Two strategies underlying the trade-off of hepatitis C virus proliferation: stay-at-home or leaving-home?

Viruses proliferate through both genome replication inside infected cells and transmission to new target cells or to new hosts. Each viral genome molecule in infected cells is used either for amplifying the intracellular genome as a template (“stay-at-home strategy”) or for packaging into progeny virions to be released extracellularly (“leaving-home strategy”). The balance between these strategies is important for both initial growth and transmission of viruses. In this study, we used hepatitis C virus (HCV) as a model system to study the functions of viral genomic RNA in both RNA replication in cells and in progeny virus assembly and release. Using viral infection assays combined with mathematical modelling, we characterized the dynamics of two different HCV strains (JFH-1, a clinical isolate, and Jc1-n, a laboratory strain), which have different viral assembly and release characteristics. We found that 1.27% and 3.28% of JFH-1 and Jc1-n intracellular viral RNAs, respectively, are used for producing and releasing progeny virions. Analysis of the Malthusian parameter of the HCV genome (i.e., initial growth rate) and the number of de novo infections (i.e., initial transmissibility) suggests that the leaving-home strategy provides a higher level of initial transmission for Jc1-n, while, in contrast, the stay-at-home strategy provides a higher initial growth rate for JFH-1. Thus, theoretical-experimental analysis of viral dynamics enables us to better understand the proliferation strategies of viruses. Ours is the first study to analyze stay-leave trade-offs during the viral life cycle and their significance for viral proliferation.


Introduction 27
Hepatitis C virus (HCV) is an RNA virus specifically infecting liver cells. HCV 28 produces progeny viruses rapidly, with ~10 12 copies sometimes observed in patients 29 [1]. Following virus entry into target cells, viral genomic RNA produces structural 30 proteins (S) and non-structural proteins (NS) (Fig. 1A). Using the genomic RNA as a 31 template, the viral non-structural proteins amplify HCV RNA ("RNA replication").
32 Genomic RNA can also be assembled with viral structural proteins into progeny 33 virions to be egressed outside of cells, creating the opportunity for transmission (in 34 this study, we call the process including particle assembly and egress "release").
35 Thus, a single HCV genomic RNA molecule can be used either for RNA replication or 36 for release, and the balance between these processes governs viral proliferation. The 44 genome from the core to NS2 was replaced by sequences from another genotype 2a 45 virus, the J6 strain) was developed as a laboratory strain to improve virus production, 46 and used for development of antiviral agents and vaccines, which requires large 47 amounts of virus [5,6]. In spite of their high sequence similarity (97% identity over the 48 whole genome), these two viruses have different virological characteristics especially 49 in terms of the release process: while JFH-1 particles assemble on lipid droplet 50 membranes, particle assembly of J6/JFH-1-chimeric lab strains is associated with 51 endoplasmic reticulum-derived membranes [2, 3]. Thus, these two related strains are 52 a useful a reference set to compare the dynamics of release and RNA replication.

53
In this study, we used a cell culture model of infection with these two HCV 54 reference strains and measured the time-course of viral production (including HCV 55 RNA inside cells and virions produced outside of the cells), infectivity of progeny HCV, 56 and infected cell numbers. We also developed a multiscale mathematical model to 57 describe intra-and inter-cellular HCV dynamics. This interdisciplinary approach 58 suggests that different strategies exist for viral proliferation: the stay-at-home strategy 59 (JFH-1) and the leaving-home strategy (Jc1-n, a J6/JFH-1-chimeric strain). We 60 discuss the relevance of these strategies for viral proliferation, while referring to [7] for 61 wider evolutionary context.

Results
63 Age-structured multiscale modeling of HCV infection

64
To describe the intracellular replication dynamics of HCV viral RNA, we used 65 the following mathematical model: 67 where is the amount of intracellular viral RNA in cells that have been infected ( ) 68 for time . The intracellular viral RNA replicates at rate , degrades at rate , and is 69 released to extracellular space at rate (Fig. 1B)

94
To assess the variability of kinetic parameters and model predictions, we 103 Experimental measurements below the detection limit were excluded in the fitting.
146 Hence, our parameter estimation captured the characteristics of the two strains well 147 and was able to quantitatively describe viral infection dynamics.

148
In our multiscale model (Eqs. (2-6)), the accumulation rate of intracellular 150 the degradation rate and the release rate (i.e., 153 bootstrap t-test) ( Table 1). The preferential accumulation of JFH-1 RNA inside cells 154 was consistent with its tendency toward gradual increased levels of intracellular RNA 155 at later time points (Fig. 2C). To further evaluate total viral RNA level considering 160 The Malthusian parameters for JFH-1 and Jc1-n were calculated as (95% CI: 1.11

161
) and (95% CI: ), respectively, and were significantly 1.04 -1.18 1.02 0.95 -1.09 162 different from one another ( by bootstrap t-test) ( Fig. 3G and Table 1). = 1.02 × 10 -3 163 Interestingly, even if Jc1-n had a larger infection rate, , and release rate, , 164 compared with JFH-1, the initial growth rate of total JFH-1 RNA was higher than that 165 of Jc1-n. This result demonstrated that the capacity to accumulate viral RNA inside 166 cells predominantly determines the initial growth rate rather than release of progeny 167 viruses.

168
169 Stay-at-home strategy or leaving-home strategy for "optimizing" HCV 170 proliferation

171
We investigated how differences between the two strains, JFH-1 and Jc1-n, 172 might be interpreted in an evolutionary perspective. As mentioned above, we 173 considered two opposing strategies: the "stay-at-home strategy" and the 182 1.27% and 3.28% for JFH-1 and Jc1-n, respectively (Fig. 4B). Notably, Jc1-n used 183 intracellular viral RNA for virus release 2.58 times faster than JFH-1, explaining the 184 rapid transmission of Jc1-n (Fig. 2C). These results indicate the preferential 185 "leaving-home" strategy of Jc1-n as compared with JFH-1, which adopts a 186 "stay-at-home" strategy.

187
To further investigate these two opposing strategies, we addressed the 188 relevance of viral RNA release rates for viral proliferation using in silico analysis. With 189 various values of the release rate of intracellular viral RNA, , we calculated the 190 Malthusian parameter for each strain as an indicator of viral fitness (Fig. 4C). Each Thus, it appears that Jc1-n is more optimized for producing 213 newly infected cells. This implies that HCV Jc1-n adopts the leaving-home strategy to 214 acquire an advantage in producing newly-infected cells. 215

Taken together, our theoretical investigation based on viral infection
216 experiments revealed that the JFH-1 strain optimizes its initial growth rate, but the 217 Jc1-n strain optimizes de novo infection. Ours is the first report to quantitatively 218 evaluate these opposing evolutionary strategies and to show their significance for 219 virus proliferation at the intracellular and intercellular levels.

221
Through a combined experimental-theoretical approach, we analyzed the 222 dynamics of the HCV life cycle using two related HCV strains, JFH-1 and Jc1-n, 223 employing different particle assembly/release strategies. We quantified the intra-and 224 inter-cellular viral dynamics of these strains by applying an age-structured multiscale 225 model to time-course experimental data from an HCV infection cell culture assay (Fig.   226 2A and  . (2-6)), and estimated 228 parameters shared between the PDE and ODE models. It is technically challenging to 229 obtain experimental measurements with age information, but thanks to the estimated 230 values of these common parameters, we managed to reconstruct age information for 231 intracellular viral RNA (Fig. 2BC). In addition, comparing the calculated Malthusian 232 parameters and the cumulative number of newly infected cells between the two 233 strains (Fig. 3FG), we found that the JFH-1 strain had a higher initial growth rate but 234 that Jc1 produced more de novo infections.

235
Based on our results, we propose two opposing strategies for viral 236 proliferation: the "stay-at-home strategy" and the "leaving-home strategy." From an 237 evolutionary perspective, JFH-1 adopts a stay-at-home strategy and preferentially 238 uses viral genomic RNA for increasing intracellular replication. In contrast, adopting a 239 leaving-home strategy, Jc1-n uses more viral genomic RNA for producing progeny 240 virions capable of new transmission events to increase the number of infected cells 241 (Fig. 4). Thus, Jc1-n infects cells 1.71 times faster and produces viral RNA from 242 infected cells 2.57 times faster than JFH-1. Our group and others reported that JFH-1 243 assembled progeny virions on the membranes of hepatic lipid droplets, while 244 J6/JFH-1 chimeric strains mainly used endoplasmic reticulum-derived membranes for 245 particle production [2, 3]. Although the molecular aspects of this difference have been 246 analyzed, its significance for viral proliferation and dynamics is not completely 247 understood. Our results raise the possibility that different subcellular locations for 248 particle assembly impact the rates of particle assembly and release, which in turn  Fig. 2A