Mathematical Model of Viral Kinetics In Vitro Estimates the Number of E2-CD81 Complexes Necessary for Hepatitis C Virus Entry
[['HCV entry into target cells is a complex process involving the interactions of the viral envelope proteins E1 and E2 and several cell surface receptors , namely , scavenger receptor class B type I ( SR-BI ) [1] , the tetraspanin CD81 [2] , [3] , and the tight junction proteins claudin-1 ( CLDN1 ) [4] and occludin [5] .', 'Several recent studies suggest a central role for CD81 in HCV entry: E2 has been shown to interact directly with SR-BI and CD81 following viral attachment to a target cell [1] , [2] .', 'Patient derived neutralizing antibodies appear to target the CD81 binding domains on E2 [6] .', 'Indeed , anti-CD81 antibodies were able to block infection in vitro [3] and in a mouse model [7] .', 'Graft reinfection following liver transplantation was observed recently to select for HCV strains capable of more efficient entry , achieved partly through mutations in the CD81 binding domains on E2 [8] .', 'Expression of human CD81 and occludin was essential for infection of genetically humanized mice [7] .', 'Besides , CLDN1 appears to mediate HCV entry through its association with CD81 [9] , [10] .', 'Consequently , the E2-CD81 interaction presents a potent target for intervention; drugs that block the E2-CD81 interaction are currently under development [11] , [12] .', 'How many E2-CD81 complexes must be formed between a virion and a target cell to enable HCV entry ?', 'Knowledge of this threshold would determine the number of E2-CD81 complexes that a drug or a vaccine must prevent from forming in order to block viral entry , thus presenting a quantitative guideline for intervention strategies targeting the E2-CD81 interaction .', 'This threshold is currently unknown .', 'Direct observation of the number of E2-CD81 complexes formed before HCV entry has not been possible .', 'Recent cell culture studies have determined the dependence of viral entry and kinetics in vitro on the CD81 expression level on target cells [10] , [13]–[18] .', 'In particular , cells expressing higher levels of CD81 were found to be more susceptible to infection [13] .', 'Further , the frequency of cells with low CD81 expression typically increased with time following the exposure of cells to HCV [14] , [15] .', 'We reasoned that analysis of these observations using a mathematical model of viral kinetics , akin to studies of HIV entry ( for example , see [19] , [20] ) , may allow estimation of the threshold number of E2-CD81 complexes necessary for HCV entry .', 'While models of HCV viral kinetics in vivo have been employed successfully to analyse patient data and elucidate guidelines for treatment [21]–[31] , models of HCV viral kinetics in vitro have just begun to be formulated .', 'Here , we constructed a mathematical model of HCV viral kinetics in vitro that mimics cell culture studies of the dependence of viral entry and kinetics on CD81 expression .', 'Model predictions captured data from several independent experiments quantitatively and yielded estimates of the threshold number of E2-CD81 complexes necessary for HCV entry .'], ['We considered in vitro experiments where a population of target cells , , with a known distribution of the CD81 expression level across cells is exposed to a population of HCVcc ( cell culture adapted ) virions , , and the progression of infection followed [13]–[15] .', 'We modelled the ensuing viral kinetics as follows ( Fig . 1 ) .', 'We first considered a single virus-cell pair with the virus attached to the cell by interactions that precede E2-CD81 binding [12] .', 'E2 and CD81 then interact to form E2-CD81 complexes .', 'We computed the mean number of these complexes formed at equilibrium , , as a function of the CD81 expression level , , on the cell .', 'Assuming that the number of complexes formed , , followed a Poisson distribution with mean , we computed the probability , , that was larger than a threshold number .', 'We assumed that viral entry ( and subsequently infection ) occurred if ( Fig . 1A ) .', 'Thus , yielded the relative susceptibility to infection of a cell with CD81 expression level .', 'We next considered the population of cells exposed to virions ( Fig . 1B ) .', 'We divided the cells into different subpopulations with distinct CD81 expression levels and hence different susceptibilities , where .', 'Cells in each subpopulation were assumed to proliferate , die , or be infected at a rate proportional to .', 'The resulting infected cells , , were lost at enhanced rates compared to due to virus-induced cytopathicity in vitro [15] , [32] .', 'Free virions were produced by infected cells and were cleared .', 'With this description , we constructed dynamical equations to predict the time-evolution of each of the uninfected and infected cell subpopulations and the population of free virions and compared our predictions with experiments ( Methods ) .'], ['Analysis of experimental data using mathematical models has provided crucial insights into disease pathogenesis and the effectiveness of drugs and established guidelines for rational optimization of therapy for HCV infection [21]–[31] , [33] , [34] .', 'The recent development of cell culture systems that allow persistent HCV infection in vitro [35]–[38] has yielded a wealth of new data on HCV replication , evolution , and the impact of drugs .', 'Analysis of this data is expected to provide further insights into HCV pathogenesis and outcomes of therapy , but has been precluded by the lack of mathematical models of HCV viral kinetics in vitro .', 'Indeed , significant efforts are underway to construct models of the intracellular replication and evolution of HCV with the aim of elucidating the activity of direct acting antiviral drugs [39]–[41] .', 'Here , we have constructed a mathematical model of HCV viral kinetics in vitro .', 'Applying it to the analysis of data from several recent cell culture studies , we obtained quantitative insights into the molecular requirements of HCV entry .', 'We estimated that depending on the binding affinity of E2 and CD81 , between 1 and 13 E2-CD81 complexes are necessary for HCVcc entry into human hepatoma-derived cells .', 'Our estimate provides a quantitative guideline for the optimal usage of drugs and vaccines that target the E2-CD81 interaction: A potent drug or vaccine must ensure that not more than 1–13 E2-CD81 complexes are formed across a virus-cell pair in order to prevent viral entry .', 'This guideline assumes significance as drugs that target the E2-CD81 interaction are under development [11] , [12] and may become part of future treatments involving direct acting antiviral agents that seek to overcome the limitations of the current interferon-ribavirin-based treatments [42] .', 'Further , a recent analysis of the HCV quasispecies in six patients who underwent liver transplantation revealed that viral strains capable of more efficient entry , achieved through modulation of the CD81 dependence of viral entry , were selected following liver transplantation [8] .', 'Blocking E2-CD81 interactions effectively , for which our estimate presents a quantitative criterion , may thus be a promising avenue to prevent graft reinfection following liver transplantation .', 'In an earlier study , Koutsoudakis et al . [13] estimated that a cell must express >70000 CD81 molecules to allow HCV entry .', 'This threshold was identified as follows .', 'In independent experiments , cells with widely varying distributions of CD81 were exposed to HCV at low MOI in culture and the percentage of cells infected at day 5 was measured .', 'This latter percentage was found to correlate well with the percentage of cells initially expressing >70000 CD81 molecules/cell .', 'For instance , with Huh7 . 5 cells , the percentage of cells infected at day 5 was 91 . 5 and the percentage expressing >70000 CD81 molecules/cell at day 0 was 93 , and with Huh7-Lunet cells , which express fewer CD81 molecules than Huh7 . 5 cells , the percentages were 11 . 2 and 17 , respectively .', 'Based on this correlation , Koutsoudakis et al . suggested 70000 CD81 molecules/cell as the threshold for entry .', 'Our present analysis suggests that the underlying viral kinetics may render this estimate an upper bound .', 'Virus-induced cytopathicity in culture would result in the loss of susceptible cells and therefore a continuous decrease in the frequency of susceptible cells with time .', 'Consequently , the percentage of cells susceptible initially is expected to be higher than the percentage of cells susceptible–and hence even higher than the percentage infected–at day 5 post-infection .', 'The higher percentage of cells susceptible initially would therefore imply a threshold smaller than 70000 molecules/cell .', 'This is also evident in the experiments of Koutsoudakis et al . , where cells with lower CD81 expression ( MFI∼50 ) than 70000 molecules/cell ( MFI∼100 ) were infected ( see Fig . 3C ) .', 'From our analysis , we identified the threshold number of E2-CD81 complexes and not CD81 molecules necessary for entry .', 'Because stochastic events can result in the formation of the requisite number of complexes , cells expressing few CD81 molecules have small but nonzero susceptibilities to infection .', 'Indeed , we found using parameters employed in Fig . 3D that a small percentage of cells expressing as few as ∼10000 CD81 molecules was infected by day 5 .', 'More recently , Zhang et al . [18] have argued that a substantially smaller expression level than suggested by Koutsoudakis et al . may suffice for entry and that CD81 may also be necessary , perhaps at higher expression levels , for post-entry events .', 'Our model does not distinguish between entry and post-entry requirements of CD81 .', 'Our estimate of 1–13 E2-CD81 complexes defines successful infection of a cell and combines the requirements for entry and any post-entry steps [17] , [18] .', 'Our model yielded good fits to data with in the range , which is higher than the value , , determined using recombinant E2 and soluble CD81 [43] .', '( Fits with were poor ( not shown ) .', ') This discrepancy may be because the binding affinity when the proteins are in solution may be different from that when the proteins are restricted to membranes [44] , recombinant E2 may not accurately mimic the true E2-CD81 interaction [45] , and/or only a fraction of the CD81 may lie outside tetraspanin-enriched microdomains and/or be associated with CLDN1 and therefore available for binding E2 [10] , [46] .', 'The E2-CD81 binding affinity in situ remains to be determined .', 'Our model yielded best-fit values of the threshold number of complexes , , that decreased as increased ( Fig . 4 ) .', 'For a given CD81 expression level , the mean number of E2-CD81 complexes formed decreased as increased , lowering susceptibility ( Eqs .', '( 4 ) and ( 5 ) ) .', 'Decreasing restored this susceptibility ( Fig . 2B inset and Fig . S6 ) , thus ensuring that the resulting viral kinetics was conserved and in agreement with data .', 'This does not imply , however , that viral strains with lower E2-CD81 affinity ( higher ) would require fewer E2-CD81 complexes for entry .', 'On the contrary , given a value of , our model predicts that a cell would be less susceptible to entry of viral strains with higher .', 'Our model was designed to mimic experiments that examined the influence of CD81 expression on viral entry and kinetics [13]–[15] .', 'In these experiments , cells with high CD81 expression were preferentially infected and lost due to virus-induced cytopathicity , and cells with low CD81 expression , refractory to infection , eventually dominated the culture , suggesting that CD81 expression limited entry .', 'Accordingly , our model assumed that other entry receptors were not limiting .', 'Our model then predicted triphasic viral kinetics in vitro , in agreement with experiments .', 'We note that the origins of the triphasic pattern here are distinct from the triphasic viral load decline in some patients undergoing combination therapy , the latter due to liver homeostatic mechanisms [25] .', 'Further , the triphasic kinetics is a short-term phenomenon ( ∼2–3 weeks ) .', 'Over longer periods , viral evolution may alter the kinetics substantially [15] , which our model does not consider .', 'Nonetheless , our model can be readily adapted to the scenario where a receptor other than CD81 is limiting and may thus serve to quantify the requirements of that receptor for HCV entry .', 'We recognize a few additional simplifications in our model .', 'First , our model ignored cell-cell transmission of infection .', 'CD81 appears to be necessary for direct cell-cell transmission [47] .', 'If the susceptibility of a cell to cell-cell transmission depends on CD81 expression in a manner similar to its susceptibility to viral entry , which remains to be ascertained , then we can show that our model with the pseudo-steady state approximation , , implicitly accounts for cell-cell transmission: the net infection rate from both modes , , is in agreement with our model ( Eq .', '( 1 ) ) with an effective infection rate constant that lumps the rate constants of infection by free virions , , and cell-cell transmission , .', 'Second , our model assumes that reaction equilibrium is attained rapidly compared to viral entry and that the diffusion of CD81 on the target cells continually replenishes the free CD81 in the virus-cell contact region lost due to binding .', 'Accordingly , our model predicts an upper bound on the mean number of E2-CD81 complexes formed in the contact region .', 'Thus , if CD81 diffusion or its binding with E2 were rate limiting , a threshold smaller than 1–13 E2-CD81 complexes is expected to describe the data we considered .', 'Finally , we ignored the splitting of cell culture at confluence .', 'We employed the data at day 3 post-infection from Koutsoudakis et al . [13] , when cells are not expected to have reached confluence .', 'Further , the best-fit parameter estimates obtained from the data of Koutsoudakis et al . [13] were close to those from Zhong et al . [15] ( Fig . 4 ) .', 'Also , accounting for splitting did not significantly alter our comparisons with the data of Tscherne et al . [14] so long as the splitting was performed after day 5 post-infection ( Fig . S7 ) .', 'Also , including a logistic term to limit the proliferation of cells as they approached confluence did not alter our parameter estimates significantly ( not shown ) .', 'Nonetheless , that model predictions described several independent experimental observations quantitatively indicated that even with the above simplifications our model captured the essential features of HCV viral kinetics in vitro successfully .', 'At the same time , the simplifications restricted model parameters to a number that allowed robust parameter estimation through fits to available data .'], ['We considered in vitro experiments where a population of uninfected cells , , is exposed to a population of HCVcc virions , .', 'We divided the cells into subpopulations , denoted , where , with cells in each subpopulation expressing CD81 in a range around molecules per unit area .', 'At the start of infection ( ) , the variation of with was determined from a known distribution , , of CD81 expression levels across cells ( Fig . 1 ) .', 'The following equations described the ensuing viral kinetics ( ) : ( 1 ) ( 2 ) ( 3 ) Here , and are the proliferation and death rates of .', 'is the infection rate of cells expressing excess CD81 .', 'is the death rate of .', 'Following observations of HCV-induced cell cycle arrest in vitro [32] , [48] , we neglected the proliferation of .', 'and are the per cell production rate and the clearance rate of free virions , respectively .', 'Here , represents the combined rate of the natural degradation of virions , the loss of viral infectivity , and the loss of virions due to entry and attachment [49] , [50] .', 'For simplicity , we assumed to be a constant .', 'To determine , we considered a cell , with CD81 expression level , closely apposed to a virion with E2 molecules per unit area .', 'We assumed , as with HIV [20] , that the E2-CD81 interactions across the virus-cell interface attain equilibrium well before viral entry .', 'If is the surface density of E2-CD81 complexes , that of unbound CD81 and that of unbound E2 molecules in the contact area , then at equilibrium , where is the equilibrium dissociation constant of E2-CD81 complexes when the proteins are restricted to membranes .', "From Bell's analysis , , where is the equilibrium dissociation constant when the proteins are in solution and is the encounter distance between the proteins for bond formation [51] .", 'The virus-cell contact area , , is small compared to the surface area of the cell .', 'Further , free CD81 can diffuse on the cell membrane and therefore be recruited to the contact area .', 'Consequently , is expected not to decrease substantially below , as suggested also by an independent reaction-diffusion model [52] .', 'In contrast , the viral surface area is comparable to and assuming E2 to be less mobile than CD81 , it follows that the surface density of E2 in the contact area obeys the species balance equation: .', 'Under the latter two constraints , the mean number of complexes formed across the virus-cell contact at equilibrium , , is given by ( 4 ) We recognized next that the E2 expression level on the virion and the virus-cell contact area are subject to stochastic variations .', 'We assumed therefore that the number of complexes formed during a virus-cell contact , , follows a Poisson distribution with mean .', 'Viral entry ( and subsequently infection ) occurred if , where is the threshold number of E2-CD81 complexes necessary for HCV entry .', 'The probability that , ( 5 ) thus yielded the relative susceptibility to infection of a cell with CD81 expression level .', '( The Poisson distribution does allow to exceed the limit of 180 set by the number of E2 molecules present on a virion , but in all our calculations remained fewer than 30 ( Fig . S8 ) so that the probability that was negligibly small .', ') Equations ( 1 ) – ( 5 ) yielded a model of HCV kinetics in vitro that accounted explicitly for the dependence of viral entry on the CD81 expression level on cells .', 'We solved model equations using a computer program written in MATLAB and computed quantities measured experimentally , namely , the time-evolution of the populations of uninfected and infected cells , and , the viral titre , , the fraction of cells infected , , the fraction of cells infected within each subpopulation , , and the populations of viable and dead cells , and , respectively .', 'We employed the following parameter values and initial conditions: corresponding to 180 E2 molecules on a virion with average diameter 50 nm [53]; , corresponding to a virus-cell contact radius of [54]; the target cell diameter was [17]; was varied over the range ( see above ) .', 'The initial CD81 expression was assumed to follow the log-normal distribution , , where and were the mean and standard deviation of .', 'For comparisons with experimental data , the initial distributions were obtained from measurements ( Fig . S2; see below ) .', 'Thus , the initial cell subpopulations , , where was the total initial target cell population .', 'We divided the range of CD81 expression levels into intervals , which determined ; finer discretisation did not improve the accuracy of our solution ( Fig . S1 ) .', 'The remaining parameters , estimated from fits to data , are listed in Tables S1 and S2 .', 'We have summarized model parameters in Table 1 .', 'We considered data from three recently published cell culture studies of HCV kinetics .', 'First , we considered data of Zhong et al . [15] , where Huh-7 . 5 . 1 cells were exposed to JFH-1 virions and the kinetics of infection followed for 21 d .', 'Specifically , we employed the data of the time-evolution of the supernatant infectivity , the population of attached ( viable ) cells , and the ratio of the populations of floating ( dead ) and attached cells , the latter two datasets with mock infection as well as with HCVcc infection ( Fig . S1 in [15] ) .', 'Second , we considered data of Koutsoudakis et al . [13] , where a 1∶1 mixture of Huh7-Lunet and Lunet/CD81 cells was exposed to HCVcc Venus-Jc1 virus and the fractions of cells infected in subpopulations with distinct CD81 expression levels were measured after 72 h .', 'The initial viral population was at an MOI of ∼5 and ∼1 TCID50/cell ( where TCID50 is the 50% tissue culture infective dose ) , respectively , in two independent experiments ( Fig . 5 in [13] ) .', 'Third , we considered the data of Tscherne et al . [14] , where Huh7 . 5 cells were exposed to J6/JFH virus and the time-evolution of the fraction of cells infected as well as of the distribution of CD81 expression levels across cells was followed for 17 d ( Fig . 8 in [14] ) .', 'The CD81 expression on cells is usually measured in terms of fluorescent intensity .', 'To convert the measurements to CD81 surface densities , we adopted the following procedure .', 'Measured distributions of the CD81 expression level on Huh-7 . 5 ( silRR ) cells were digitized from Zhang et al . [55] and on Huh-7 . 5 cells from Koutsoudakis et al . [13] .', 'The log-normal distribution , , yielded good fits to the data ( Fig . S9 ) .', 'The best-fit parameter values ( 95% CI ) were =\u200a4 . 29 ( 4 . 27–4 . 31 ) and =\u200a0 . 45 ( 0 . 43–0 . 47 ) for the data of Zhang et al . and =\u200a6 . 1 ( 6 . 07–6 . 12 ) and =\u200a0 . 7 ( 0 . 67–0 . 72 ) for the data of Koutsoudakis et al . Because the same cell lines were used , the underlying distributions of CD81 expression are expected to be similar in the two experiments .', 'The differences in the reported fluorescence intensities may therefore be attributed to different conversions of CD81 surface density to fluorescence intensity in the two measurements .', 'To unravel the underlying distribution , we assumed that log fluorescence intensity was linearly proportional to log CD81 surface density ( or that the fluorescence intensity had a power law dependence on the surface density ) .', 'Thus , a cell with CD81 surface density would yield a fluorescence intensity and in the measurements of Zhang et al . and Koutsoudakis et al . , respectively , such that and , where a , b , c , and d are constants .', 'To determine the latter constants , we employed the following observations: The mean surface density of CD81 for the Huh-7 cell line is 2×105 molecules/cell [3] , which corresponded to a fluorescence intensity of 48 units in the measurements of Zhang et al . , so that .', 'Also , Koutsoudakis et al . measured the number of CD81 molecules/cell corresponding to a fluorescence intensity of 100 units and found it to be 7×104 molecules/cell , so that .', 'Further the above relationship between fluorescence intensity and CD81 expression also implied that and .', 'Solving the latter equations using the best-fit parameters above , we obtained a\u200a=\u200a2 . 8 , b\u200a=\u200a2 . 43 , c\u200a=\u200a3 . 96 and d\u200a=\u200a1 . 56 , which enabled conversion of measured fluorescence intensities to CD81 surface densities , ( Fig . S2 ) .', 'The measured distributions in terms of counts versus fluorescence intensities were then converted to probability distributions , versus , by normalizing the counts such that the areas under the versus curves equalled unity .', 'The resulting distributions ( Fig . S2 ) were employed as the initial CD81 distributions for our fits to data ( Fig . 3 ) .', 'We digitized data using Engauge digitizer and fit model predictions to data using the nonlinear regression tool NLINFIT in MATLAB .']]
['Introduction', 'Results', 'Discussion', 'Methods']
['Interaction between the hepatitis C virus ( HCV ) envelope protein E2 and the host receptor CD81 is essential for HCV entry into target cells .', 'The number of E2-CD81 complexes necessary for HCV entry has remained difficult to estimate experimentally .', 'Using the recently developed cell culture systems that allow persistent HCV infection in vitro , the dependence of HCV entry and kinetics on CD81 expression has been measured .', 'We reasoned that analysis of the latter experiments using a mathematical model of viral kinetics may yield estimates of the number of E2-CD81 complexes necessary for HCV entry .', 'Here , we constructed a mathematical model of HCV viral kinetics in vitro , in which we accounted explicitly for the dependence of HCV entry on CD81 expression .', 'Model predictions of viral kinetics are in quantitative agreement with experimental observations .', 'Specifically , our model predicts triphasic viral kinetics in vitro , where the first phase is characterized by cell proliferation , the second by the infection of susceptible cells and the third by the growth of cells refractory to infection .', 'By fitting model predictions to the above data , we were able to estimate the threshold number of E2-CD81 complexes necessary for HCV entry into human hepatoma-derived cells .', 'We found that depending on the E2-CD81 binding affinity , between 1 and 13 E2-CD81 complexes are necessary for HCV entry .', 'With this estimate , our model captured data from independent experiments that employed different HCV clones and cells with distinct CD81 expression levels , indicating that the estimate is robust .', 'Our study thus quantifies the molecular requirements of HCV entry and suggests guidelines for intervention strategies that target the E2-CD81 interaction .', 'Further , our model presents a framework for quantitative analyses of cell culture studies now extensively employed to investigate HCV infection .']
['The interaction between the hepatitis C virus ( HCV ) envelope protein E2 and the host cell surface receptor CD81 is critical for HCV entry into hepatocytes and presents a promising drug and vaccine target .', 'Yet , the number of E2-CD81 complexes that must be formed between a virus and a target cell to enable viral entry remains unknown .', 'Direct observation of the E2-CD81 complexes preceding viral entry has not been possible .', 'We constructed a mathematical model of HCV viral kinetics in vitro and using it to analyze data from recent cell culture studies obtained estimates of the threshold number of E2-CD81 complexes necessary for HCV entry .', 'We found that depending on the E2-CD81 binding affinity , between 1 and 13 complexes are necessary for HCV entry into human hepatoma-derived cells .', 'Our study thus presents new , quantitative insights into the molecular requirements of HCV entry , which may serve as a guideline for intervention strategies targeting the E2-CD81 interaction .', 'Further , our study shows that HCV viral kinetics in vitro can be described using a mathematical model , thus facilitating quantitative analyses of the wealth of data now emanating from cell culture studies of HCV infection .']
['medicine', 'infectious diseases', 'theoretical biology', 'gastroenterology and hepatology', 'biology', 'computational biology']