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1.
Fig. 8.

Fig. 8. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

Phosphomimetic Ala-to-Asp substitutions restore production of infectious virus by AWA and 4SA mutant RNAs. Shown are infectious virus yields from cells transfected with H77S.3 and H77S.3/4SA RNAs (A) and H77S.3, H77S.3/DWD, and H77S.3/4SD mutants (B). Results shown are the means ± SE from two transfections with independent RNA transcripts. At the bottom are immunoblots of NS5A and core protein expression with actin (A) and GAPDH loading controls (B).

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.
2.
Fig. 4.

Fig. 4. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

Infectious virus titer of cell culture supernatant fluids and intracellular lysates of JFH-1 and JFH-1/H5Ad3 RNA-transfected cells on day 3 after transfection. Lysates were prepared by multiple freeze-thaw cycles. Shown are the means ± SE from duplicate transfections. Percentages are the proportions of the total virus yield released into supernatant (Sup) culture fluids (i.e., virus in supernatant/virus in supernatant + virus in lysate).

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.
3.
Fig. 2.

Fig. 2. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

(A) Virus yields (extracellular culture fluids) from RNA-transfected cells after intergenotypic (genotype 1a→2a and genotype 2a→1a) exchanges of NS5A or NS5A domain III. Culture supernatants obtained at day 3 after transfection were assayed for infectious virus. Shown are the mean yields from each chimera in triplicate transfections ± standard deviations (SD). (B) ELISA for the HCV core protein present within supernatant culture fluids of RNA-transfected cells. Data shown represent mean values ± SE determined from duplicate experiments.

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.
4.
Fig. 6.

Fig. 6. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

Inhibition of virus production by DMAT, an inhibitor of CK II. (A) Following transfection of the indicated RNA, cells were treated with the indicated concentration of DMAT for 48 h. The medium was then replaced with fresh medium (no drug), followed 24 h later by the harvesting of supernatant fluids for virus titration. Means ± SE were calculated from duplicate experiments. (B) Immunoblots for NS5A, NS2, NS3, and GAPDH from cell lysates prepared 72 h after transfection. (C) Cytotoxic effects of DMAT assessed by a WST-1 cellular proliferation assay. Means ± SE were calculated from triplicate experiments.

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.
5.
Fig. 9.

Fig. 9. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

Sequence logo depiction of amino acid sequence conservation within domain III of NS5A among genotype 1a (224 sequences), genotype 1b (357 sequences), and genotype 2a (18 sequences) strains of HCV from the European HCV Database (euHCVdb) (). Sequences were aligned with MUSCLE (), with minor manual modifications, and logos were generated with WebLogo (). The height of each single-character amino acid code is proportional to the representation of that amino acid at each position. The sequence extending from Ser-432 to Thr-442, which contains potential phosphoacceptor residues in H77S, is boxed in red. To show how representative the H77S sequence is of other genotype 1 viruses, residues identical to those in H77S are shown in blue in the genotype 1a and 1b sequences, while residues that differ from those in H77S are shown in gray. Residues identical to JFH-1 are shown in red in the genotype 2a sequence.

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.
6.
Fig. 3.

Fig. 3. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

Ectopically expressed NS5A proteins. (A) Expression vectors encoding various NS5A molecules, each with an N-terminal Myc tag, were transfected into Huh-7.5 cells along with a second vector expressing Gaussia luciferase as a transfection control. Cell lysates were prepared 72 h later and subjected to SDS-PAGE followed by immunoblotting with the 9E10 monoclonal antibody or an anti-Myc antibody. The results shown are representative of data from replicate experiments. (B) 9E10 and anti-Myc antibodies bound by NS5A proteins in parallel immunoblots of cell lysates were detected with an infrared fluorescent probe and quantified by using an Odyssey fluorescent scanner. Results for each antibody were normalized to the amount bound by the JFH-1 protein. The results shown represent the means ± SE from 3 separate transfection experiments. The JFH-1/H5Ad3 protein demonstrated a greater binding of 9E10 than of anti-Myc, but there are no significant differences in the recognitions of JFH-1 and H77S NS5A by these antibodies. The equivalent abundance when the NS5A proteins were probed by anti-Myc antibody suggests that there are no significant differences in the intrinsic stabilities of these proteins. Differences in transfection efficiency, monitored by assessing luciferase activity, were minimal.

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.
7.
Fig. 5.

Fig. 5. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

Replication and infectious virus yields from the structural protein chimera HJ3-5 with or without exchange of domain III of NS5A from H77S virus. (A) Schematic showing the organization of the HJ3-5 intergenotypic chimeric RNA and the related NS5A domain III (d3) swap with the H77S sequence. The H77S sequence (core-NS2) is shown as open boxes, while the JFH-1-derived sequence (NS3-NS5B) is shaded; noncoding RNA segments are from JFH-1. The asterisks indicate the location of compensatory mutations (E1 and NS3) that promote yields of infectious virus from the chimera (). In HJ3-5/ΔE1-p7 and ΔE1-p7/H5Ad3, the E1-p7 sequence has been deleted. (B) Infectious virus production. Shown are the means ± ranges of yields of infectious virus from chimeric RNAs, calculated from duplicate transfections. (C) ELISA for core protein secreted by cells transfected with HJ3-5 and HJ3-5/H5Ad3 RNAs. Means ± SE were calculated from duplicate experiments. (D) Immunoblots for the HCV core protein in lysates of HJ3-5 and HJ3-5/H5Ad3 RNA-transfected cells. Actin served as a loading control. Also shown are immunoblots for NS5A in lysates of cells transfected with the related ΔE1-p7 mutant RNAs; GAPDH was the loading control. (E) Real-time RT-PCR measurements of HCV RNA replication. The relative HCV RNA copy number represents the copy number for each construct relative to a replication-lethal GND mutant, normalized to the value present 4 h after transfection. Results shown represent the means ± SE calculated from triplicate RT-PCR assays.

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.
8.
Fig. 1.

Fig. 1. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

Construction and evaluation of replication properties of NS5A chimeras. (A) Amino acid sequences of NS5A domain III in H77S and JFH-1 viruses. The amino acid sequence identity between these two domains is 46.2%. (B) Schematic diagram of the NS5A chimeras constructed for these studies. The entire NS5A sequence or NS5A domain III was exchanged between H77S and JFH-1 to assess the impact on infectious virus production and viral RNA replication. (C) Replication properties of transfected H77S (left) and JFH-1 (right) chimeric RNAs. The abundance of in vitro-transcribed genomic RNA relative to that of a replication-defective mutant with a GND mutation in NS5B was determined at various times following transfection by using a real-time RT-PCR assay. Shown are mean values ± standard errors (SE) from triplicate RT-PCR assays. (D) Immunoblots of lysates of HCV RNA-transfected cells showing the abundance of core and NS5A in comparison to actin and GAPDH loading controls, respectively. Faint bands are indicated to their left as follows: “x,” H77S NS5A; “o,” H77S/J5Ad3 NS5A; “s,” additional hyperphosphorylated NS5A species (see the text); “*,” nonspecific band. The predicted molecular masses of the H77S, JFH-1, and chimeric NS5A proteins, prior to any posttranslational modification, are shown between the NS5A and GAPDH blots. Pred. NS5A m.w., predicted NS5A molecular weight (in thousands).

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.
9.
Fig. 7.

Fig. 7. From: Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III.

Impact of Ala substitutions at potential Ser/Thr phosphoacceptor site residues in domain III of H77S on viral RNA replication and infectious virus production. (A) Possible Ser/Thr phosphoacceptor sites in the C-terminal region of domain III of the NS5A proteins of JFH-1 and H77S viruses. At the top, the H77S and JFH-1 sequences are aligned: Ser residues previously found to be important for the NS5A-core interaction and the assembly and release of infectious JFH-1 virus by Masaki et al. () (red box) and Ser-457, identified by Tellinghuisen et al. () as a site of CK II phosphorylation (red arrow), are highlighted. Within the related H77S sequence, Ser-438 and Thr-442 are possible sites of CK II phosphorylation predicted by the NetPhos 1.0 server. Below are the C-terminal NS5A sequences of the single- and multiple-Ala-substitution mutants studied. Potential Ser/Thr phosphoacceptor sites studied are shown in red, while Ala substitutions ablating these sites in the mutants are shown in boldface type. (B) Potential phosphorylation sites near the C terminus of domain III of H77S NS5A were mutated to Ala to assess their role(s) in infectious virus production. Shown are the mean virus yields following the transfection of the mutant RNAs, normalized to that of the H77S virus (100% = mean of 160 FFU/ml) ± SE from two independent transfections. (C) HCV RNA replication compared to the replication-lethal GND mutant measured by a real-time RT-PCR assay. The means ± SE were calculated from triplicate RT-PCR assays. (D) Immunoblots for HCV NS5A, core, and GAPDH in lysates of RNA-transfected cells. Pred. m.w., predicted molecular weight (in thousands).

Seungtaek Kim, et al. J Virol. 2011 July;85(13):6645-6656.

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