Effects of HCV on basal and tat-induced HIV LTR activation

Abstract

Hepatitis C virus (HCV) co-infection occurs in ∼30-40% of the HIV-infected population in the US. While a significant body of research suggests an adverse effect of HIV on HCV replication and disease progression, the impact of HCV on HIV infection has not been well studied. Increasing data suggest that hepatocytes and other liver cell populations can serve as reservoirs for HIV replication. Therefore, to gain insight into the impact of HCV on HIV, the effects of the HCV Core protein and infectious hepatitis C virions were evaluated on basal and Tat-induced activation of the HIV long terminal repeat (LTR) in hepatocytes. The HIV LTR was highly induced by the HIV transactivator protein Tat in hepatocytes. Activation varied according to the number of NF-kB binding sites present in the LTRs from different HIV subtypes. Involvement of the NF-kB binding pathway in LTR activation was demonstrated using an NF-kB inhibitor and deletion of the NF-kB binding sites. TNFα, a pro-inflammatory cytokine that plays an important role in HIV pathogenesis, also induced LTR activity in hepatocytes. However, HIV LTR activity was suppressed in hepatocytes in the presence of HCV Core protein, and the suppressive effect persisted in the presence of TNFα. In contrast, infectious hepatitis C virions upregulated HIV LTR activation and gene transcription. Core-mediated suppression remained unaltered in the presence of HCV NS3/4A protein, suggesting the involvement of other viral/cellular factors. These findings have significant clinical implications as they imply that HCV could accelerate HIV disease progression in HIV/HCV co-infected patients. Such analyses are important to elucidate the mechanisms by which these viruses interact and could facilitate the development of more effective therapies to treat HIV/HCV co-infection.

Figure 1. Basal and Tat-induced HIV LTR activation in Huh7.5 cells (A), HepG2 cells (B), and Huh7/β-gal cells (C).

Huh7.5 and HepG2 cells were transfected with an HIV subtype B LTR (LTR-B) luciferase construct in the presence or absence of a Tat-expressing vector. 100 ng of each DNA was transfected per well of a 96-well plate. A luciferase assay was performed at 48 hours post-transfection to quantify LTR activation and was expressed as relative luciferase activity. Huh7/β-gal cells were transfected with or without a Tat expression vector. At 48 hours post-transfection, blue cells were counted after β-gal staining. White bars denote basal (no Tat) LTR activity, and black bars denote Tat-mediated LTR activation.

Figure 2. HCV Core-mediated suppression of HIV LTR activation.

A dose-response experiment was performed in Huh7.5 cells for basal (A), as well as Tat-mediated HIV LTR activity (B). Huh7.5 cells (∼2×10⁴ cells per well) were seeded in a 96-well plate and co-transfected with 100 ng of HIV LTR-B luciferase construct or the delNFkB construct (hatched bars) and 20 ng, 100 ng, or 500 ng of an HCV Core expression vector with or without 100 ng of the Tat expression vector. The pCI control vector was used to equilibrate the total amount of DNA per well as well as a negative control. Luciferase assay was performed at 48 hours post-transfection and expressed as relative luciferase activity. Similar experiments were performed in 293T (C) and Jurkat cells (D). For Jurkats, ∼2×10⁶ cells were seeded per well of 24-well plate and co-transfected with 500 ng each of LTR-B or Tat and 250 ng, 500 ng, or 1000 ng for HCV Core using the transfection reagent TransIT-Jurkat (MIRUSBIO). White bars denote basal (no Tat) LTR activity, and black bars denote Tat-mediated LTR activation. A dose-response experiment with HCV NS3/4A was performed in Huh7.5 cells for basal as well as Tat-induced HIV LTR activation (E). The effect of HCV Core was tested in the presence or absence of HCV NS3/4A on basal (F) and Tat-induced LTR activation (G).

Figure 3. TNFα-mediated HIV LTR activation in Huh7.5 cells.

Subtype B, subtype C, and subtype E LTR activation were tested in the absence or presence of increasing concentrations of TNFα (10 ng/mL, 30 ng/mL, and 100 ng/mL) (A). Differences in number of NF-kB binding sites according to the HIV LTR subtypes (B). LTR-B (or delNFkB – denoted by hatched bars) activation was detected in Huh7.5 cells in the absence or presence of the NF-kB inhibitor PDTC at concentrations of 5 µM, 25 µM, and 125 µM (C). TNFα-mediated (100 ng/mL) LTR activation was inhibited in the presence of the NF-kB inhibitor PDTC (100 µM) (D).

Figure 4. HCV Core-mediated suppression of HIV LTR activation in the presence of TNFα.

LTR-B-transfected Huh7.5 cells were co-transfected with or without HCV Core and were treated with increasing concentration of TNFα (0 ng/mL, 100 ng/mL, or 500 ng/mL) (A). 293T (B) and Jurkat cells (C) were transfected with LTR-B and co-transfected with or without HCV Core in the presence or absence of TNFα. White bars denote the LTR-B only condition, while black bars denote the LTR-B+Core condition.

Figure 5. Dose-dependent increase in HIV LTR activation in HCV-infected Huh7.5 cells.

The TCID₅₀ of JFH1 virus harvested from the HCV_JFH1 cell line was 2.68×10⁶/mL per using a previously described methodology . The cells were infected with JFH1 virus at 3 ng/mL and 7.5 ng/mL concentrations of Core protein denoted as HCV+ and HCV++, respectively, and were transfected with HIV LTR-B in the absence (A) or presence of HIV Tat (B). White bars denote basal and black bars denote Tat-mediated LTR activation.

Figure 6. Effects of infectious HCV on HIV transcription.

JFH1 virus infected or uninfected Huh7.5 cells were transfected with or without the pNL4-3luc.R⁻E⁻ vector (A). Increased HIV transcription (as measured by CD24 expression) in HCV-infected cells compared to HCV-uninfected cells (B); HCV infected or uninfected Huh7.5 cells were transfected with the HIV expression vector pNL4-3HSA.R⁻E⁻ containing the CD24 antigen as described in the methods section. The respective numbers indicate as follows: 1– HCV-uninfected Huh7.5 cells with CD24 antibody; 2– HCV-infected Huh7.5 cells with CD24 antibody; 3– HCV-uninfected Huh7.5 cells expressing pNL4-3.HSA-R⁻E⁻ with CD24 antibody; 4– HCV-infected Huh7.5 cells expressing pNL4-3.HSA-R⁻E⁻ with CD24 antibody.