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

Abstract

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.

Keywords: SARS-CoV-2; coinfection; coronavirus; superinfection exclusion (SIE); virus-virus interaction.

Fig 1

SARS-CoV-2 reporter viruses replicate with similar kinetics. Multicycle growth kinetics of SARS-CoV-2 reporter viruses were assessed by infecting VAT cell monolayers with viruses at a multiplicity of infection of 0.01 plaque-forming unit (PFU) per cell and harvesting the supernatant at the time points indicated. Virus titers were then calculated from plaque assays on VAT cells. The data represent the mean and standard deviation (n = 3). The lower limit of quantification is represented by a dashed line. Differences between SARS-CoV-2 ZsGreen and mCherry growth were non-significant (P > 0.05) at each time point as assessed using a paired, non-parametric Wilcoxon test.

Fig 2

Cells infected with SARS-CoV-2 reporter viruses can be distinguished by confocal microscopy and flow cytometry. (A) Images of cells infected with SARS-CoV-2 reporter viruses taken using a high-resolution confocal microscope. VAT cells were simultaneously infected at a multiplicity of infection (MOI) of 0.5 PFU/cell of each virus and fixed at 24 hpi. Images were obtained using a ×63 objective. Scale bar = 20 µm. (B) Flow cytometric analysis of VAT cells separately infected with SARS-CoV-2 reporter viruses. Cells were infected at an MOI of 2 fluorescence-forming units per cell (FFU/cell) of either ZsGreen- or mCherry-expressing viruses and harvested for analysis 16 hpi. Gate frequencies as percentage of total cells are shown.

Fig 3

SIE restricts infection of SARS-CoV-2 mCherry in VAT cells previously infected with SARS-CoV-2 ZsGreen (A) Schematic of the experimental investigation of SARS-CoV-2 SIE. (B) Percentage of positive cells during individual infection of SARS-CoV-2 ZsGreen and mCherry reporter viruses. VAT cells were infected with an MOI of 2 FFU/cell at the time points indicated and harvested for flow cytometry at 16 hpi. Data represent mean and SD (n = 2). (C) Representative flow cytometry plots of cells infected with reporter viruses. VAT cells were first infected with SARS-CoV-2 ZsGreen, before secondary infection at the time points indicated with SARS-CoV-2 mCherry. (D) Kinetics of onset of SIE determined by flow cytometric analysis. Data represent mean and SD (n = 5). (E) Percentage of coinfected cells as the time between infection events is increased. Significance determined by non-parametric Friedman multiple comparison test. *P < 0.05, ***P < 0.0005, ****P < 0.0001.

Fig 4

SIE restricts infection of SARS-CoV-2 ZsGreen in VAT cells previously infected with SARS-CoV-2 mCherry. (A) Schematic of the experimental investigation of SARS-CoV-2 SIE. (B) Percentage of positive cells during individual infection of SARS-CoV-2 ZsGreen and mCherry viruses. Measurement of singly infected VAT cells with an MOI of 2 FFU/cell tagged viruses harvested for flow cytometry at 16 hpi. Data represent mean and SD (n = 2). (C) Representative flow cytometry plots of cells infected with reporter viruses. VAT cells were first infected with SARS-CoV-2 mCherry, before secondary infection at the time points indicated with SARS-CoV-2 ZsGreen. (D) Kinetics of onset of SIE determined by flow cytometric analysis. Data represent mean and SD (n = 5). (E) Percentage of coinfected cells as the time between infection events is increased. Significance determined by non-parametric Friedman multiple comparison test. **P < 0.005, ****P < 0.0001.

Fig 5

Kinetics of onset of SIE by SARS-CoV-2 can be described by an exponential decay model. The kinetics of SIE onset were calculated from data in Fig. 3 and 4 when the secondary virus was (A) SARS-CoV-2 mCherry or (B) SARS-CoV-2 ZsGreen. RFU and GFU per cell were calculated from the proportions of green, red, and coinfected cells (see text for details). Data points represent the mean and SD (n = 5). The solid line represents the line of best fit; the dotted line represents the exponential decay model fit.

Fig 6

SIE spatially restricts interactions between viruses from separate SARS-CoV-2 foci of infection. Monolayers of VAT cells were infected with ZsGreen- and mCherry-tagged SARS-CoV-2 viruses, overlayed with 0.6% Avicel, fixed at 24 hpi, and imaged using a Celigo fluorescent microscope. Scale bar = 250 µm. (A) Representative images of plaques and merged images of green and red channels (B) Representative images of foci interaction, single-channel images, and merged-channel image (C) A binary threshold was applied to images of foci to distinguish cells expressing either the ZsGreen, mCherry, or both fluorophores together. The threshold applied to the image in panel B is shown. (D) The percentage of coinfected areas in comparison to the total image area was calculated from images taken at 24 hpi. The mean and SD of 10 individual fields of view from one experiment are shown.