Mutants are separated by their AAA, CGR and RPH/Q amino acid motifs. The color bar corresponds to a range of P2/P1 values [log(P2/P1) = 1 to −4]. Dictionary of secondary structure of proteins (DSSP) provides an overview of helices (zigzag lines) and unstructured loops (straight lines). (Top) Alignment of core amino acid sequences of HCV strains from this study (strain FNX24) and deposited structure (PDB code 2LIF). (Bottom) Ribbon diagrams of annotated structure, along with a tentative model of the peptide’s orientation within the endoplasmic reticulum (ER). (B) Heat map of P2/P1 values in the p7 protein. DSSP secondary structure indicates a turn (arc) toward the C-terminal end of the protein. PDB code 2M6X.

FIG 3

Screening data set for hot spots tolerating small insertions. (A) Insertion mutants’ P2/P1 ratios across the HCV genome. Only data points that fall within a cluster of ≥3 consecutive insertion mutants with nonzero P2/P1 ratios were plotted. We discarded all insertion mutants with P1/P0 values of <0.1 and P2/P0 values of <0.1. At the top is a cartoon depicting HCV genome organization. (B) Engineering infectious epitope-tagged HCV. The graph shows the fitness of cloned epitope-tagged viruses. Fitness is the ratio of mutant virus infectivity to the wild-type infectivity, as determined by a limiting dilution assay of supernatants taken from transfected Huh-7.5.1 cells. Numbers (349 to 7177) refer to the genome position after which the sequence encoding the respective epitope tag was inserted. TC, tetracysteine.

FIG 4

Validation of NS4B’s role in later steps of the viral life cycle. (A) Huh-7.5.1 cells were transfected with individually cloned mutants containing insertions identified in Table 2. Controls were FNX24 parental virus (wild-type), an E1E2 deletion mutant (most of the E1 and E2 coding regions were deleted, making this mutant replication competent but assembly deficient), and a Pol mutant (the mutant’s polymerase motif contains a GDD-to-GNN mutation, making the mutant genome replication defective). Three days after transfection of mutant RNA genomes, cells were fixed and processed for an immunofluorescence assay using an anti-NS5A antibody. The Hoechst dye provides a nuclear counterstain. Scale bar: 20 µm. (B) Infectivity of cell culture supernatants taken from cells transfected with NS4B insertion mutants. We collected cell supernatants at multiple time points after transfection of RNA genomes and determined supernatant infectivity by limiting dilution assay.