Meredith LW, Farquhar MF, Tarr AW and McKeating JA.
Hepatology 2014, in press
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Type I IFN stimulates the expression of a number of interferon-stimulated genes (ISGs) that inhibit HCV genome translation and replication and protect neighboring uninfected cells from viral infection. Once IFN pathways are activated the downstream response has enormous breadth, as noted by the many hundreds of ISGs.
It has been shown that the amount of HCV RNA present declines in a bi-phasic pattern after IFN therapy, with the initial faster phase representing a decrease in viral production from infected cells, whilst the slower and more variable second phase is attributed to the loss of infected cells via the stimulated immune system. Clinically, there are multiple outcomes observed following IFN therapy, including rapid, partial and non-response, where a rapid virological response is frequently associated with a cure.
Reports of IFN-mediated signaling generally study cellular responses within minutes of receptor engagement. However, studies investigating the anti-viral effects of IFN measure HCV RNA and proteins several days post treatment. We demonstrate that treating HCV infected Huh-7 cells with IFNα for 1h reduces the infectivity of extracellular particles with no detectable effect on particle secretion. We failed to detect soluble anti- viral effectors released from IFN treated cells and purified particles show reduced infectivity, demonstrating a direct effect of IFNα on extracellular HCV particles. Studies with lentiviral HCV pseudoparticles (HCVpp) demonstrate that IFN alters E2 glycoprotein conformation that limits receptor-dependent internalization. This is the first report of such a rapid and direct effect of IFNα to reduce particle specific infectivity in the absence of of any detectable impact on viral RNA load, raising questions on the interpretation of early phase IFN responses that are solely based on viral RNA measurements
Background: Interferon-alpha (IFNα) has been used to treat chronic hepatitis C infection for over 20 years with varying efficacy depending on the infecting viral genotype. The mechanism of action of IFNα is not fully understood, but is thought to target multiple stages of the HCV lifecycle, inhibiting viral transcription and translation leading to a degradation of viral RNA and protein expression in the infected cell. IFNα induces the expression of an array of interferon-stimulated genes within minutes of receptor engagement, however, the impact of these early responses on the viral lifecycle are unknown. Results: We demonstrate that IFNα inhibits the genesis of infectious extracellular HCV particles within 2h of treating infected cells, with minimal effect on the intracellular viral burden. Importantly, this short duration of IFNα treatment of infected cells significantly reduced cell-free and cell-to-cell dissemination. The secreted viral particles showed no apparent change in protein content or density, demonstrating that IFNα inhibits particle infectivity but not secretion rates. To investigate whether particles released from IFNα treated cells have a reduced capacity to establish infection we utilized HCV lentiviral pseudotypes (HCVpp) and demonstrated a defect in cell entry. Using a panel of monoclonal antibodies targeting the E2 glycoprotein we demonstrate that IFNα alters glycoprotein conformation and receptor utilization. Conclusions: These observations show a previously unreported and rapid effect of IFNα on HCV particle infectivity that inhibits de novo infection events. Evasion of this response may be a contributing factor in whether a patient achieves early or rapid virological response, a key indicator of progression to sustained virological response or clearance of viral infection.
Figure 1. IFNα rapidly reduces extracellular virus infectivity.
(A)The fold change in 64 ISGs in HCV infected cells treated with IFNα (1000 IU/mL for 1h) compared to untreated cells.
(B) HCV infected Huh-7.5 cells were treated with an increasing dose of IFNα for 1h. The amount of extracellular virus released and the amount of virus present in cellular lysates after another hour were measured by inoculating naïve Huh-7/5 target cells. Control target cells were treated with a Jak/Stat inhibitor (InSolution JAK Inhibitor 1, 10µM) for 1h prior to infecting with the virus harvested from cells treated with the highest dose of IFNα. A clear inhibition of infectious virus release, but not of intracellular virus infectivity, is apparent
(C)The infectivity of ultracentrifugation purified extracellular virus was measured in the presence or absence of Jak/Stat inhibitor. These data suggest that the loss of infectivity is a property of the released virion rather than a factor in the cell supernatent.
Figure 2. IFNα does not perturb HCV particle composition or density.
The lipid density of the treated and untreated virus was assessed using 10-60% Iodixanol gradient centrifugation: the graphs depict the (A)infectivity and (B), HCV RNA content of each fraction from IFN-treated or untreated cells.
Figure 3. IFNα inhibits both cell-free and cell-to-cell HCV transmission.
(A) HCV infected Huh-7.5 cells were treated with IFNα (1000 IU/mL) for 1h, washed thoroughly to remove IFNα, labeled with CMFDA and co-cultured with naïve Huh-7.5 cells. 1h post co-culture anti-HCV Ig was added to neutralize extracellular virus and at the indicated time points an anti-CD81 mAb was added to block all subsequent transmission events. After 24h the levels of the infectious virus in the extracellular media were measured (A) and de novo infection events determined by flow cytometry (B). Results represent the mean and standard deviation of 3 experiments, and significance calculated using Student’s T-tests (* p<0.05, ** p<0.01, *** p<0.005) comparing treated sample to untreated controls.
Figure 4. IFNα perturbs HCV glycoprotein conformation.
HCVpp-H77 expressing 293T cells were treated with IFNα (1000 IU/mL) for 1h, washed and incubated for a further hour. A panel of mAbs targeting linear E1E2 epitopes in regions A, B and C of E2, a mAb specific for E1 (3/80w) and a control polyclonal patient derived anti-HCV Ig were assessed for binding to the cell surface (left) or to released HCVpp (right) by flow cytometry or ELISA, respectively. IFNα reduced the binding of several antibodies targeting the HVR/region A (6/16, 6/82A, 9/86A, 2/69A) and region B (7/16B, 6/1A), but had no significant effect on mAbs binding region C. These results confirm that IFNα induces a conformational change in the viral E2 glycoprotein that limits particle entry. Results are the mean and standard deviation of 3 experiments, significance was estimated using paired T-tests and corrected for multiple comparisons (* p<0.05, ** p<0.01, *** p<0.005).