During murine cytomegalovirus (mCMV) latency in the lungs, most of the viral genomes are transcriptionally silent at the major immediate-early locus, but rare and stochastic episodes of desilencing lead to the expression of IE1 transcripts. This low-frequency but perpetual expression is accompanied by an activation of lung-resident effector-memory CD8 T cells specific for the antigenic peptide 168-YPHFMPTNL-176, which is derived from the IE1 protein. These molecular and immunological findings were combined in the "silencing/ desilencing and immune sensing hypothesis" of cytomegalovirus latency and reactivation. This hypothesis proposes that IE1 gene expression proceeds to cell surface presentation of the IE1 peptide by the major histocompatibility complex (MHC) class I molecule L d and that its recognition by CD8 T cells terminates virus reactivation. Here we provide experimental evidence in support of this hypothesis. We generated mutant virus mCMV-IE1-L176A, in which the antigenic IE1 peptide is functionally deleted by a point mutation of the C-terminal MHC class I anchor residue Leu into Ala. Two revertant viruses, mCMV-IE1-A176L and the wobble nucleotide-marked mCMV-IE1-A176L*, in which Leu is restored by back-mutation of Ala codon GCA into Leu codons CTA and CTT, respectively, were constructed. Pulmonary latency of the mutant virus was found to be associated with an increased prevalence of IE1 transcription and with events of IE3 transactivator splicing. In conclusion, IE1-specific CD8 T cells recognize and terminate virus reactivation in vivo at the first opportunity in the reactivated gene expression program. The perpetual gene expression and antigen presentation might represent the driving molecular force in CMV-associated immunosenescence.After resolution of productive primary infection, in particular by CD8 T cells, cytomegaloviruses (CMVs) establish lifelong latent infections in their respective hosts (for reviews, see references 29, 31, 32, 52, 75, 83-85, and 87). Reactivation of latent human CMV (hCMV) to productive, cytopathogenic infection is still a health risk in immunocompromised patients (9, 57). Hematoablative therapy of leukemias, followed by bone marrow transplantation (BMT) or hematopoietic stem cell transplantation, is associated with a risk of CMV disease resulting from reactivation of latent donor and/or recipient CMV (15, 23). Among the manifestations of CMV disease in humans, interstitial pneumonia is the most dreaded because of its high fatality rate (79). Lungs were also identified as a major organ site of murine CMV (mCMV) disease, latency, and recurrence (4,43,70,78).Studies in the BALB/c mouse model of CMV infection in the BMT recipient have focused on the lungs for investigating mechanisms of immune control, latency, and reactivation (reviewed in references 25, 75, and 83). In this model, control of productive lung infection and prevention of disseminated viral pneumonia proved to be critically dependent upon the efficient reconstitution of CD8 T cells that infiltrated the lungs...

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Low public awareness of cytomegalovirus (CMV) results from the only mild and transient symptoms that it causes in the healthy immunocompetent host, so that primary infection usually goes unnoticed. The virus is not cleared, however, but stays for the lifetime of the host in a non-infectious, replicatively dormant state known as 'viral latency'. Medical interest in CMV results from the fact that latent virus can reactivate to cytopathogenic, tissue-destructive infection causing life-threatening end-organ disease in immunocompromised recipients of solid organ transplantation (SOT) or hematopoietic cell transplantation (HCT). It is becoming increasingly clear that CMV latency is not a static state in which the viral genome is silenced at all its genetic loci making the latent virus immunologically invisible, but rather is a dynamic state characterized by stochastic episodes of transient viral gene desilencing. This gene expression can lead to the presentation of antigenic peptides encoded by 'antigenicity-determining transcripts expressed in latency (ADTELs)' sensed by tissue-patrolling effector-memory CD8 T cells for immune surveillance of latency [In Reddehase et al., Murine model of cytomegalovirus latency and reactivation, Current Topics in Microbiology and Immunology, vol 325. Springer, Berlin, pp 315-331, 2008]. A hallmark of the CD8 T cell response to CMV is the observation that with increasing time during latency, CD8 T cells specific for certain viral epitopes increase in numbers, a phenomenon that has gained much attention in recent years and is known under the catchphrase 'memory inflation.' Here, we provide a unifying hypothesis linking stochastic viral gene desilencing during latency to 'memory inflation.'

Interstitial pneumonia is a major clinical manifestation of primary or recurrent cytomegalovirus (CMV) infection in immunocompromised recipients of a bone marrow transplant. In a murine model, lungs were identified as a prominent site of CMV latency and recurrence. Pulmonary latency of murine CMV is characterized by high viral genome burden and a low incidence of variegated immediate-early (IE) gene expression, reflecting a sporadic activity of the major IE promoters (MIEPs) and enhancer. The enhancer-flanking promoters MIEP1/3 and MIEP2 are switched on and off during latency in a ratio of ϳ2:1. MIEP1/3 latencyassociated activity generates the IE1 transcript of the ie1/3 transcription unit but not the alternative splicing product IE3 that encodes the essential transactivator of early gene expression. Splicing thus appeared to be an important checkpoint for maintenance of latency. In accordance with previous work of others, we show here that signaling by the proinflammatory cytokine tumor necrosis factor alpha (TNF-␣) activates IE1/3 transcription in vivo. As an addition to current knowledge, Poisson distribution analysis revealed an increased incidence of IE1/3 transcriptional events as well as a higher amount of transcripts per event. Notably, TNF-␣ promoted the splicing to IE3 transcripts, but transcription did not proceed to the M55/gB early gene. Moreover, the activated transcriptional state induced by TNF-␣ did not predispose latently infected mice to a higher incidence of virus recurrence after hematoablative treatment. In conclusion, TNF-␣ is an important inductor of IE gene transcriptional reactivation, whereas early genes downstream in the viral replicative cycle appear to be the rate-limiting checkpoint(s) for virus recurrence.

In human cytomegalovirus (hCMV) infection, hematopoietic progenitor cells of the myeloid differentiation lineage are a recognized cellular site of virus latency (for more-recent reviews, see references 75 and 94), and cell differentiationdependent as well as cytokine-mediated viral gene desilencing by chromatin remodeling is discussed as the triggering event leading to virus reactivation (for a review, see reference 7). Although hematopoietic stem cell or bone marrow transplantation (BMT) is frequently associated with hCMV reactivation and recurrence in recipients after hematoablative leukemia/ lymphoma therapy, the incidence of virus recurrence and disease is highest in the combination of an hCMV-negative donor (D