Single-cell analysis of VACV infection reveals pathogen-driven timing of early and late phases and host-limited dynamics of virus production.

The extent and origin of variation in the replication dynamics of complex DNA viruses is not well-defined. Here, we investigate the vaccinia virus (VACV) infection cycle at the single-cell level, quantifying the temporal dynamics of early and post(dna)-replicative phase gene expression across thousands of infections. We found that viral factors determine the initiation time of these phases, and this is influenced by the multiplicity of infection (MOI). In contrast, virus production dynamics are largely constrained by the host cell. Additionally, between-cell variability in infection start time and virus production rate were strongly influenced by MOI, providing evidence for cooperativity between infecting virions. Blocking programmed cell death by pan-caspase inhibition increased infection frequency but not virus production at the population level due to a concurrent attenuation of per-cell virus yield, suggesting a dual role for caspase signaling in VACV infection. Our findings provide key insights into the pivotal factors influencing heterogeneity in the infection cycle of a large DNA virus at the single-cell level.