1. HIV Increases HCV Replication in a TGF-β1–Dependent Manner
Wenyu Lin, Ethan M. Weinberg, Andrew W. Tai, Lee F. Peng, Mark A. Brockman, Kyung–Ah Kim, Sun Suk Kim, Carolina B. Borges, Run–Xuan Shao, Raymond T. Chung 
Gastroenterology 
Volume 134, Issue 3, Pages (March 2008)
DOI: /j.gastro
Copyright © 2008 AGA Institute Terms and Conditions

2. Figure 1
(A) HIV gp120 increases HCV replication in Huh7-2-3 cells. To test whether HIV proteins had an effect on HCV replication, we incubated a panel of purified recombinant HIV-1 proteins (10 μg/mL), including SF162 gp120 (SF gp120) (CCR5-tropic), CN54 gp120 (CN gp120) (CXCR4-tropic), Gag, Rev, and Tat with 5000 Huh7-2-3 cells/well in a 96-well plate for 48 hours. The protein lysates were harvested and HCV replication was assayed by HCV core protein expression using the Trak-C core antigen ELISA. **P < .01 for SF gp120 and CN gp120 versus PBS. (B) HIV gp120 enhances HCV replication in OR6 cells. The OR6 cell is an HCV replicon that stably harbors full-length genotype 1b HCV RNA and coexpresses Renilla luciferase. To further assess the effects of HIV proteins on HCV replication in another HCV replication model, OR6 cells (3000 cells/well) were incubated with each HIV protein (final concentration of 10 μg/mL) in a 96-well plate for 48 hours. A total of 50 ng/mL of pegylated IFN was used as an HCV inhibition control. HCV replication was monitored by measuring RLU. **P < .01 for SF gp120 and CN gp120 versus PBS; #P < .001 for all samples vs. PEG-IFN. (C) Inactivated HIV increases HCV replication in HCV replicon OR6 cells. To determine whether inactivated HIV whole virus has an effect on HCV replication, OR6 cells (3000 cells/well) were plated in a 96-well plate with heat-inactivated HIV CXCR4-tropic (NL4-3, black bars) virus, CCR5-tropic (BaL, gray bars) virus, or HIV-negative cell culture supernatant (white bars) for 48 hours. The heat-inactivated viruses were diluted by 4-fold from 1:4 to 1:64 (11.25 to 0.70 ng/mL p24 for NL4-3; 8.75 to 0.55 ng/mL p24 for BaL). HCV replication was determined by RLU. *P < .05 or **P < .01 versus negative control supernatant. (D) gp120 increases HIV replication in the JFH1 model. To assess the effects of HIV proteins on HCV replication in the JFH1 infectious model, Huh7.5.1 cells were infected with JFH-1 virus on day 1. HIV gp120 or Gag protein was added to the JFH-1 infected cells on day 3. Forty-eight hours later, protein lysates were prepared and HCV replication was assayed by HCV core protein expression using the Trak-C core antigen ELISA. **P < .01 for SF gp120 and #P < .001 for CN gp120 versus PBS. (E) HIV gp120 and inactivated HIV increase HCV RNA replication in OR6 cells. #P < .001 for gp120 and HIV versus PBS control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

3. Figure 2
(A) Anti-CCR5 or CXCR4 neutralizing antibodies abrogate a gp120-mediated increase of HCV replication in Huh7-2-3 cells. To determine whether the increase in HCV replication observed with HIV gp120 was dependent on a chemokine receptor, HCV replicon Huh7-2-3 cells (5000 cells/well) were preincubated with control MSIgG (white bars), anti-CCR5 monoclonal antibody (gray bars), or anti-CXCR4 monoclonal antibody (black bars) for 1 hour. All 3 antibodies were used at 5 μg/mL. The antibody-treated cells were then washed with PBS and incubated with PBS, SF gp120, Gag, CN gp120, Tat, and Rev for 48 hours. HCV replication was monitored by HCV core antigen enzyme immunoassay (*P < .05 for SF gp120 or CN gp120 vs PBS). (B) Anti-CXCR4 and anti-CCR5 antibody abrogate the effect of gp120 and inactivated HCV on HCV replication in OR6 cells. To confirm the results obtained in Huh cells in a second independent HCV replicon cell line, OR6 cells were preincubated with 5 μg/mL of MSIgG or neutralizing antibody to CCR5 (gray bars) or CXCR4 (black bars) for 1 hour. Unbound antibodies were washed off with PBS before adding HIV proteins. Antibody-treated OR6 cells were incubated with PBS, 1:16 diluted inactivated CCR5 or CXCR4-tropic HIV supernatant, HIV-negative supernatant, CN gp120, SF gp120, Gag, or Pol for 48 hours. HCV replication was monitored by RLU (*P < .05 vs MSIgG, PBS-treated control cells; **P < .01 vs MSIgG, PBS-treated control cells).
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
(A) Inactivated HIV and gp120 do not further block pISRE-mediated type I IFN-α signaling in JFH1 cells. To assess the effects of inactivated HIV and gp120 proteins on type I IFN-α signaling, we infected pISRE-Huh7.5.1 cells with JFH1 for 6 hours and then incubated them for 48 hours with inactivated HIV, HIV proteins, or negative controls. A total of 100 IU/mL pegylated IFN was then added to the cells for 6 hours and ISRE-stimulated transcription was determined by measuring relative firefly luciferase activity unit (RFLU). (B) Inactivated HIV does not further impair STAT1 proteins in JFH1-infected cells. We analyzed whether inactivated HIV has an effect on the type I IFN signaling pathway protein STAT1. Huh cells were first infected with JFH1 for 6 hours. Inactivated HIV or negative controls were then incubated with Huh7.5.1 cells or JFH1-infected cells for 48 hours. Pegylated IFN (100 IU/mL) was added to selected cells for 6 hours. STAT1, HCV core protein, and β-actin were detected by Western blotting. Lanes 1–4: Huh7.5.1 cells. Lane 1, CXCR4-tropic HIV; lane 2, CCR5-tropic HIV; lane 3, HIV-negative control supernatant; lane 4, 10% fetal bovine serum. Lanes 5–8: JFH1-infected Huh7.5.1 cells. Lane 5, CXCR4 HIV; lane 6, CCR5 HIV; lane 7, negative HIV; lane 8, 10% fetal bovine serum.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
(A) Inactivated HIV and gp120 proteins increase TGF-β1 expression in OR6 cells and cured OR6 cells. Heat-inactivated HIV (1:16 dilution) or HIV proteins (10 μg/mL) were incubated with HCV replicon OR6 cells (black bars) or cured OR6 cells (white bars) in a 96-well plate (3000 cells/well) for 48 hours. TGF-β1 levels were measured by TGF-β1 enzyme-linked immunosorbent assay. #P < .001 for HIV versus serum-free medium in cured OR6 cells; **P < .01 for HIV versus serum-free medium in OR6 cells. (B) Inactivated HIV and HIV gp120 proteins increase TGF-β1 expression in JFH1-infected Huh7.5.1 cells. Uninfected Huh7.5.1 cells (white bars) or Huh7.5.1 cells infected with JFH1 for 48 hours (black bars) were incubated with heat-inactivated HIV (1:16 dilution) or HIV proteins (10 μg/mL) in serum-free medium (10,000 cells/well) for an additional 48 hours. TGF-β1 levels were measured by the TGF-β1 enzyme-linked immunosorbent assay. #P < .001 for HIV versus serum-free medium in Huh7.5.1 cells; **P < .01 for HIV vs serum-free medium in JFH1 cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

6. Figure 5
(A) TGF-β1 has a dose-dependent effect on HCV replication. To assess the effects of TGF-β1 on HCV replication, OR6 cells (3000 cells/well) were incubated with serial dilutions of human TGF-β1 (32 ng/mL to 0 ng/mL) in serum-free medium in a 96-well plate for 48 hours. HCV replication was monitored by measuring RLU. *P < .05 (1 ng/mL) and **P < .01 (2–32 ng/mL) compared with 0 ng/mL TGF-β1 control. (B) Higher concentrations of TGF-β1 decrease OR6 cell viability. To assess the effects of TGF-β1 on cell viability, OR6 cells (3000 cells/well) were incubated with serial dilutions of human TGF-β1 (32 ng/mL to 0 ng/mL) in serum-free medium in a 96-well plate for 48 hours. Cell viability was monitored by assessing Cell Titer-Glo luminescence units (CTLU) normalized to the 0 ng/mL TGF-β1 treatment, which was considered 100% CTLU. **P < .01 (16 and 32 ng/mL) versus 0 ng/mL TGF-β1 control. (C) The correlation between HCV replication and cell viability in the presence of TGF-β1. To normalize the effect of TGF-β1 on HCV replication by its effect on cell viability, we divided OR6 HCV replication (RLU from A) by OR6 cell viability (CTLU from B) in each TGF-β1 treatment. *P < .05 (1 ng/mL), **P < .01 (2 and 4 ng/mL), and #P < .001 (8–32 ng/mL) compared with 0 ng/mL TGF-β1 control. (D) Neutralizing antibody to TGF-β1 abrogates the effects of TGF-β1 on HCV replication in OR6 cells. To study the mechanism by which human TGF-β1 increases HCV replication, OR6 cells were preincubated with PBS (white bars), 5 μg/mL of MSIgG (black bars), or 5 μg/mL of neutralizing antibody to TGF-β1 (gray bars) for 1 hour. Excess antibody was washed off with PBS before adding human TGF-β1. The washed OR6 cells were incubated with serial dilutions of human TGF-β1 (32 ng/mL to 0 ng/mL) in serum-free medium in a 96-well plate for 48 hours. HCV replication was monitored by RLU. *P < .05 (1 ng/mL) and **P < .01 (2–32 ng/mL) compared with control PBS-incubated or MSIgG-incubated cells without added TGF-β1 (0 ng/mL).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Neutralizing antibody to TGF-β1 blocks the effect of HIV on HCV replication in OR6 cells. To determine whether gp120 or heat-inactivated HIV enhanced HCV replication through TGF-β1, OR6 cells were preincubated with PBS (white bars), MSIgG at 5 μg/mL (black bars), or neutralizing antibody to TGF-β1 at 5 μg/mL (gray bars) for 1 hour. The preincubated OR6 cells were washed with PBS. The washed OR6 cells were incubated with recombinant HIV proteins (final concentration of 10 μg/mL) or HIV supernatant (1:16) in serum-free medium in a 96-well plate for 48 hours. HCV replication was monitored by RLU. Quadruplicate testing was performed for each of 2 independent experiments (n = 8). #P < .001 for PBS-incubated or for MSIgG-incubated and HIV-treated cells compared with HIV-negative control in PBS-incubated or MSIgG-incubated cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions