FIG. 2.

Simulation of subgenomic HCV RNA replication in Huh-7 cells. To explore the impact of each unknown parameter on subgenomic HCV kinetics, from time of transfection (t = 0) to steady state, we varied one or more chosen parameters within a given range, while all other parameters were maintained fixed as follows: k₁ = 80 h⁻¹ molecule⁻¹, k_Pin = 0.2 h⁻¹, k_Pout = 0.2 h⁻¹, k_Ein = 1.3 × 10⁻⁵ h⁻¹, k_c = 0.6 h⁻¹, k₂ = 100 h⁻¹, k_4p = k_4m = 1.7 h⁻¹, k₃ = 0.02 h⁻¹ molecule⁻¹, k₅ = 4 h⁻¹ molecule⁻¹, μ_Ip = 0.04 h⁻¹, μ_E = 0.04 h⁻¹, μ_p^cyt = 10 h⁻¹, μ_p = 0.07 h⁻¹, μ_ds = 0.06 h⁻¹, μ_Ids = 0.13 h⁻¹, μ_E^cyt = 0.06 h⁻¹, μ_Tc = 0.015 h⁻¹, R_ibo^Tot = 700 ribosome complexes, and R_P^cyt(0) = 500 plus-strand RNA copies. (A) Within a range of 1 to 1,000 available ribosome complexes per cell (R_ibo^Tot), the model reached steady-state characteristics when 500 < R_ibo^Tot < 1,000. We found that the steady-state HCV NS5B polymerase level (green line) increases with higher R_ibo^Tot numbers. The total plus- and minus-strand RNAs at steady state are shown as black and red lines, respectively. (B) We chose k_Ein and μ_Ids within the ranges 5 × 10⁻⁶ to 2 × 10⁻⁴ h⁻¹and 0.01 to 0.9 h⁻¹, respectively. We found that the ratio (R_P + R_Ip + R_ds + R_Ids)/(R_P^cyt + T_c), which is the ratio of total plus-strand RNAs inside and outside of the VMS (thick line), and the ratio of total plus-strand RNA to total minus-strand RNA (R_P^Tot/R_M^Tot, thin line) increase with the ratio μ_Ids/k_Ein between the rate of replicative intermediate degradation and the rate of polymerase transport into the VMS. To obtain a 1:1 ratio of (R_P + R_Ip + R_ds + R_Ids)/(R_P^cyt + T_c) and a 10:1 ratio of R_P^Tot/R_M^Tot, the ratio μ_Ids/k_Ein needs to be about 10⁴. A ratio of ∼6 of total plus-strand RNA to total minus-strand RNA in VMS is found with many different μ_Ids and k_Ein rates (dashed line). (C) We chose k₅ and k₃ within the ranges 0.8 to 4.0 h⁻¹ and 0.004 to 0.02 h⁻¹, respectively. The ratio of total plus-strand RNA to total minus-strand RNA increases with the ratio k₅/k₃ of formation rates of minus-strand to plus-strand replication complexes. While k₅/k₃ = 1 does not allow for a 10:1 total plus-strand-to-minus-strand asymmetry (thick line), and 6:1 total plus-strand-to-minus-strand asymmetry in VMS (thin line), k₅/k₃ ∼ 200 will. (D) We checked whether the total plus-strand-to-minus-strand asymmetry can be generated with different synthesis rates of plus- and minus-strand RNA, i.e., k_4p > k_4m. We assumed the same formation rates (k₅ = k₃ = 0.02 h⁻¹) of plus-strand and double-strand replicative intermediate complexes (R_Ip and R_Ids, respectively). Interestingly, although 50- to 500-fold faster synthesis rates for the plus-strand RNA than for the minus-strand RNA generate larger numbers of R_Ip complexes (thick red line) than of R_Ids complexes (thick black lines), the total plus-strand RNA (thin black line) is approximately equal to the total minus-strand RNA (thin red line). Free plus-strand RNA (R_P) is represented in blue. The curve for R_ds superimposes on the curve for R_M^Tot since almost all of the minus-strand RNA is in double-stranded complexes. (E) With 10 subgenomic plus-strand RNAs successfully transfected [R_P^cyt(0) = 10 per cell (thin lines)], total plus-strand RNA (black line), minus-strand RNA (red line), and NS5B molecules (green line) reach steady state in about 300 h. However, with R_P^cyt(0) = 500 (thick lines), the steady state is attained in about 50 h. The total plus-strand RNA level at steady state is not affected by different initial R_P^cyt numbers. (F) We tested how the plus-strand RNA copy numbers (1 to 1,000) at time of transfection, R_P^cyt(0), affect the time to attain steady state. Only when R_P^cyt(0) was >7 was a steady state attained, whereas R_P^cyt(0) at <7 led to elimination of viral RNA (not shown). Higher initial R_P^cyt numbers leads to faster attainment of steady-state.