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Roizman's definition of herpesviral latency, which applies also to cytomegaloviruses (CMVs), demands maintenance of reactivation-competent viral genomes after clearance of productive infection. It is more recent understanding that failure to complete the productive viral cycle for virus assembly and release does not imply viral gene silencing at all genetic loci and all the time. It rather appears that CMV latency is transcriptionally "noisy" in that silenced viral genes get desilenced from time to time in a stochastic manner, leading to "transcripts expressed in latency" (TELs). If a TEL happens to code for a protein that contains a CD8 T cell epitope, protein processing can lead to the presentation of the antigenic peptide and restimulation of cognate CD8 T cells during latency. This mechanism is discussed as a potential driver of epitope-selective accumulation of CD8 T cells over time, a phenomenon linked to CMV latency and known as "memory inflation" (MI). So far, expression of an epitope-encoding TEL was shown only for the major immediate-early (MIE) gene m123/ie1 of murine cytomegalovirus (mCMV), which codes for the prototypic MI-driving antigenic peptide YPHFMPTNL that is presented by the MHC class-I molecule Ld. The only known second MI-driving antigenic peptide of mCMV in the murine MHC haplotype H-2d is AGPPRYSRI presented by the MHC-I molecule Dd. This peptide is very special in that it is encoded by the early (E) phase gene m164 and by an overlapping immediate-early (IE) transcript governed by a promoter upstream of m164. If MI is driven by presentation of TEL-derived antigenic peptides, as the hypothesis says, one should find corresponding TELs. We show here that E-phase and IE-phase transcripts that code for the MI-driving antigenic peptide AGPPRYSRI are independently and stochastically expressed in latently infected lungs.

Acute infection with murine cytomegalovirus (mCMV) is controlled by CD8+ T cells and develops into a state of latent infection, referred to as latency, which is defined by lifelong maintenance of viral genomes but absence of infectious virus in latently infected cell types. Latency is associated with an increase in numbers of viral epitope-specific CD8+ T cells over time, a phenomenon known as “memory inflation” (MI). The “inflationary” subset of CD8+ T cells has been phenotyped as KLRG1+CD62L- effector-memory T cells (iTEM). It is agreed upon that proliferation of iTEM requires repeated episodes of antigen presentation, which implies that antigen-encoding viral genes must be transcribed during latency. Evidence for this has been provided previously for the genes encoding the MI-driving antigenic peptides IE1-YPHFMPTNL and m164-AGPPRYSRI of mCMV in the H-2d haplotype. There exist two competing hypotheses for explaining MI-driving viral transcription. The “reactivation hypothesis” proposes frequent events of productive virus reactivation from latency. Reactivation involves a coordinated gene expression cascade from immediate-early (IE) to early (E) and late phase (L) transcripts, eventually leading to assembly and release of infectious virus. In contrast, the “stochastic transcription hypothesis” proposes that viral genes become transiently de-silenced in latent viral genomes in a stochastic fashion, not following the canonical IE-E-L temporal cascade of reactivation. The reactivation hypothesis, however, is incompatible with the finding that productive virus reactivation is exceedingly rare in immunocompetent mice and observed only under conditions of compromised immunity. In addition, the reactivation hypothesis fails to explain why immune evasion genes, which are regularly expressed during reactivation in the same cells in which epitope-encoding genes are expressed, do not prevent antigen presentation and thus MI. Here we show that IE, E, and L genes are transcribed during latency, though stochastically, not following the IE-E-L temporal cascade. Importantly, transcripts that encode MI-driving antigenic peptides rarely coincide with those that encode immune evasion proteins. As immune evasion can operate only in cis, that is, in a cell that simultaneously expresses antigenic peptides, the stochastic transcription hypothesis explains why immune evasion is not operative in latently infected cells and, therefore, does not interfere with MI.

Members of the genus Bartonella are fastidious Gram-negative facultative intracellular bacteria that are typically transmitted by arthropod vectors. Several Bartonella spp. have been found to cause culture-negative endocarditis in humans. Here, we report the case of a 75-year old German woman with prosthetic valve endocarditis due to Bartonella washoensis. The infecting agent was characterized by sequencing of six housekeeping genes (16S rRNA, ftsZ, gltA, groEL, ribC, rpoB) applying a multilocus sequence typing (MLST) approach. The 5097 bp of the concatenated housekeeping gene sequence from the patient were 99.0% identical to a B. washoensis strain from a red squirrel (Sciurus vulgaris orientis) from China. 39% (24/62) of red squirrel (S. vulgaris) samples from the Netherlands were positive for the B. washoensis gltA gene variant detected in the patient. This suggests that the red squirrel is the reservoir host for human infection in Europe.

Expansion of the CD8 T-cell memory pool, also known as ‘memory inflation’, for certain but not all viral epitopes in latently infected host tissues is a special feature of the immune response to cytomegalovirus. The Ld-presented murine cytomegalovirus (mCMV) immediate–early (IE) 1 peptide is the prototype of an epitope that is associated with memory inflation. Based on the detection of IE1 transcripts in latently infected lungs it was previously proposed that episodes of viral gene expression and antigenic activity due to desilencing of a limited number of viral genes may drive epitope-specific memory inflation. This would imply direct antigen presentation through latently infected host tissue cells rather than cell death-associated cross-presentation of viral antigens derived from productively infected cells through uninfected, professional antigen-presenting cells (profAPCs). To address the role of bone marrow-derived profAPCs in CD8 T-cell priming and memory to mCMV, we have used here a combined sex-mismatched and MHC class-I mismatched dual-marker bone marrow chimera model in which presentation of the IE1 epitope is restricted to donor-derived sry +Ld+ cells of haematopoietic differentiation lineages. Successful CD8 T-cell priming specific for the Ld- and Dd-presented inflationary epitopes IE1 and m164, respectively, but selective failure in IE1 epitope-specific memory inflation in these chimeras indicates different modes of antigen presentation involved in CD8 T-cell priming and memory inflation. These data suggest that memory inflation during mCMV latency requires expression of the epitope-presenting MHC class-I molecule by latently infected non-haematopoietic host tissue cells and thus predicts a role for direct antigen presentation in memory inflation.

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor–GFP reporter mice, we never observed reactivation originating from CX3CR1+ monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b+ and CX3CR1+ subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

Gene m164 of murine cytomegalovirus belongs to the large group of 'private' genes that show no homology to those of other cytomegalovirus species and are thought to represent 'host adaptation' genes involved in virus-host interaction. Previous interest in the m164 gene product was based on the presence of an immunodominant CD8 T-cell epitope presented at the surface of infected cells, despite interference by viral immune-evasion proteins. Here, we provide data to reveal that the m164 gene product shows unusual features in its cell biology. A novel strategy of mass-spectrometric analysis was employed to map the N terminus of the mature protein, 107 aa downstream of the start site of the predicted open reading frame. The resulting 36.5 kDa m164 gene product is identified here as an integral type-I membrane glycoprotein with exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane with an outstandingly high lateral membrane motility, actually 100 times higher than those published for cellular ER-resident proteins. Notably, gp36.5/m164 does not contain any typical ER-retention/retrieval signals, such as the C-terminal motifs KKXX or KXKXX, and does not pass the Golgi apparatus. Instead, it belongs to the rare group of viral glycoproteins in which the transmembrane domain (TMD) itself mediates direct ER retention. This is the first report relating TMD usage of an ER-resident transmembrane protein to its lateral membrane motility as a paradigm in cell biology. We propose that TMD usage for ER retention facilitates free and fast floating in ER-related membranes and between ER subdomains.

CD8 T cells control cytomegalovirus (CMV) infection in bone marrow transplantation recipients and persist in latently infected lungs as effector memory cells for continuous sensing of reactivated viral gene expression. Here we have addressed the question of whether viral immunoevasins, glycoproteins that specifically interfere with antigen presentation to CD8 T cells, have an impact on viral latency in the murine model. The data show that deletion of immunoevasin genes in murine CMV accelerates the clearance of productive infection during hematopoietic reconstitution and leads to a reduced latent viral genome load, reduced latency-associated viral transcription, and a lower incidence of recurrence in lung explants.

Hematopoietic stem cell transplantation (HSCT) bears a risk of reactivating latent cytomegalovirus (CMV) in either the transplanted hematopoietic donor cells or in parenchymal and stromal tissue cells of the immunocompromised recipient, or in both. While reactivated human CMV in recipients of organ transplantations is frequently the virus variant of the donor, this is not usually the case in HSCT recipients. Here we have used experimental sex-mismatched HSCT in the BALB/c mouse model to test if latent murine CMV from CMV-immune donors is transmitted with bone marrow cells to naive immunocompromised recipients.

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.

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 advent of cloning herpesviral genomes as bacterial artificial chromosomes (BACs) has made herpesviruses accessible to bacterial genetics and has thus revolutionised their mutagenesis. This opened all possibilities of reverse genetics to ask scientific questions by introducing precisely accurate mutations into the viral genome for testing their influence on the phenotype under study or to create phenotypes of interest. Here, we report on our experience with using BAC technology for a designed modulation of viral antigenicity and immunogenicity with focus on the CD8 T-cell response. One approach is replacing an intrinsic antigenic peptide in a viral carrier protein with a foreign antigenic sequence, a strategy that we have termed “orthotopic peptide swap”. Another approach is the functional deletion of an antigenic peptide by point mutation of its C-terminal MHC class-I anchor residue. We discuss the concepts and summarize recently published major scientific results obtained with immunological mutants of murine cytomegalovirus.

Publisher Summary Cytomegaloviruses (CMVs) are conditional pathogens that are strictly species specific and are usually well controlled in their respective mammalian hosts by the effector mechanisms of both innate and adaptive immunity. Human CMV (hCMV) is mostly acquired perinatally as well as in early childhood and is transmitted, for instance, through breast milk and saliva. Whilst the immune response in an immunocompetent host prevents an overt CMV disease and rapidly terminates the productive acute infection, viral genome is maintained in most tissues for the life span of the infected host in a state known as viral latency. Latency implies that infectious virions are no longer produced so that the host is no longer infectious. Furthermore, CMV diseases with multiple organ manifestations such as interstitial pneumonia, hepatitis, adrenalitis, gastrointestinal disease and bone marrow failure result from primary or recurrent infection of the immunocompromised or immunologically immature host. The chapter also describes murine CMV.

Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy )-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients' tissues. The viral genome was found to localize primarily to sry -negative CD11b − CD11c − CD31 + CD146 + cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry -positive CD146 + cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 10 4 LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M11 2 - M 1 13/e1 , M55/gB , or M86/MCP . Importantly, in an LSEC transfer model, infectious virus reactivated from recipients' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver.

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 acis-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.

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.

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 host's 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.

Cytomegalovirus (CMV) poses a threat to the therapy of hematopoietic malignancies by hematopoietic stem cell transplantation, but efficient reconstitution of antiviral immunity prevents CMV organ disease. Tumor relapse originating from a minimal residual leukemia poses another threat. Although a combination of risk factors was supposed to enhance the incidence and severity of transplantation-associated disease, a murine model of a liver-adapted B-cell lymphoma has previously shown a survival benefit and tumor growth inhibition by nonlethal subcutaneous infection with murine CMV. Here we have investigated the underlying antitumoral mechanism. Virus replication proved to be required, since inactivated virions or the highly attenuated enhancerless mutant mCMV-ΔMIEenh did not impact the lymphoma in the liver. Surprisingly, the dissemination-deficient mutant mCMV-ΔM36 inhibited tumor growth, even though this virus fails to infect the liver. On the other hand, various strains of herpes simplex viruses consistently failed to control the lymphoma, even though they infect the liver. A quantitative analysis of the tumor growth kinetics identified a transient tumor remission by apoptosis as the antitumoral effector mechanism. Tumor cell colonies with cells surviving the CMV-induced “apoptotic crisis” lead to tumor relapse even in the presence of full-blown tissue infection. Serial transfer of surviving tumor cells did not indicate a selection of apoptosis-resistant genetic variants. NK cell activity of CD49b-expressing cells failed to control the lymphoma upon adoptive transfer. We propose the existence of an innate antitumoral mechanism that is triggered by CMV infection and involves an apoptotic signal effective at a distant site of tumor growth.