SARS-CoV-2 cellular coinfection is limited by superinfection exclusion.

The coinfection of individual cells is a requirement for exchange between two or more virus genomes, which is a major mechanism driving virus evolution. Coinfection is restricted by a mechanism known as superinfection exclusion (SIE), which prohibits the infection of a previously infected cell by a related virus after a period of time. SIE regulates coinfection for many different viruses, but its relevance to the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was unknown. In this study, we investigated this using a pair of SARS-CoV-2 variant viruses encoding distinct fluorescent reporter proteins. We show for the first time that SARS-CoV-2 coinfection of individual cells is limited temporally by SIE. We defined the kinetics of the onset of SIE for SARS-CoV-2 in this system, showing that the potential for coinfection starts to diminish within the first hour of primary infection and then falls exponentially as the time between the two infection events is increased. We then asked how these kinetics would affect the potential for coinfection with viruses during a spreading infection. We used plaque assays to model the localized spread of SARS-CoV-2 observed in infected tissue and showed that the kinetics of SIE restrict coinfection-and therefore sites of possible genetic exchange-to a small interface of infected cells between spreading viral infections. This indicates that SIE, by reducing the likelihood of coinfection of cells, likely reduces the opportunities for genetic exchange between different strains of SARS-CoV-2 and therefore is an underappreciated factor in shaping SARS-CoV-2 evolution. IMPORTANCE: Since SARS-CoV-2 first emerged in 2019, it has continued to evolve, occasionally generating variants of concern. One of the ways that SARS-CoV-2 can evolve is through recombination, where genetic information is swapped between different genomes. Recombination requires the coinfection of cells; therefore, factors impacting coinfection are likely to influence SARS-CoV-2 evolution. Coinfection is restricted by SIE, a phenomenon whereby a previously infected cell becomes increasingly resistant to subsequent infection. Here we report that SIE is activated following SARS-CoV-2 infection and reduces the likelihood of coinfection exponentially following primary infection. Furthermore, we show that SIE prevents coinfection of cells at the boundary between two expanding areas of infection, the scenario most likely to lead to recombination between different SARS-CoV-2 lineages. Our work suggests that SIE reduces the likelihood of recombination between SARS-CoV-2 genomes and therefore likely shapes SARS-CoV-2 evolution.