HCV Defective Genomes Promote Persistent Infection by Modulating the Viral Life Cycle

Abstract

Defective interfering (DI) RNAs have been detected in several human viruses. HCV in-frame deletions mutants (IFDMs), missing mainly the envelope proteins, have been found in patient sera and liver tissues. IFDMs replicate independently and can be trans-packaged into infectious virions in the presence of full length viral genome. So far, their biological role is unclear. In this study, we have isolated and cloned IFDMs from sera samples and liver tissues of patients infected with HCV genotypes 1b, 2a, and 3a. IFDMs were present in up to 26% of samples tested. Using the in vitro HCV cell culture system, co-expression of the wild type (wt) HCV replicon with HCV IFDMs RNA resulted in increased HCV replication. Additionally, co-transfection of the HCV full length genome RNA and a defective mutant missing the envelope region led to increased viral release, collectively suggesting an important biological role for IFDMs in the virus life cycle. Recently, exosomes, masters of intercellular communication, have been implicated in the transport of HCV viral genomes. We report for the first time that exosomal RNA isolated from HCV sera samples contains HCV defective genomes. We also demonstrate that inhibition of exosomal biogenesis and release influences HCV viral replication. Overall, we provide evidence that the presence of HCV IFDMs affects both viral replication and release. IFDMs exploit exosomes as means of transport, a way to evade the immune system, to spread more efficiently and possibly maintain persistent infection.

Keywords: defective genomes; exosomes; hepatitis C; viral persistence; viral replication.

FIGURE 1

Detection of HCV natural mutants, containing large in-frame deletions in sera samples and liver tissue of HCV-infected individuals with different genotype. (A) Nested PCR amplicons from HCV 3a-infected sera samples were examined on 1% (w/v) agarose gel. Lanes 1, 3, 9, 10 correspond to sera samples containing IFDMs. The remaining lines 2, 4, 5, 6, 7, 8, 11 correspond to sera samples containing full length genomes. Positive controls are depicted in lanes C1, JFH1 full length; C2, JFH1 ΔE1E2. Line M, molecular size marker 1 Kb. (B) PCR amplicons from 1b and 2a HCV samples were similarly examined, using 1 Kb marker, where the IFDMs were observed on 1, 2, 3, 6 lanes for 1b and on 1, 2, 4 lanes for 2a. (C) Nested-PCR amplification products from liver tissue samples of HCV-infected patients (genotypes 1b and 2a). (D) Schematic representation of the defective and full length genomes’ architecture identified in the sera samples and liver tissues of HCV patients chronically infected with genotypes 3a and 1b.

FIGURE 2

Effect of HCV defective genomes on viral replication and release. (A) Effect of IFDMs on HCV replication. The pFK-I₃₄₁PI-Luc/NS3-3′/JFH1–replicating construct (replicon wt) was electroporated in Huh7.5 cells simultaneously with different RNA IFDMs constructs at a ratio 1:1. pHPI 8388 clone (containing the FL HCV sequence) served as a control. The majority of the IFDMs used were HCV genotype 3a (8381, 8387, 8389) and 1b (32A). Cells were collected at 48 h, lyzed and luciferase activity was measured and normalized to total protein. The value for replicon wt was arbitrarily set as 100% and all other values are a percentage of this (seven independent experiments). (B) Quantity of viral RNA (viral copies/mL) from replicon wt, control (containing the FL HCV sequence) and IFDMs 8381, 8387, 8389, 32A measured in the cell culture supernatants of the above experiment with the SACACE Real Time PCR kit for HCV detection. (C) HCV Ag (Core) was measured in cell lysates and supernatants from replicon wt, control (containing the FL HCV sequence) and IFDM HCV 3a (8389) using the Abbott ARCHITECT HCV Ag assay. ^∗p-value ≤ 0.05; ^∗∗p-value ≤ 0.005; ^∗∗∗p-value ≤ 0.001.

FIGURE 3

Prevalence of IFDMs in the HCV in vitro cell culture system. (A) Electroporation experiments were performed in Huh7.5 cells using RNA JFH1 or JFH1 ΔE1E2 at ratios 1:1. The 1% (w/v) electrophoretogram depicts amplicons from isolated IFDMs, following RNA isolation and RT-PCR analysis. M, Molecular size marker. (B) HCV Ag (Core) was measured at 48 post-transfection in cell lysates and supernatants using the Abbott ARCHITECT HCV Ag assay. (C) Quantity of viral RNA (viral copies/mL) was measured in the above cell culture supernatants using the SACACE Real Time PCR kit for HCV detection. ANOVA analysis showed statistical significance between samples. ^∗p-value ≤ 0.05; ^∗∗p-value ≤ 0.005.

FIGURE 4

Identification and function of exosomal IFDMs. (A) Exosomes were isolated, with the Exosome Precipitation Solution (serum plasma; Macherey-Nagel), from sera samples of three distinct groups, healthy blood donors (Healthy), chronic HCV patients with full length genomes (wt) and HCV patients with IFDMs of genotype 3a (IFDMs). Exosomes were characterized by Western blotting analysis using antibodies against exosomal markers CD63, CD9 and Hsp90. (B) In a representative experiment, exosomal RNA (Exo RNA) was subjected to nested PCR analysis and run on a 1% (w/v) agarose gel against a molecular size marker (M). Bold and thin arrows indicate the IFDM and FL sequences, respectively. (C) pFK-I₃₄₁PI-Luc/NS3-3′/JFH1-replicating construct was co-electroporated with representative IFDMs RNA constructs in Huh7.5 cells in the presence of the exosomal inhibitor GW4869. 5 μM of the inhibitor GW4869 (Sigma) were added to the electroporated cells, in order to block exosomes’ release. Cells were collected at 48 h, lyzed and luciferase was measured and normalized to total protein. The values for replicon wt/replicon wt+inhibitor were arbitrarily set as 100% and all other values are a percentage of these. ^∗p-value ≤ 0.05; ^∗∗p-value ≤ 0.005; ^∗∗∗p-value ≤ 0.001.