Christian O. Simon

CD8 T Cells Control Cytomegalovirus Latency by Epitope-Specific Sensing of Transcriptional Reactivation

ABSTRACT 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 derivedfrom 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 Ld 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.

Cytomegalovirus Misleads Its Host by Priming of CD8 T Cells Specific for an Epitope Not Presented in Infected Tissues

Cytomegaloviruses (CMVs) code for several proteins that inhibit the presentation of antigenic peptides to CD8 T cells. Although the molecular mechanisms of CMV interference with the major histocompatibility complex class I pathway are long understood, surprisingly little evidence exists to support a role in vivo. Here we document the first example of the presentation of an antigenic peptide being blocked by a CMV immune evasion protein in organs relevant to CMV disease. Although this Db-restricted peptide, which is derived from the antiapoptotic protein M45 of murine CMV (mCMV), is classified as an immunodominant peptide based on response magnitude and long-term memory, adoptive transfer of M45 epitope-specific CD8 T cells did not protect against infection with wild-type mCMV. Notably, the same cells protected C57BL/6 mice infected with an mCMV mutant in which immune evasion protein m152/gp40 is deleted. These data indicate that direct presentation or cross-presentation of an antigenic peptide by professional antigen-presenting cells can efficiently prime CD8 T cells that fail in protection against CMV organ disease because m152/gp40 prevents presentation of this peptide in pathogenetically relevant tissue cells.

Murine Model of Cytomegalovirus Latency and Reactivation

Efficient resolution of acute cytopathogenic cytomegalovirus infection through innate and adaptive host immune mechanisms is followed by lifelong maintenance of the viral genome in host tissues in a state of replicative latency, which is interrupted by episodes of virus reactivation for transmission. The establishment of latency is the result of aeons of co-evolution of cytomegaloviruses and their respective host species. Genetic adaptation of a particular cytomegalovirus to its specific host is reflected by private gene families not found in other members of the cytomegalovirus group, whereas basic functions of the viral replicative cycle are encoded by public gene families shared between different cytomegaloviruses or even with herpesviruses in general. Private genes include genes coding for immunoevasins, a group of glycoproteins specifically dedicated to dampen recognition by the hosts innate and adaptive immune surveillance to protect the virus against elimination. Recent data in the mouse model of cytomegalovirus latency have indicated that viral replicative latency established in the immunocompetent host is a dynamic state characterized by episodes of viral gene desilencing and immune sensing of reactivated presentation of antigenic peptides at immunological checkpoints by CD8 T cells. This sensing maintains viral replicative latency by triggering antiviral effector functions that terminate the viral gene expression program before infectious viral progeny are assembled. According to the immune sensing hypothesis of latency control, immunological checkpoints are unique for each infected individual in reflection of host MHC (HLA) polymorphism and the proteome(s) of the viral variant(s) harbored in latency.

Role for Tumor Necrosis Factor Alpha in Murine Cytomegalovirus Transcriptional Reactivation in Latently Infected Lungs

ABSTRACT 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 ∼2:1. MIEP1/3 latency-associated 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-α) 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-α 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-α did not predispose latently infected mice to a higher incidence of virus recurrence after hematoablative treatment. In conclusion, TNF-α 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.

Subdominant CD8 T-Cell Epitopes Account for Protection against Cytomegalovirus Independent of Immunodomination

ABSTRACT Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-TM) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-ΔIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule Ld and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule Dd, are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-TM cells primed by mCMV-ΔIDE and the corresponding revertant virus mCMV-revΔIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-TM cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-TM cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.

Processing and presentation of murine cytomegalovirus pORFm164-derived peptide in fibroblasts in the face of all viral immunosubversive early gene functions.

ABSTRACT CD8 T cells are the principal effector cells in the resolution of acute murine cytomegalovirus (mCMV) infection in host organs. This undoubted antiviral and protective in vivo function of CD8 T cells appeared to be inconsistent with immunosubversive strategies of the virus effected by early (E)-phase genes m04, m06, and m152. The so-called immune evasion proteins gp34, gp48, and gp37/40, respectively, were found to interfere with peptide presentation at different steps in the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation in fibroblasts. Accordingly, they were proposed to prevent recognition and lysis of infected fibroblasts by cytolytic T lymphocytes (CTL) during the E phase of viral gene expression. We document here that the previously identified MHC class I Dd-restricted antigenic peptide 257AGPPRYSRI265 encoded by gene m164 is processed as well as presented for recognition by m164-specific CTL during the E and late phases of viral replication in the very same cells in which the immunosubversive viral proteins are effectual in preventing the presentation of processed immediate-early 1 (m123-exon 4) peptide 168YPHFMPTNL176. Thus, while immunosubversion is a reality, these mechanisms are apparently not as efficient as the term immune evasion implies. The pORFm164-derived peptide is the first noted peptide that constitutively escapes the immunosubversive viral functions. The most important consequence is that even the concerted action of all immunosubversive E-phase proteins eventually fails to prevent immune recognition in the E phase. The bottom-line message is that there exists no immune evasion of mCMV in fibroblasts.

Viral latency drives ‘memory inflation’: a unifying hypothesis linking two hallmarks of cytomegalovirus infection

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.’

The Immune Evasion Paradox: Immunoevasins of Murine Cytomegalovirus Enhance Priming of CD8 T Cells by Preventing Negative Feedback Regulation

ABSTRACT Cytomegaloviruses express glycoproteins that interfere with antigen presentation to CD8 T cells. Although the molecular modes of action of these “immunoevasins” differ between cytomegalovirus species, the convergent biological outcome is an inhibition of the recognition of infected cells. In murine cytomegalovirus, m152/gp40 retains peptide-loaded major histocompatibility complex class I molecules in a cis-Golgi compartment, m06/gp48 mediates their vesicular sorting for lysosomal degradation, and m04/gp34, although not an immunoevasin in its own right, appears to assist in the concerted action of all three molecules. Using the Ld-restricted IE1 epitope YPHFMPTNL in the BALB/c mouse model as a paradigm, we provide here an explanation for the paradox that immunoevasins enhance CD8 T-cell priming although they inhibit peptide presentation in infected cells. Adaptive immune responses are initiated in the regional lymph node (RLN) draining the site of pathogen exposure. In particular for antigens that are not virion components, the magnitude of viral gene expression providing the antigens is likely a critical parameter in priming efficacy. We have therefore focused on the events in the RLN and have related priming to intranodal viral gene expression. We show that immunoevasins enhance priming by downmodulating an early CD8 T-cell-mediated “negative feedback” control of the infection in the cortical region of the RLN, thus supporting the model that immunoevasins improve antigen supply for indirect priming by uninfected antigen-presenting cells. As an important consequence, these findings predict that deletion of immunoevasin genes in a replicative vaccine virus is not a favorable option but may, rather, be counterproductive.

Transactivation of Cellular Genes Involved in Nucleotide Metabolism by the Regulatory IE1 Protein of Murine Cytomegalovirus Is Not Critical for Viral Replicative Fitness in Quiescent Cells and Host Tissues

ABSTRACT Despite its high coding capacity, murine CMV (mCMV) does not encode functional enzymes for nucleotide biosynthesis. It thus depends on cellular enzymes, such as ribonucleotide reductase (RNR) and thymidylate synthase (TS), to be supplied with deoxynucleoside triphosphates (dNTPs) for its DNA replication. Viral transactivation of these cellular genes in quiescent cells of host tissues is therefore a parameter of viral fitness relevant to pathogenicity. Previous work has shown that the IE1, but not the IE3, protein of mCMV transactivates RNR and TS gene promoters and has revealed an in vivo attenuation of the mutant virus mCMV-ΔIE1. It was attractive to propose the hypothesis that lack of transactivation by IE1 and a resulting deficiency in the supply of dNTPs are the reasons for growth attenuation. Here, we have tested this hypothesis with the mutant virus mCMV-IE1-Y165C expressing an IE1 protein that selectively fails to transactivate RNR and TS in quiescent cells upon transfection while maintaining the capacity to disperse repressive nuclear domains (ND10). Our results confirm in vivo attenuation of mCMV-ΔIE1, as indicated by a longer doubling time in host organs, whereas mCMV-IE1-Y165C replicated like mCMV-WT and the revertant virus mCMV-IE1-C165Y. Notably, the mutant virus transactivated RNR and TS upon infection of quiescent cells, thus indicating that IE1 is not the only viral transactivator involved. We conclude that transactivation of cellular genes of dNTP biosynthesis is ensured by redundancy and that attenuation of mCMV-ΔIE1 results from the loss of other critical functions of IE1, with its function in the dispersal of ND10 being a promising candidate.

Murine Cytomegalovirus Major Immediate-Early Enhancer Region Operating as a Genetic Switch in Bidirectional Gene Pair Transcription

ABSTRACT Enhancers are defined as DNA elements that increase transcription when placed in any orientation relative to a promoter. The major immediate-early (MIE) enhancer region of murine cytomegalovirus is flanked by transcription units ie1/3 and ie2, which are transcribed in opposite directions. We have addressed the fundamental mechanistic question of whether the enhancer synchronizes transcription of the bidirectional gene pair (synchronizer model) or whether it operates as a genetic switch, enhancing transcription of either gene in a stochastic alternation (switch model). Clonal analysis of cytokine-triggered, transcription factor-mediated MIE gene expression from latent viral genomes provided evidence in support of the switch model.