Stochastic Episodes of Latent Cytomegalovirus Transcription Drive CD8 T-Cell "Memory Inflation" and Avoid Immune Evasion.

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.

Evaluation of a laboratory-based high-throughput SARS-CoV-2 antigen assay for non-COVID-19 patient screening at hospital admission.

Several rapid antigen tests (RATs) for the detection of SARS-CoV-2 were evaluated recently. However, reliable performance data for laboratory-based, high-throughput antigen tests are lacking. Therefore and in response to a short-term shortage of PCR reagents, we evaluated DiaSorin's LIAISON SARS-CoV-2 antigen test in comparison to RT-qPCR, and concerning the application of screening non-COVID-19 patients on hospital admission. Applying the manufacturer-recommended cut-off of 200 arbitrary units (AU/mL) the specificity of the LIAISON Test was 100%, the overall analytical sensitivity 40.2%. Lowering the cut-off to 100 AU/mL increased the sensitivity to 49.7% and decreased the specificity to 98.3%. Confining the analysis to samples with an RT-qPCR result < 25 Ct resulted in a sensitivity of 91.2%. The quality of the LIAISON test is very similar to that of good RATs described in the literature with the advantage of high throughput and the disadvantage of relatively long analysis time. It passes the WHO quality criteria for rapid antigen tests and is characterized by particularly high specificity. The LIAISON test can therefore be used for the same applications as recommended for RATs by the WHO. Due to limited sensitivity, the LIAISON test should only be used for screening, if PCR-based assays are not available.

Revisiting CD8 T-cell 'Memory Inflation': New Insights with Implications for Cytomegaloviruses as Vaccine Vectors.

Murine models of cytomegalovirus (CMV) infection have revealed an exceptional kinetics of the immune response. After resolution of productive infection, transient contraction of the viral epitope-specific CD8 T-cell pool was found to be followed by a pool expansion specific for certain viral epitopes during non-productive 'latent' infection. This phenomenon, known as 'memory inflation' (MI), was found to be based on inflationary KLRG1+CD62L- effector-memory T cells (iTEM) that depend on repetitive restimulation. MI gained substantial interest for employing CMV as vaccine vector by replacing MI-driving CMV epitopes with foreign epitopes for generating high numbers of protective memory cells specific for unrelated pathogens. The concept of an MI-driving CMV vector is questioned by human studies disputing MI in humans. A bias towards MI in experimental models may have resulted from systemic infection. We have here studied local murine CMV infection as a route that is more closely matching routine human vaccine application. Notably, KLRG1-CD62L+ central memory T cells (TCM) and conventional KLRG1-CD62L- effector memory T cells (cTEM) were found to expand, associated with 'avidity maturation', whereas the pool size of iTEM steadily declined over time. The establishment of high avidity CD8 T-cell central memory encourages one to pursue the concept of CMV vector-based vaccines.

Cytomegalovirus-Associated Inhibition of Hematopoiesis Is Preventable by Cytoimmunotherapy With Antiviral CD8 T Cells.

Reactivation of latent cytomegalovirus (CMV) in recipients of hematopoietic cell transplantation (HCT) not only results in severe organ manifestations, but can also cause "graft failure" resulting in bone marrow (BM) aplasia. This inhibition of hematopoietic stem and progenitor cell engraftment is a manifestation of CMV infection that is long known in clinical hematology as "myelosuppression." Previous studies in a murine model of sex-chromosome mismatched but otherwise syngeneic HCT and infection with murine CMV have shown that transplanted hematopoietic cells (HC) initially home to the BM stroma of recipients but then fail to further divide and differentiate. Data from this model were in line with the hypothesis that infection of stromal cells, which constitute "hematopoietic niches" where hematopoiesis takes place, causes a local deficiency in essential hematopoietins. Based on this understanding, one must postulate that preventing infection of stromal cells should restore the stroma's capacity to support hematopoiesis. Adoptively-transferred antiviral CD8+ T cells prevent lethal CMV disease by controlling viral spread and histopathology in vital organs, such as liver and lungs. It remained to be tested, however, if they can also prevent infection of the BM stroma and thus allow for successful HC engraftment. Here we demonstrate that antiviral CD8+ T cells control stromal infection. By tracking male donor-derived sry+ HC in the BM of infected female sry- recipients, we show the CD8+ T cells allow for successful donor HC engraftment and thereby prevent CMV-associated BM aplasia. These data provide a further argument for cytoimmunotherapy of CMV infection after HCT.

Transcripts expressed in cytomegalovirus latency coding for an antigenic IE/E phase peptide that drives "memory inflation".

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.

Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors.

Human cytomegalovirus (HCMV) represents a major cause of clinical complications during pregnancy as well as immunosuppression, and the licensing of a protective HCMV vaccine remains an unmet global need. Here, we designed and validated novel Sendai virus (SeV) vectors delivering the T cell immunogens IE-1 and pp65. To enhance vector safety, we used a replication-deficient strain (rdSeV) that infects target cells in a nonproductive manner while retaining viral gene expression. In this study, we explored the impact that transduction with rdSeV has on human dendritic cells (DCs) by comparing it to the parental, replication-competent Sendai virus strain (rcSeV) as well as the poxvirus strain modified vaccinia Ankara (MVA). We found that wild-type SeV is capable of replicating to high titers in DCs while rdSeV infects cells abortively. Due to the higher degree of attenuation, IE-1 and pp65 protein levels mediated by rdSeV after infection of DCs were markedly reduced compared to those of the parental Sendai virus recombinants, but antigen-specific restimulation of T cell clones was not negatively affected by this. Importantly, rdSeV showed reduced cytotoxic effects compared to rcSeV and MVA and was capable of mediating DC maturation as well as secretion of alpha interferon and interleukin-6. Finally, in a challenge model with a murine cytomegalovirus (MCMV) strain carrying an HCMV pp65 peptide, we found that viral replication was restricted if mice were previously vaccinated with rdSeV-pp65. Taken together, these data demonstrate that rdSeV has great potential as a vector system for the delivery of HCMV immunogens.IMPORTANCE HCMV is a highly prevalent betaherpesvirus that establishes lifelong latency after primary infection. Congenital HCMV infection is the most common viral complication in newborns, causing a number of late sequelae ranging from impaired hearing to mental retardation. At the same time, managing HCMV reactivation during immunosuppression remains a major hurdle in posttransplant care. Since options for the treatment of HCMV infection are still limited, the development of a vaccine to confine HCMV-related morbidities is urgently needed. We generated new vaccine candidates in which the main targets of T cell immunity during natural HCMV infection, IE-1 and pp65, are delivered by a replication-deficient, Sendai virus-based vector system. In addition to classical prophylactic vaccine concepts, these vectors could also be used for therapeutic applications, thereby expanding preexisting immunity in high-risk groups such as transplant recipients or for immunotherapy of glioblastomas expressing HCMV antigens.

Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice.

Reactivation of human cytomegalovirus (HCMV) can cause severe disease in recipients of hematopoietic stem cell transplantation. Although preclinical research in murine models as well as clinical trials have provided 'proof of concept' for infection control by pre-emptive CD8 T-cell immunotherapy, there exists no predictive model to experimentally evaluate parameters that determine antiviral efficacy of human T cells in terms of virus control in functional organs, prevention of organ disease, and host survival benefit. We here introduce a novel mouse model for testing HCMV epitope-specific human T cells. The HCMV UL83/pp65-derived NLV-peptide was presented by transgenic HLA-A2.1 in the context of a lethal infection of NOD/SCID/IL-2rg-/- mice with a chimeric murine CMV, mCMV-NLV. Scenarios of HCMV-seropositive and -seronegative human T-cell donors were modeled by testing peptide-restimulated and T-cell receptor-transduced human T cells, respectively. Upon transfer, the T cells infiltrated host tissues in an epitope-specific manner, confining the infection to nodular inflammatory foci. This resulted in a significant reduction of viral load, diminished organ pathology, and prolonged survival. The model has thus proven its potential for a preclinical testing of the protective antiviral efficacy of HCMV epitope-specific human T cells in the evaluation of new approaches to an immunotherapy of CMV disease.

Identification of an atypical CD8 T cell epitope encoded by murine cytomegalovirus ORF-M54 gaining dominance after deletion of the immunodominant antiviral CD8 T cell specificities.

Control of murine cytomegalovirus (mCMV) infection is mediated primarily by CD8 T cells, with four specificities dominating in BALB/c mice. Functional deletion of the respective immunodominant epitopes (IDEs) in mutant virus Δ4IDE revealed a still efficient control of infection. In a murine model of hematopoietic cell transplantation and infection with Δ4IDE, an mCMV-specific open reading frame (ORF) library screening assay indicated a strong CD8 T cell reactivity against the ORF-M54 product, the highly conserved and essential mCMV homolog of human CMV DNA polymerase UL54, which is a known inducer of in vivo protection against mCMV by DNA immunization. Applying bioinformatic algorithms for CD8 T cell epitope prediction, the top-scoring peptides were used to stimulate ex vivo-isolated CD8 T cells and to generate cytolytic T cell lines; yet, this approach failed to identify M54 epitope(s). As an alternative, a peptide library consisting of 549 10-mers with an offset of two amino acids (aa), covering the complete aa-sequence of the M54 protein, was synthesized and used for the stimulation. A region of 12 aa proved to encompass an epitope. An 'alanine walk' over this antigenic 12-mer and all possible 11-, 10- and 9-mers derived thereof revealed aa-residues critical for antigenicity, and terminal truncations identified the H-2D(d) presented 8-mer M5483-90 as the optimal epitope. An increased frequency of the corresponding CD8 T cells in the absence of the 4 IDEs indicated immunodomination by the IDE-specific CD8 T cells as a mechanism by which the generation of M54-specific CD8 T cells is inhibited after infection with wild-type mCMV.

Mast cells as rapid innate sensors of cytomegalovirus by TLR3/TRIF signaling-dependent and -independent mechanisms.

The succinct metaphor, 'the immune system's loaded gun', has been used to describe the role of mast cells (MCs) due to their storage of a wide range of potent pro-inflammatory and antimicrobial mediators in secretory granules that can be released almost instantly on demand to fight invaders. Located at host-environment boundaries and equipped with an arsenal of pattern recognition receptors, MCs are destined to be rapid innate sensors of pathogens penetrating endothelial and epithelial surfaces. Although the importance of MCs in antimicrobial and antiparasitic defense has long been appreciated, their role in raising the alarm against viral infections has been noted only recently. Work on cytomegalovirus (CMV) infection in the murine model has revealed MCs as players in a novel cross-talk axis between innate and adaptive immune surveillance of CMV, in that infection of MCs, which is associated with MC degranulation and release of the chemokine CCL5, enhances the recruitment of protective CD8 T cells to extravascular sites of virus replication, specifically to lung interstitium and alveolar epithelium. Here, we have expanded on these studies by investigating the conditions for MC activation and the consequent degranulation in response to host infection. Surprisingly, the data revealed two temporally and mechanistically distinct waves of MC activation: an almost instant indirect activation that depended on TLR3/TRIF signaling and delayed activation by direct infection of MCs that did not involve TLR3/TRIF signaling. Cell type-specific Cre-recombination that yielded eGFP-expressing reporter virus selectively originating from MCs identified MC as a new in vivo, first-hit target cell of productive murine CMV infection.

The p36 isoform of murine cytomegalovirus m152 protein suffices for mediating innate and adaptive immune evasion.

The MHC-class I (MHC-I)-like viral (MHC-Iv) m152 gene product of murine cytomegalovirus (mCMV) was the first immune evasion molecule described for a member of the ß-subfamily of herpesviruses as a paradigm for analogous functions of human cytomegalovirus proteins. Notably, by interacting with classical MHC-I molecules and with MHC-I-like RAE1 family ligands of the activatory natural killer (NK) cell receptor NKG2D, it inhibits presentation of antigenic peptides to CD8 T cells and the NKG2D-dependent activation of NK cells, respectively, thus simultaneously interfering with adaptive and innate immune recognition of infected cells. Although the m152 gene product exists in differentially glycosylated isoforms whose individual contributions to immune evasion are unknown, it has entered the scientific literature as m152/gp40, based on the quantitatively most prominent isoform but with no functional justification. By construction of a recombinant mCMV in which all three N-glycosylation sites are mutated (N61Q, N208Q, and N241Q), we show here that N-linked glycosylation is not essential for functional interaction of the m152 immune evasion protein with either MHC-I or RAE1. These data add an important functional detail to recent structural analysis of the m152/RAE1g complex that has revealed N-glycosylations at positions Asn61 and Asn208 of m152 distant from the m152/RAE1g interface.

Immune control in the absence of immunodominant epitopes: implications for immunotherapy of cytomegalovirus infection with antiviral CD8 T cells.

Adoptive transfer of virus-specific donor-derived CD8 T cells is a therapeutic option to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic cell transplantation. Due to their high coding capacity, human as well as animal CMVs have the potential to encode numerous CD8 T cell epitopes. Although the CD8 T cell response to CMVs is indeed broadly specific in that it involves epitopes derived from almost every open reading frame when tested for cohorts of immune CMV carriers representing the polymorphic MHC/HLA distribution in the population, the response in any one individual is directed against relatively few epitopes selected by the private combination of MHC/HLA alleles. Of this individually selected set of epitopes, few epitopes are 'immunodominant' in terms of magnitude of the response directed against them, while others are 'subdominant' according to this definition. In the assumption that 'immunodominance' indicates 'relevance' in antiviral control, research interest was focused on the immunodominant epitopes (IDEs) and their potential use in immunotherapy and in vaccines. The murine model has provided 'proof of concept' for the efficacy of CD8 T cell therapy of CMV infection. By experimental modulation of the CD8 T cell 'immunome' of murine CMV constructing an IDE deletion mutant, we have used this established cytoimmunotherapy model (a) for evaluating the actual contribution of IDEs to the control of infection and (b) for answering the question whether antigenicity-determining codon polymorphisms in IDE-encoding genes of CMV strains impact on the efficacy of CD8 T cell immunotherapy in case the donor and the recipient harbor different CMV strains.

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.'

Antigen-presenting cells of haematopoietic origin prime cytomegalovirus-specific CD8 T-cells but are not sufficient for driving memory inflation during viral latency.

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 L(d)-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(+)L(d+) cells of haematopoietic differentiation lineages. Successful CD8 T-cell priming specific for the L(d)- and D(d)-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.

A novel transmembrane domain mediating retention of a highly motile herpesvirus glycoprotein in the endoplasmic reticulum.

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.

Immune evasion proteins enhance cytomegalovirus latency in the lungs.

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.

Synergism between the components of the bipartite major immediate-early transcriptional enhancer of murine cytomegalovirus does not accelerate virus replication in cell culture and host tissues.

Major immediate-early (MIE) transcriptional enhancers of cytomegaloviruses are key regulators that are regarded as determinants of virus replicative fitness and pathogenicity. The MIE locus of murine cytomegalovirus (mCMV) shows bidirectional gene-pair architecture, with a bipartite enhancer flanked by divergent core promoters. Here, we have constructed recombinant viruses mCMV-DeltaEnh1 and mCMV-DeltaEnh2 to study the impact of either enhancer component on bidirectional MIE gene transcription and on virus replication in cell culture and various host tissues that are relevant to CMV disease. The data revealed that the two unipartite enhancers can operate independently, but synergize in enhancing MIE gene expression early after infection. Kick-start transcription facilitated by the bipartite enhancer configuration, however, did not ultimately result in accelerated virus replication. We conclude that virus replication, once triggered, proceeds with a fixed speed and we propose that synergism between the components of the bipartite enhancer may rather increase the probability for transcription initiation.

Liver sinusoidal endothelial cells are a site of murine cytomegalovirus latency and reactivation.

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 M112-M113/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.

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.

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-DeltaIE1. 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-DeltaIE1, 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-DeltaIE1 results from the loss of other critical functions of IE1, with its function in the dispersal of ND10 being a promising candidate.

Hematopoietic stem cell transplantation with latently infected donors does not transmit virus to immunocompromised recipients in the murine model of cytomegalovirus infection.

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.

CD8 T cells control cytomegalovirus latency by epitope-specific sensing of transcriptional reactivation.

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.