Unmasking complex kinetics in viral entry by inferring hypoexponential models.

Single-event completion times, such as are estimated in viral entry, offer both promise and challenge to kinetic interpretation. The promise is that they constrain underlying kinetic models much more efficiently than bulk kinetics, but the challenge is that completion times alone can incompletely determine complex reaction topologies. Gamma distributions or mechanistic models have often been used to estimate kinetic parameters for such data, but the gamma distribution relies on homogenous processes to explain the rate-limiting behavior of the system. Here, we introduce hypoexponential analysis to estimate heterogeneous kinetic processes. We demonstrate that hypoexponential models can indeed estimate rate constants separated by two to three orders of magnitude. We then apply this approach to measurements of SARS-CoV-2 entry, showing that the presence of the ACE2 receptor for SARS-CoV-2 reduces the number of rate-limiting steps but does not change the rates of these kinetic processes. We propose a kinetic model whereby SARS-CoV-2 entry is driven by a mixture of ACE2-accelerated and ACE2-independent spike protein activation events. Inferring such models requires the capability to detect heterogeneous kinetic processes, provided by robust estimation of hypoexponential distributions.