Cellular co-infection can modulate the efficiency of influenza A virus production and shape the interferon response

Abstract

15 During viral infection, the numbers of virions infecting individual cells can vary significantly over 16 time and space. The functional consequences of this variation in cellular multiplicity of infection 49 Cellular co-infection plays an important, yet poorly defined role in shaping the outcome of 50 influenza A virus (IAV) infection. By facilitating reassortment between incoming viral genomes, 51 cellular co-infection can give rise to new viral genotypes with increased fitness or emergence 52 potential (1). Cellular co-infection can also enhance the replicative potential of the virus by 53promoting the complementation and multiplicity reactivation of the semi-infectious particles that 54 constitute the bulk of influenza virus populations (2-4). Despite its clear importance, the 55 prevalence and the specific functional consequences of cellular co-infection during IAV infection 56 remain largely unknown. 57 58We and others have previously shown that cellular co-infection can be common in vivo (5,6). 59There is growing evidence that IAV replication and spread is focal and thus that the distribution 60 of individual virions across cells and tissues is highly spatially structured, resulting in foci of high 61 cellular multiplicity of infection (MOI) (7-10). Given the dynamic distribution of virions over time 62and space during infection, it appears likely that the MOIs of individual infected cells can vary 63 significantly. This raises the question whether this variation in the number of virions that infect a 64 cell has distinct phenotypic consequences. If so, it could have significant implications for 65understanding IAV infection dynamics as two viral populations of identical size and genome 66 sequence could give rise to divergent infection outcomes if the dispersal patterns of virions (and 67 thus the MOI distribution across cells) differs. 68 69Several previous studies have suggested that the number of virions that enter a given cell 70 (referred to throughout as "cellular MOI" or "viral input") may affect replication kinetics and 71 interferon (IFN) induction (11-14); however, the phenotypic consequences of cellular MOI during 72 IAV infection have not yet been rigorously or comprehensively quantified. Here, we combine 73 precise single-cycle infection experiments with statistical model fitting to reveal that cellular MOI 74 significantly alters virus production rates, the host response to infection, and the potential for 75 further superinfection. In doing so, we precisely define the functional forms that govern the 76 relationships between the amount of viral input and the phenotypes of infected cells, information 77 that will aid future efforts to quantitatively model IAV infection. Altogether, these results reveal 78and define an unappreciated role for cellular co-infection in shaping the outcome of IAV infection. 79 80 RESULTS 81To define how variation in cellular MOI affects viral replication dynamics and the host response 82to infection, we infected either MDCK or A549 cells with A/Puerto Rico/8...

Single cell heterogeneity in influenza A virus gene expression shapes the innate antiviral response to infection

“…This suggests that the number of viral genomes entering individual cells is likely quite variable. We recently showed that this variability in cellular MOI can have distinct phenotypic consequences, both for viral replication dynamics and for IFN induction [21]. Altogether, it appears likely that the heterogeneity that we describe here underrepresents what would be observed in vivo.…”

“…To assess the effects of viral population heterogeneity on the host response to infection, we examined the combined viral and host transcriptional profiles from thousands of single infected cells. To focus on the effects of viral heterogeneity, we wanted to remove the variability that could arise from variation in cellular MOI [21]. To ensure that the vast majority of infected cells were each infected with a single virion, we infected A549 cells with either the 2009 H1N1 pandemic strain A/California/07/2009 (Cal07), or the seasonal human H3N2 strain A/Perth/ 16/2009 (Perth09) at an MOI of 0.01, and blocked secondary spread in the culture through the addition of NH 4 Cl [22].…”

Cooperative Virus-Virus Interactions: An Evolutionary Perspective

“…This antiviral response imposes a time window for viruses to produce and release progeny. Increased percell yield has been demonstrated for VSV [68], influenza A virus (IAV) [69][70][71][72], infectious bursal disease virus (IBDV) [51], and vaccinia virus [73].…”

Heterogeneity in viral replication dynamics shapes the antiviral response

“…Moreover, several studies used infections with an MOI>1 (Doğanay et al, 2017; Zawatzky et al, 1985; Zhao et al, 2012), resulting in considerable variation in the number of virions that infect a single cell. Because MOI affects the rate of virus replication (Martin et al, 2020; Schulte and Andino, 2014), it is challenging to disentangle heterogeneity in viral replication rates from variation in the number of infecting virions in infections with MOI>1. Thus, to study the effect of viral replication rates on innate immune activation, highly sensitive live-cell read-outs are required to precisely determine the moment of infection by individual viruses and the timing and strength of antiviral response activation in single cells.…”