Figure 1.
Measurement of HCV replication dynamics.
(A) Schematic representation of the subgenomic HCV luciferase reporter replicon used for the study. The 5′-non-translated region (NTR) contains the HCV internal ribosome entry site (IRES), controlling translation of the firefly luciferase gene (Luc). The non-structural proteins of the HCV isolate JFH1 are under control of the encephalomyocarditis (EMCV) virus IRES, and are followed by the orthologous 3′- NTR of JFH1. (B&C) Quantitative assessment of the HCV replication dynamics upon instantaneous (t = 0 h) electro-transfection into (B) high permissive Huh7-Lunet cells or (C) low permissive Huh-7 low passage cells. The top panel shows a Northern blot analysis of the viral plus- and minus-strand RNA. The lower panel shows a graph of the Northern blot signals quantified by phosphor imaging (plus-strand RNA: blue lines; minus-strand RNA: red lines), as well as the corresponding luciferase activity (RLU, yellow lines). Luciferase activity and plus-strand RNA are normalized to the input values (2 h and 0 h, respectively; one representative experiment is shown. Lines in the plots are for illustrative purposes and connect data points, but are not results of mathematical modeling.
Figure 2.
Model development and model selection.
(A) Model simulation of our calibrated base model (comprising the model by Dahari et al. [24], with an added initial processing step for transfected RNA and cis-triggered formation of the replication compartment,) compared to experimental data for high-permissive Huh 7-Lunet cells. Black: Polyprotein, red: plus-strand RNA, blue: minus-strand RNA. (B) Different hypotheses for the involvement of a host function at all feasible steps in the viral lifecycle were assessed to explain differences observed in the replication dynamics of Huh7-Lunet and Huh-7 lp cells. For each hypotheses, the base model was calibrated simultaneously to data from high- and low-permissive cell lines, allowing only parameters to differ between the two cell lines that are involved in the respective process. The table shows resulting residual squared errors (χ2) and computed values of the Akaike Information Criterion, a measure that balances goodness-of-fit with the degrees of freedom of a model. Time courses for the individual fits are shown in supplementary figure S1. (C) Graphical illustration of the final model. The main steps are: (1) viral RNA enters the cell, e.g. via transfection (in our subgenomic replicon experiments) or via receptor mediated endocytosis (in a natural infection setting). RNA then undergoes some structural preprocessing (eq. 1), leading to an increased stability and availability to the translation machinery (as Rpcyt, eq. 2). (2) Ribosomes bind the viral RNA, forming translation complexes (Tc, eq. 3) and translate it into a polyprotein (P, eq. 4); (3) the polyprotein is subsequently cleaved into the mature viral proteins (Ecyt) with rate kc (eq. 5); (4) viral proteins then induce the formation of a membranous replication compartment (RC), into which actively translated plus-strand RNA (Tc), viral NS proteins (Ecyt) and one or more host factors (subsumed as HF) enter with rate kPin, forming the plus-strand replication initiation complex (Rip, eq. 7); (5) complementary minus-strand is then transcribed with rate k4m, and the complex dissociates into dsRNA (Rds, eq. 8) and viral polymerase (E, eq. 9); (6) dsRNA and polymerase can then re-associate (RIds, eq. 10) with rate k5 and synthesize progeny plus-strand genomes (Rp) at rate k4p (eq. 11); (7) eventually, new positive strand RNA (Rp) is liberated from the replication vesicles into the cytoplasm at rate kpout (eq. 11 and 2) or, alternatively, can remain within the vesicles for further genome replication (initiating at rate k3), and is ultimately degraded.
Figure 3.
Experimentally measured and model predicted time courses of viral RNA replication.
Experimental data (symbols) and results of model simulation (lines) over 80 hours, showing the dynamics of viral replication in (A) high permissive Huh7-Lunet and (B) low permissive Huh-7 lp cells. Solid blue lines and symbols: viral plus-strand RNA; dashed red lines and symbols: viral minus-strand RNA; dotted black lines and symbols: rescaled luciferase activity (i.e. polyprotein molecule numbers). Experimental data represent mean values +/− two standard deviations from three independent replicates. Note the logarithmic scale of the y-axes. Model predictions were obtained after calibration of model parameters to the data.
Figure 4.
Validation of model predictions.
(A+B) Model validation using replication deficient HCV RNA (NS5BΔGDD) in high- and low permissive cells. (A) Plus-strand RNA concentration and (B) protein translation (luciferase activity) were measured. Solid lines indicate model predictions. (C+D) Model validation using chimeric NTR HCV replicons. Exchange of 5′-NTR (green symbols) specifically inhibits initiation of plus-strand synthesis, 3′-NTR exchange (brown symbols) inhibits initiation of minus-strand synthesis. Luciferase measurements are shown as means +/− two standard deviations of two independent experiments. Lines represent model predictions. (D) Comparison of model prediction and literature data [22] for resulting plus- to minus-strand RNA ratios.
Figure 5.
Global sensitivity analysis of the replication model.
Sensitivity analysis was performed using the extended Fourier Amplitude Test (eFAST) at (A) 4 hours and (B) 72 hours. Shown are eFAST total order sensitivity indices for plus strand RNA; sensitivities for minus strand RNA and viral protein can be found in supplementary figure S4. The dotted blue line indicates the level of a negative control parameter that does not occur in any of the equations. Sensitivities lower or equal to this negative control should not be considered significantly different from zero [39].
Figure 6.
Analysis of the importance of a distinct replicative compartment (RC).
(A) Protective effect of replication vesicles: replication dynamics (plus strand RNA shown) at different degradation rates (μRC) of viral RNA inside of the replicative compartment (RC). Actual values for μRC and μPcyt obtained from model calibration are marked in the figure. Different degradation rates are depicted on the y-axis, resulting time courses for positive strand RNA molecules are color-coded along the x-axis. At μRC = μPcyt, viral RNA replication becomes unstable, and efficient replication cannot be sustained. (B) The plot shows the resulting sum of residual squared errors (χ2) when simultaneously varying the degradation rates μRC and μPcyt. The plot shows that χ2 increases over five-fold when μRC and μPcyt attain similar magnitudes. (C) Effect of host factor (HF) expression levels on the steady state levels of viral RNA and protein. Plus-strand RNA steady state levels (red line) respond linearly to concentration changes of HF in the range of 1–100 HF “molecules”. Viral polyprotein levels (blue line) show a bi-phasic steady state behavior with an exponential response for up to approx. 20 HF “molecules”, showing saturation thereafter. Note that HF is a hypothetical species likely comprising different host cellular proteins and/or processes; “molecules” therefore does not reflect physical molecule numbers.
Table 1.
Parameter estimates obtained from model calibration.
Figure 7.
Gene expression profiling of differently permissive Huh-7 cells.
(A) Relative permissiveness for HCV replication of eight different Huh7 derived cell lines. Permissiveness was normalized to Huh-7 p28 cells. (B) Scatterplot of host gene expression in high permissive Huh7-Lunet versus low permissive Huh-7 lp cells. Off-diagonal elements are differentially expressed and are potential candidates underlying the difference in replication efficiency. Colors encode the distance from the diagonal. A selection of strongly differentially expressed genes is labeled with gene symbols. (C) Eight different cell lines with different replication permissiveness (see panel A) were used, and replication efficiency was correlated with host gene expression. A linear model was fitted to predict replication permissiveness from gene expression data, and goodness of fit assessed using ANOVA. Shown are resulting p-values, plotted over the log- fold-change of expression between Huh7-Lunet and Huh-7 lp cells. Shown are genes with p-values<0.2 and a log-fold-change of more than 0.3 or less than −0.3. Seventeen genes that were previously shown to be functionally linked to HCV replication or to directly interact with viral proteins are highlighted in red and labeled.
Table 2.
Established HCV host factors identified in transcriptomic analysis.