A Single Herpes Simplex Virus 1 Genome Reactivates from Individual Cells

Latent infection is a characteristic feature of herpesviruses' life cycle. Herpes simplex virus 1 is a common human pathogen that establishes lifelong latency in peripheral neurons. Symptomatic or asymptomatic periodic reactivations from the latent state allow the virus to replicate and spread among individuals. The latent viral genomes are found as several quiescent episomes inside the infected nuclei; however, it is not clear if and how many latent genomes are able to reactivate together. To address this question, we developed a quiescent infection assay, which provides a quantitative analysis of the number of genomes reactivating per cell, in cultured immortalized fibroblasts. We found that, almost always, only one viral genome reactivates per cell. We showed that different timing of entry to quiescence did not result in a significant change in the probability of reactivating. Reactivation from this quiescent state allowed only limited intergenomic recombination between two viral strains compared to lytic infection. Following coinfection with a mutant that is unable to reactivate, only coreactivation with a reactivation-proficient recombinant can provide the opportunity for the mutant to reactivate. We speculate that each individual quiescent viral genome has a low and stochastic chance to reactivate in each cell, an assumption that can explain the limited number of genomes reactivating per cell. IMPORTANCE Herpesviruses are highly prevalent and cause significant morbidity in the human and animal populations. Most individuals who are infected with herpes simplex virus (HSV-1), a common human pathogen, will become lifelong carriers of the virus, as HSV-1 establishes latent (quiescent) infections in the host cells. Reactivation from the latent state leads to many of the viral symptoms and to the spread of the virus among individuals. While many triggers for reactivation were identified, how many genomes reactivate from an individual cell and how are these genomes selected remain understudied. Here, we identify that, in most cases, only one genome per cell reactivates. Mutated HSV-1 genomes require coinfection with another strain to allow coreactivation. Our findings suggest that the decision to reactivate is determined for each quiescent genome separately and support the notion that reactivation preferences occur at the single-genome level.