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Results: 5

1.
FIG. 2.

FIG. 2. From: Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a .

Replication levels in 11 unique clonal cell lines harboring a genotype 1a replicon without a luciferase reporter. After transfection with the 1a H77 neo replicon, individual G418-resistant colonies were isolated, expanded, and tested in an NS3 protease activity assay (as a marker for HCV replication levels). For comparative purposes, the genotype 1b luciferase replicon cell line Huh-Luc was also tested in the assay. RFU, relative fluorescence units.

Margaret Robinson, et al. Antimicrob Agents Chemother. 2010 August;54(8):3099-3106.
2.
FIG. 4.

FIG. 4. From: Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a .

Robust luciferase expression in 1a luciferase cell clones. Stable cell lines replicating either the 1a H77 Rluc-Neo, 1a H77 Gluc-Neo, or 1a SF9 Rluc-Neo replicon were assayed for luciferase activity. In the nomenclature of the cell lines, 51C and 57C refer to the cured cell line that the cells were derived from, H77 and SF9 refer to the strain of HCV the replicons were constructed with, Gluc and Rluc refer to the reporter gene (Gaussia luciferase or Renilla luciferase), and the terminal number indicates the clone number. Error bars show standard deviations. RLU, relative light units.

Margaret Robinson, et al. Antimicrob Agents Chemother. 2010 August;54(8):3099-3106.
3.
FIG. 1.

FIG. 1. From: Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a .

Design of novel 1a reporter replicons. HCV replicons used to generate novel, stable genotype 1a replicon cell lines. (A) A neomycin phosphotransferase (Neo)-encoding H77 1a strain replicon. (B) An H77 1a strain replicon encoding a Gaussia luciferase (Gluc)-Neo fusion reporter. (C) An H77 1a strain replicon encoding a Renilla luciferase (Rluc)-Neo fusion reporter. (D) An SF9 1a strain replicon encoding an Rluc-Neo fusion reporter. A black segment indicates an HCV core sequence. A spotted segment indicates an SF9 sequence. “L” indicates the P1496L adaptive mutation. “I” indicates the S2204I adaptive mutation. “K” indicates the E1202K adaptive mutation. “Y” indicates the D1431Y adaptive mutation. The 5′ and 3′ nontranslated regions (NTR) and encephalomyocarditis virus (EMCV) IRES segments are indicated.

Margaret Robinson, et al. Antimicrob Agents Chemother. 2010 August;54(8):3099-3106.
4.
FIG. 5.

FIG. 5. From: Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a .

Antiviral response is consistent across multiple genotype 1a replicon cell lines. Novel genotype 1a luciferase-encoding replicon cell lines were seeded in 96-well plates and treated with the indicated HCV inhibitors for three days. After drug treatment, the dose-response for each antiviral was quantified by measuring luciferase activity in duplicate, and EC50s were calculated by nonlinear regression. EC50s are plotted on the y axis, the antiviral compounds on the x axis, and the cell lines are indicated by the symbols showing the data points (○, 1A 51C-H77-Gluc1; ⋄, 1a 51C-H77-Gluc2; ▿, 1a 51C-H77-Gluc3; •, 1a 57C-H77-RlucP; ♦, 1a 57C-SF9-Rluc1; ▾, 1a 57C-SF9-Rluc2; ▪, 1a 57C-SF9-Rluc3). Each data point represents the arithmetic mean of the results of two independent experiments. Horizontal lines represent the average EC50 across all seven cell lines.

Margaret Robinson, et al. Antimicrob Agents Chemother. 2010 August;54(8):3099-3106.
5.
FIG. 3.

FIG. 3. From: Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a .

The cured cell lines are highly permissive to genotype 1a replication but also support replication of genotypes 1b and 2a. (A) RNA encoding the genotype 1a replicon pH/SG-neo(L+I) was electroporated into the Huh7-Lunet (top left) or the 51C cured cell line (top right). After 3 weeks of selection with G418, resistant colonies, which harbor the genotype 1a replicon, were visualized with crystal violet. The 51C cell line (top right) gave rise to a significantly greater number of stable replicon clones than the Huh7-Lunet cell line (top left). Confirming that 51C cells are cured of the HCV replicon, electroporation without RNA (Mock) resulted in fully G418-sensitive cells for both cell lines (bottom). (B) Genotype 1a [pH/SG-GlucNeo(L+I)], 1b (pFK-rep PI-luc/5.1), and 2a replicons (pLucNeo2a) were transfected into either 57C cured (white bars) or Lunet cells (black bars), and luciferase levels at 3 days posttransfection were measured. To control for transfection efficiency, data were normalized to luciferase levels at 4 hours posttransfection. For each genotype, replication in Lunet cells is displayed as a percentage of the replication in 57C cured cells. Error bars show standard deviations.

Margaret Robinson, et al. Antimicrob Agents Chemother. 2010 August;54(8):3099-3106.

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