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

Remenyi R, Qi H, Su SY, Chen Z, Wu NC, Arumugaswami V, Truong S, Chu V, Stokelman T, Lo HH, Olson CA, Wu TT, Chen SH, Lin CY, Sun R - MBio (2014)

Bottom Line: To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures.Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, USA.

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Related in: MedlinePlus

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.
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fig4: 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.

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.


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

Remenyi R, Qi H, Su SY, Chen Z, Wu NC, Arumugaswami V, Truong S, Chu V, Stokelman T, Lo HH, Olson CA, Wu TT, Chen SH, Lin CY, Sun R - MBio (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196222&req=5

fig4: 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.
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.

Bottom Line: To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures.Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, USA.

Show MeSH
Related in: MedlinePlus