Herpesvirus Replication Compartments Originate with Single Incoming Viral Genomes

ABSTRACT Previously we described a method to estimate the average number of virus genomes expressed in an infected cell. By analyzing the color spectrum of cells infected with a mixture of isogenic pseudorabies virus (PRV) recombinants expressing three fluorophores, we estimated that fewer than seven incoming genomes are expressed, replicated, and packaged into progeny per cell. In this report, we expand this work and describe experiments demonstrating the generality of the method, as well as providing more insight into herpesvirus replication. We used three isogenic PRV recombinants, each expressing a fluorescently tagged VP26 fusion protein (VP26 is a capsid protein) under the viral VP26 late promoter. We calculated a similar finite limit on the number of expressed viral genomes, indicating that this method is independent of the promoter used to transcribe the fluorophore genes, the time of expression of the fluorophore (early versus late), and the insertion site of the fluorophore gene in the PRV genome (UL versus US). Importantly, these VP26 fusion proteins are distributed equally in punctate virion assembly structures in each nucleus, which improves the signal-to-noise ratio when determining the color spectrum of each cell. To understand how the small number of genomes are distributed among the replication compartments, we used a two-color fluorescent in situ hybridization assay. Most viral replication compartments in the nucleus occupy unique nuclear territories, implying that they arose from single genomes. Our experiments suggest a correlation between the small number of expressed viral genomes and the limited number of replication compartments. IMPORTANCE Herpesviruses use nuclear factors and architecture to replicate their DNA genomes in the host nuclei. Viral replication compartments are distinct nuclear foci that appear during productive infection. We have recently developed a method that uses three viral recombinants (each expressing a different fluorescent protein) to quantify the number of incoming viral genomes that are expressed and replicated in each cell. We found that fewer than seven herpesvirus genomes can be expressed and replicated. Here we have expanded and improved upon our method and demonstrated that the phenomenon of limited genome expression is independent of the recombinants used. We correlated the small number of genomes expressed to the limited number of replication compartments by demonstrating that most replication compartments originate with a single genome. The distinction among replication compartments is maintained even when most of the nucleus is filled with viral DNA, implying that nuclear architecture constrains the compartments.