Counihan NA, Rawlinson SM, Lindenbach BD. 2011. Physique?S1, PDF file, 0.2 MB mbo005142011sf1.pdf (177K) GUID:?D0F05EEE-08C8-466E-9EB2-BD4053E41341 Physique?S2: Mapping tolerated insertions to the structure of p7 viroporin and N terminus of NS2. (A) Surface views of annotated p7 viroporin structure (PDB code 2M6X). Gray indicates a lack of the Calcitetrol respective insertion mutant in pool 0. We colored amino acid residues in the p7 structure according to the warmth map in Fig.?2B. Functional data for AAA, CGR, and RPH/Q frames Calcitetrol are shown separately. Surface transparency was set to 65% for top and bottom views to reveal ribbon representations inside viroporin. Yellow dotted circles show area tolerating insertions for three reading frames. (B) Helical segments (zigzag lines), turns (arcs) and unstructured loops (straight lines) were deduced from NMR analyses (PDB codes 2JY0, 2KWT, and 2KWZ) of NS2 peptides (aa 1 to 27, 27 to 59, and 60 to 99). (Bottom) Tentative model of NS2 topology within the ER membrane. Download Physique?S2, PDF file, 0.2 MB mbo005142011sf2.pdf (261K) GUID:?1B3FBCCF-F3BF-4610-B5EE-0D067A0AD302 Physique?S3: Immunofluorescence assay of cells infected with epitope-tagged HCV. Huh-7.5.1 cells were infected with indicated epitope-tagged viruses. Three?days after contamination, we fixed and processed cells for immunofluorescence using antibodies against the inserted epitope tag and either NS5A or E2 viral proteins. Hoechst dye staining the nuclei of both infected and uninfected cells. Images were acquired by epifluorescence microscopy. Level bar: 10 m. Download Physique?S3, PDF file, 0.2 MB mbo005142011sf3.pdf (180K) GUID:?FC1CD211-F890-48E2-B143-1ED604434064 Physique?S4: Four-color fluorescence microscopy of cells infected with NS5A-FLAG computer virus. (A) (Right) Overview of an infected cell, with the nucleus marked by a dashed collection. We acquired multiplexed images with specific filters for each fluorochrome and pseudocolored each channel as indicated: lipid droplets in gray, core in green, E2 in reddish, and NS5A-FLAG in blue. Bar, 10?m. The box indicates a perinuclear area rich in lipid droplets and expressing viral proteins. (Left) Zoom-in of the boxed region. Bar, 1?m. (B) To better visualize localization of each viral protein with regard to lipid droplets, these zoom-in views show only two channels at a time. Here, we pseudocolored lipid droplets in reddish and each of the three viral proteins in green (NS5A, E2, and core). Arrows show areas made up of spatially overlapping signals in the NS5A, E2, and core channels. Download Physique?S4, PDF file, 0.1 MB mbo005142011sf4.pdf (74K) GUID:?39FCB7AD-E411-409F-A5FB-45315BF060D1 Table?S1: List of primers and antibodies used in this study. Table?S1, DOCX file, 0.02 MB. mbo005142011st1.docx (18K) GUID:?7D6E9EFD-DD25-4C4D-8CC6-E691B3549C8D 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 computer Rabbit Polyclonal to SHD 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 experts, 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 made up of one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered computer virus pools, and simultaneously assayed the large quantity 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 power of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we Calcitetrol 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 experienced 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 useful new resources for the research community that can direct the attention of investigators toward unidentified functions of individual protein domains. IMPORTANCE Our insertional mutagenesis library provides a resource that illustrates Calcitetrol 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. Experts 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.