The capacity of human cytomegalovirus (HCMV) to establish and maintain a latent infection from which it can later reactivate ensures its widespread distribution in the population, but the mechanisms enabling maintenance of latency in the face of a robust immune system are poorly understood. We examined the role of the HCMV UL111A gene, which encodes homologs of the immunosuppressive cytokine interleukin-10 in the context of latent infection of myeloid progenitor cells. A UL111A deletion virus was able to establish, maintain, and reactivate from experimental latency in a manner comparable with parental virus, but major histocompatibility complex class II levels increased significantly on the surfaces of cells infected with the deletion virus. Importantly, there was an increase in both allogeneic and autologous peripheral blood mononuclear cells and CD4 ؉ Tcell responses to UL111A deletion virusinfected myeloid progenitors, indicating that loss of the capacity to express IntroductionHuman cytomegalovirus (HCMV) is a species-specific ␤-herpesvirus that infects a majority of the world's population. 1 During primary productive infection, both innate and adaptive immune responses are activated, and replicating virus is eventually cleared from the host. However, the virus is able to evade complete immune clearance by establishing and maintaining a life-long latent infection in hematopoietic cells, specifically those of the myeloid lineage. [2][3][4][5][6][7] During latency, detectable infectious virus production ceases, the viral genome is maintained as an extrachromosomal plasmid 8 at a low viral genome copy number, 7 and only a subset of viral genes remain transcriptionally active. [9][10][11][12][13][14] Reactivation from latency results in reinitiation of the full replicative cycle, with production of new infectious virus.Reactivation of HCMV from latency during states of reduced immune surveillance has profound implications for the management of patients with hematologic and immune deficiency disorders. In these clinical contexts, HCMV reactivation can lead to tissue infection causing major morbidity and mortality. As a result, the presence of HCMV seropositivity and thus of latent virus in donors or recipients of unrelated stem cell transplantations is an adverse prognostic feature despite major advances in HCMV monitoring and management. 15,16 Reactivation of HCMV is also of importance in seropositive patients with nontransplantation cellular immunodeficiencies, such as those resulting from human immunodeficiency virus infection, 17 or in seropositive patients with iatrogenically induced immunodeficiency, such as that resulting from treatment with the anti-CD52 antibody Campath. 18,19 There is no known therapy for the elimination of virus that persists during latency. Instead, where necessary, medical treatment has concentrated on the preemptive treatment or the prophylaxis of viral reactivation. Even here, no single strategy prevents reactivation while universally avoiding unnecessary therapy. There is an urge...

Immune control of human cytomegalovirus (HCMV) infection can be mediated by CD8 + cytolytic T lymphocytes (CTL). Adoptive transfer of antiviral CTL confers protection against HCMV reactivation and disease. The tegument protein pp65 and the immediate-early 1 protein (IE1) are recognized to be major CTL targets, even though during productive infection the viral immunoevasion proteins gpUS2-11 act to suppress major histocompatibility complex (MHC) class I-restricted antigen presentation. Thus it was not clear how infected cells could be labelled with antigenic peptides in the face of immunoevasion. We show here that the immunodominant peptide pp65 NLV was presented by MHC class I in cells infected with a gpUS2-11-competent virus. Presentation of pp65 NLV was still detectable at 96 h post-infection, although at low levels. Partial suppression of pp65 NLV presentation was dependent on the ability of the infecting strain to express gpUS2-11. MHC class I-restricted antigen presentation in HCMV-infected cells (encoding gpUS2-11) exhibited specificity for pp65-derived peptides, as infected fibroblasts did not present the IE1-derived nonapeptide IE1 TMY . Remarkably, infected cells could restore pp65 NLV peptide presentation after acid removal of MHC class I despite gpUS2-11 expression. This recovery was shown to be dependent on proteasome functionality. In contrast to IE1, pp65 peptides are loaded on MHC class I molecules to be transported to the cell surface at early and late times after infection in the face of gpUS2-11-mediated immunoevasion. pp65 is therefore the first example of an HCMV protein only incompletely subjected to gpUS2-11-mediated immunoevasion. INTRODUCTIONInfection with human cytomegalovirus (HCMV) affects more than 50 % of the human population. Individuals with immature or compromised immune defence functions are more likely to encounter severe clinical disease conditions. In healthy individuals, however, HCMV is normally efficiently controlled by the immune system (Pass, 2001), and in particular by HMCV-specific CD8 + cytoxic T lymphocytes (CTL) (Reddehase et al., 1985;Reusser et al., 1991). The CTL response is primed against short peptides that are proteolytically processed from intracellular viral proteins and presented on the cell surface by major histocompatibility complex (MHC) class I molecules (Kloetzel, 2004). For this, proteins are labelled by ubiquitination to be targeted to proteasomes for degradation. Peptides of appropriate length are released from proteasomes and processed further at their amino terminus by cytosolic aminopeptidases. Ultimately they are translocated to the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP), where they associate with MHC class I. In some instances, ER-resident proteases contribute to the trimming of antigenic peptides (reviewed by Kloetzel, 2004).The HCMV genome encodes over 150 proteins, which can potentially serve as target antigens for CTL (Chee et al., 1990). Recent studies showed that memory CTL isolated from HC...

Direct protein delivery is an emerging technology in vaccine development and gene therapy. We could previously show that subviral dense bodies (DB) of human cytomegalovirus (HCMV), a beta-herpesvirus, transport viral proteins into target cells by membrane fusion. Thus these non-infectious particles provide a candidate delivery system for the prophylactic and therapeutic application of proteins. Here we provide proof of principle that DB can be modified genetically. A 55 kDa fusion protein consisting of the green fluorescent protein and the neomycin phosphotransferase could be packed in and delivered into cells by recombinant DB in a functional fashion. Furthermore, transfer of protein into fibroblasts and dendritic cells by DB was efficient, leading to exogenous loading of the MHC-class I antigen presentation pathway. Thus, DB may be a promising basis for the development of novel vaccine strategies and therapeutics based on recombinant polypeptides.

Human cytomegalovirus (HCMV) has evolved strategies to counteract its surveillance by the immune system. Mitigation of antiviral immune responses is considered critical for establishment of viral latency and for spread. Recently, a gene encoding an interleukin-10 homologue (cmvIL-10) has been discovered in the HCMV genome. Using recombinant cmvIL-10, several mostly immunosuppressive functions of the molecule have been described. However, the role of cmvIL-10 in the context of viral infection was not addressed. To be able to analyze this issue, we generated cmvIL- 10-negative viral mutants. Using these mutants, we tested whether the expression of cmvIL-10 by infected cells would render bystander antigen-presenting cells less efficient in their capacity to present antigenic peptides in the context of MHC class I. To test this, CTL clones specific for the viral nonapeptides P65(495-503) and IE1(297-305) were used as tools. Culture supernatant from fibroblasts infected with cmv-IL10-negative viruses was supplemented with increasing concentrations of recombinant cmvIL-10. Treatment of human THP-1 cells with these conditioned media did not impair their capacity to present HCMV-derived nonapeptides in the context of MHC-class I, even when high concentrations of cmvIL-10 were used. To investigate whether close cell contact was important, fibroblasts were infected with either wild-type HCMV or cmvIL-10 null mutants and were cocultured with nonpermissive lymphoblastoid cell lines, serving as target cells. No correlation was found between the ability of HCMV strains to express the cmvIL-10 gene and the capacity of neighboring LCL to present peptides in the context of MHC class I. Consequently, we propose that cmvIL- 10 expressed in the context of HCMV infection has no direct impact on MHC class I-restricted antigen presentation of noninfected bystander cells.

Control of human cytomegalovirus (HCMV) infection is predominantly mediated by cytolytic CD8+ T lymphocytes (CTL). Among the roughly 200 HCMV-encoded polypeptides, the tegument protein pp65 (ppUL83) and the nonstructural IE1 protein are considered to be dominant CTL targets. Yet the importance of CTL against IE1 for protective immunity against HCMV reactivation and disease has remained elusive. Analyses have been difficult, as all MHC class I presented peptides of IE1 defined so far are located in parts of the protein that are variable between viral strains. In this study a conserved decameric peptide from IE1 (P6, IE1(354-363)) that bound to HLA-A2 was identified. Using peptide-pulsed, HLA-matched stimulator cells, CTL lines which recognized P6 after exogenous loading as well as after endogenous processing could repeatedly be generated. However, memory CTL directed against P6 were not readily detectable by ex vivo ELISPOT analysis in peripheral blood mononuclear cells of healthy seropositive individuals, indicating that this peptide represents a quantitatively subdominant determinant during latent HCMV infection. Using the conserved HLA-A2 presented peptide P6 will enable more detailed studies on the role of IE1-specific CTL in patients suffering from various HCMV-related disease conditions and investigation of the role of such cells for immune control of HCMV. Since IE1 is the first viral protein to be expressed after reactivation from latency, P6 may also serve as an important component of a future recombinant HCMV vaccine.

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Primary human cytomegalovirus (HCMV) infection during pregnancy is a frequent cause of fatal damage in populations with low prevalence of HCMV. Differentiation of primary vs. recurrent HCMV infection is an important issue in prenatal counseling. Antibodies specific for viral glycoproteins become detectable only with considerable delay with relation to HCMV infection or IgG seroconversion. Thus, lack of glycoprotein specific (gp-specific) antibodies can serve as a convenient indicator to identify those pregnant women that bear an elevated risk for HCMV transplacental transmission and fetal sequelae. In the opposite case, presence of gp-specific antibodies virtually excludes HCMV primary infection several weeks before sampling. However, no standardized screening assay for HCMV gp-specific antibodies had been available thus far. For this reason, an ELISA based on procaryotically expressed fragments of HCMV glycoprotein B (gB; gpUL55) was developed. Small fragments of gB from two different laboratory strains, encompassing the antigenic domain 2 (AD2) sufficed for sensitive and specific detection of gp-specific antibodies. The gB-ELISA titers correlated with titers of virus neutralizing antibodies in serum samples from primary or recurrent HCMV infections. Seroconversion kinetics of the gB-ELISA in samples from patients with primary HCMV infection closely paralleled the delay in seroconversion of gp-specific antibodies as determined by neutralization assay. Thus this assay provides a diagnostic tool that is easy to perform and can significantly add to available methods for the timely identification of primary HCMV infection during pregnancy. In addition, the gB-ELISA may be helpful in other clinical settings for the differentiation of primary HCMV infection from diseases caused by other pathogens.

The phosphoprotein 65 (pp65) of human cytomegalovirus is a prominent target of the antiviral CD8 T lymphocyte response. This study focused on investigating the properties of pp65 that render it a privileged antigen. It was found that pp65 was metabolically stable. The tegument protein was introduced into MHC class I presentation following its delivery via non-replicating dense bodies. No ubiquitination was found on particle-associated pp65. Proof was obtained that pp65 was a nucleocytoplasmic shuttle protein, using heterokaryon analyses. Based on this finding, inhibition experiments showed that presentation of particle-derived pp65 by HLA-A2 was sensitive to the impairment of the CRM1-mediated nuclear export pathway. The data support the idea that particle-derived pp65 can serve as a nuclear reservoir for proteasomal processing and MHC class I presentation, following its CRM1-dependent nuclear export. The presentation of pp65-derived peptides was also impaired by CRM1-inhibition following de novo synthesis of the tegument protein. However, pp65 protein levels were also reduced when blocking CRM1-mediated export after transient expression. This indicated that pp65 expression rather than direct interference with its own nuclear export was responsible for its reduced presentation in this case. The functionality of CRM1-mediated nuclear export is thus important for the presentation of pp65-derived peptides in the context of MHC class I on organ cells, both after exogenous uptake and after de novo synthesis of the tegument protein, but different mechanisms may account for either case.