We and others have consistently observed that plaques formed upon infection with wild-type (or mutant rescue) MHV68 are often quite heterogeneous in size. One possible explanation for this is asynchronous entry into the lytic cycle, in which smaller plaques arise from later entry into the lytic cycle. In this regard, we sought to determine whether the large plaque phenotype of MHV68.DHS might be due to enhanced entry into the lytic cycle, signified by an increased percentage of infected cells expressing early lytic proteins relative to the wild-type virus. To test this hypothesis, we acquired a version of MHV68 (MHV68-YFP) which constitutively expresses YFP from a CMV promoter, regardless of whether infected cells are in the lytic or latent phase [29] . Thus, YFP expression serves as a general marker of infection, whereas lytically infected cells are identified by immunofluorescence-based detection of the early lytic protein muSOX. NIH 3T3 cells were infected at an MOI of 1 with either MHV68-YFP or a version of MHV68-YFP bearing the muSOX R443I mutation (MHV68-YFP.DHS), and the percentage of cells co-expressing muSOX and YFP was calculated at 18 hours post infection. A similar number of YFP-expressing cells were detected in cultures infected with MHV68-YFP and MHV68-YFP.DHS, indicating equivalent efficiency of initial infection ( Figure 5C & data not shown). However, there was an approximately three-fold increase in the percent of cells coexpressing both YFP and muSOX following infection with MHV68-YFP.DHS compared to MHV68-YFP (30.1% versus 11.0%; Figure 5C ). A similar trend was observed when the YFPand muSOX-expressing cell populations were analyzed by flow cytometry following infection with these viruses ( Figure S1 ). Also in agreement with these findings were increased levels of the viral ORF54 transcript following a MHV68.DHS infection relative to MHV68.WT or MHV68.MR ( Figure S2 ). Thus, enhanced and/ or more rapid expression of lytic genes occurs in cells infected with MHV68.DHS, perhaps escalating entry into the lytic cycle and leading to larger plaques.