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RESEARCH PRODUCT

Patchwork Pattern of Transcriptional Reactivation in the Lungs Indicates Sequential Checkpoints in the Transition from Murine Cytomegalovirus Latency to Recurrence

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The lungs are a relevant organ site of primary and recurrent human cytomegalovirus (hCMV) disease (for overviews, see references 21, 22, 31, 34, 39, and 44). Murine CMV (mCMV) can serve us as a model for studying CMV pneumonia in acute infection (6, 27, 33, 37) as well as for studying viral latency, reactivation, and recurrence in the lungs (2, 17, 18, 42, 43). We have shown recently that transcription from the major immediate-early (MIE) transcription unit ie1-ie3 (hereafter referred to as ie1/3), which is driven by a strong MIE promoter-enhancer (MIEPE) (3), occurs during pulmonary latency of mCMV but fails to initiate the productive cycle (17). Notably, the paralogous MIEPE of hCMV can functionally replace the MIEPE of mCMV for productive infection in vitro (1) and in vivo (4), suggesting that regulation of MIE gene expression via the enhancer element has some degrees of freedom and that a more stringent control is operative at subsequent checkpoints. During productive infection, a 2.75-kb ie1 gene mRNA (2,305 nucleotides of exons 1, 2, 3, and 4 of ie1/3) and a 2.75-kb ie3 gene mRNA (2,303 nucleotides of exons 1, 2, 3, and 5 of ie1/3) specifying the mCMV IE1 (12) and IE3 (26) proteins, respectively, are thought to be generated from a precursor transcript by differential splicing (10–12, 26). While viral genomes were found to be evenly distributed in the latently infected lungs (17), a mosaic of transcriptionally active and transcriptionally silent pieces of lung tissue indicated that MIE transcriptional activity at any given time during latency was focal, randomly distributed, and, most probably, temporary. Notably, in the transcriptionally active lung tissue pieces, IE1 mRNA was generated selectively, while IE3 mRNA was missing (17). For the sake of clarity it should be emphasized that we refer to transcriptional activity with specific respect to transcription of the ie1/3 transcription unit, which is controlled by the MIEPE and is implicated in the initiation of the productive cycle. One must certainly consider the possibility of transcription occurring elsewhere in the latent viral genome. Representational difference analysis comparing transcription in latently infected lung tissue with that in normal lung tissue has indeed already indicated expression of mCMV genes outside the ie1/3 transcription unit, and the identities of these transcripts are currently under investigation (43). The absence of IE3 mRNA explains the absence of gB early-late gene transcripts as well as of infectious virus, because the 88-kDa IE3 protein of mCMV (26), the functional analog of the hCMV 86-kDa IE2 protein (5, 14; reviewed in reference 41), is the main transactivator of early gene expression initiating the productive cycle. Specifically, previous work by Messerle et al. has demonstrated efficient activation of the e1 promoter by IE3 alone but not by IE1 alone, whereas IE1 did enhance the activity when coexpressed with IE3 (26). Accordingly, recurrent infection measured 14 days after immunoablative, genotoxic treatment was in fact associated with the generation of IE3 mRNA in addition to IE1 mRNA (17). We therefore concluded that transcription of the ie1/3 transcription unit, and thus an “on position” of the MIEPE, is a primary condition for virus recurrence and that there exists an additional, subsequent control point at the level of cotranscriptional processing defining the levels of IE1 and IE3 mRNAs. Actually, since the MIEPE was on or off during latency, cotranscriptional processing appears to provide a more stringent control of the latent state. In essence, these previous data have implied that IE3 rather than IE1 mRNA is indicative of productive reactivation. If this is true, the generation of IE3 mRNA should correlate with recurrence of infectious virus. The original aim of the present work was to investigate the temporal association between the generation of IE3 mRNA and virus recurrence in the lungs and to estimate the incidences of transcriptional reactivation and virus recurrence after immunoablative, genotoxic treatment. One could envisage two alternative mechanisms of CMV reactivation and recurrence. We refer to these mechanisms as the model of spontaneous reactivation and the model of induced reactivation. The model of spontaneous reactivation assumes that MIEPE activity, which drives IE1/3 precursor transcription, occurs randomly, either spontaneously or as a result of endogenous and random signalling, and that recurrence of infectious virus is precluded in immunocompetent mice by antiviral effector cells eliminating cells in which reactivation leads to the presentation of antigenic viral peptides. This mechanism was previously our own favored idea (17, 42) and was proposed recently based on the kinetics of mCMV recurrence after combined NK-cell and T-cell depletion in latently infected B-cell-deficient mice (28). If this model applies, we should find an accumulation of reactivations over time after withdrawal of immune control. By contrast, the model of induced reactivation assumes that reactivation involves an external signalling that has to switch on the productive viral cycle. In that case, reactivation should be a quantal event that follows the rule of all or nothing. Here we present data supporting the model of induced reactivation. Moreover, the pattern of transcriptional activity in the lungs after induction of reactivations reveals a hitherto unknown complexity of regulation involving multiple, sequential checkpoints on the way from mCMV latency to recurrence.