Human cytomegalovirus (HCMV) encodes four glycoproteins, termed gpUS2, gpUS3, gpUS6 and gpUS11 that interfere with MHC class I biosynthesis and antigen presentation. Despite gpUS2-11 expression, however, HCMV infection is efficiently controlled by cytolytic CD8 T lymphocytes (CTL). To address the role of gpUS2 and gpUS11 in antigen presentation during viral infection, HCMV mutants were generated that expressed either gpUS2 or gpUS11 alone without coexpression of the three other proteins. Fibroblasts infected with these viruses showed reduced HLA-A2 and HLA-B7 surface expression. Surprisingly, however, CTL directed against the tegument protein pp65 and the regulatory IE1 protein still recognized and lysed mutant virus infected fibroblasts. Yet, suppression of IE1 derived peptide presentation by gpUS2 or gpUS11 was far more pronounced. The results show that gpUS2 and gpUS11 alone only incompletely protect HCMV infected fibroblasts from CTL recognition and underline the importance of studying infected cells to elucidate HCMV immune evasion.

Polyethylenimines are cationic polymers with potential as delivery vectors in gene therapy and with proven antimicrobial activity. However, the antiviral activity of polyethylenimines has not been addressed in detail thus far. We have studied the inhibitory effects of a linear 25-kDa polyethylenimine on infections with human papillomaviruses and human cytomegaloviruses. Preincubation of cells with polyethylenimine blocked primary attachment of both viruses to cells, resulting in a significant reduction of infection. In addition, the dissemination of human cytomegalovirus in culture cells was efficiently reduced by recurrent administration of polyethylenimine. Polyethylenimine concentrations required for inhibition of human papillomavirus and cytomegalovirus did not cause any cytotoxic effects. Polyethylenimines and their derivatives may thus be attractive molecules for the development of antiviral microbicides.

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...

Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is proficient at establishing lifelong persistence within the host in part due to immune modulating genes that limit immune recognition. HCMV encodes at least five glycoproteins within its unique short (US) genomic region that interfere with MHC class I antigen presentation, thus hindering viral clearance by cytotoxic T lymphocytes (CTL). Specifically, US3 retains class I within the endoplasmic reticulum (ER), while US2 and US11 induce class I heavy chain destruction. A cooperative effect on class I down-regulation during stable expression of HCMV US2 and US3 has been established. To address the impact of US3 on US11-mediated MHC class I downregulation, the fate of class I molecules was examined in US3/US11-expressing cells and virus infection studies. Co-expression of US3 and US11 resulted in a decrease of surface expression of class I molecules. However, the class I molecules in US3/US11 cells were mostly retained in the ER with an attenuated rate of proteasome destruction. Analysis of class I levels from virusinfected cells using HCMV variants either expressing US3 or US11 revealed efficient surface class I down-regulation upon expression of both viral proteins. Cells infected with both US3 and US11 expressing viruses demonstrate enhanced retention of MHC class I complexes within the ER. Collectively, the data suggests a paradigm where HCMV-induced surface class I downregulation occurs by diverse mechanisms dependent on the expression of specific US genes. These results validate the commitment of HCMV to limiting the surface expression of class I levels during infection.

Exogenous introduction of particle-associated proteins of human cytomegalovirus (HCMV) into the major histocompatibility complex (MHC) class I presentation pathway by subviral dense bodies (DB) is an effective way to sensitize cells against CD8 T-cell (CTL) recognition and killing. Consequently, these particles have been proposed as a platform for vaccine development. We have developed a strategy to refine the antigenic composition of DB. For proof of principle, an HCMV recombinant (RV-VM3) was generated that encoded the immunodominant CTL determinant IE1 TMY from the IE1 protein in fusion with the major constituent of DB, the tegument protein pp65. To generate RV-VM3, a bacterial artificial chromosome containing the HCMV genome was modified by applying positive/negative selection based on the expression of the bacterial galactokinase in conjunction with l Red-mediated homologous recombination. This method allowed the efficient and seamless insertion of the DNA sequence encoding IE1 TMY in frame into the pp65 open reading frame (UL83) of the viral genome. RV-VM3 expressed its fusion protein to high levels. The fusion protein was packaged into DB and into virions. Its delivery into fibroblasts by these viral particles led to the loading of the MHC class I presentation pathway with IE1 TMY and to efficient killing by specific CTLs. This demonstrated that a heterologous peptide, not naturally present in HCMV particles, can be processed from a recombinant, DB-derived protein to be subsequently presented by MHC class I. The results presented here provide a rationale for the optimization of a vaccine based on recombinant DB. INTRODUCTIONInfection with human cytomegalovirus (HCMV; family Herpesviridae, subfamily Betaherpesvirinae) may cause significant morbidity and mortality in individuals with immature or compromised immune-defence functions, but proceeds asymptomatically in most healthy adults (Pass, 2001). Prevention of HCMV infection or disease by a vaccine has been ranked as a high-priority goal (Stratton et al., 2001). Although several vaccine approaches have been developed, none have yet entered routine clinical practice (Pepperl-Klindworth & Plachter, 2006;Plotkin, 2004;Schleiss & Heineman, 2005;Zhong & Khanna, 2007). We have demonstrated that HCMV dense bodies (DB) provide a promising basis for vaccine development (Pepperl et al., 2000;Pepperl-Klindworth et al., 2002). DB are enveloped, subviral particles devoid of capsids and viral DNA. They are released from infected fibroblast cultures and enter cells presumably via the normal HCMV entry processes. The major constituent of DB is pp65 (ppUL83) (Varnum et al., 2004), which is a prominent target of the CD4 and CD8 T-cell (CTL) responses following natural infection (Beninga et al., 1995;McLaughlin-Taylor et al., 1994;Wills et al., 1996). Although pp65 by itself appears to be an attractive antigen for the design of a vaccine, other HCMV proteins may also be important and should thus be considered (Reddehase, 2002). One of these, as far as the CTL response is co...

Immunoevasive proteins (“evasins”) of human CMV (HCMV) modulate stability and localization of MHC class I (MHC I) molecules, and their supply of antigenic peptides. However, it is largely unknown to what extent these evasins interfere with recognition by virus-specific CD8 T cells. We analyzed the recognition of HCMV-infected cells by a panel of CD8 T cells restricted through one of nine different MHC I allotypes. We employed a set of HCMV mutants deleted for three or all four of the MHC I modulatory genes US2, US3, US6, and US11. We found that different HCMV evasins exhibited different allotype-specific patterns of interference with CD8 T cell recognition of infected cells. In contrast, recognition of different epitopes presented by the same given MHC I allotype was uniformly reduced. For some allotypes, single evasins largely abolished T cell recognition; for others, a concerted action of evasins was required to abrogate recognition. In infected cells whose Ag presentation efficiency had been enhanced by IFN-γ pretreatment, HCMV evasins cooperatively impared T cell recognition for several different MHC I allotypes. T cell recognition and MHC I surface expression under influence of evasins were only partially congruent, underscoring the necessity to probe HCMV immunomodulation using specific T cells. We conclude that the CD8 T cell evasins of HCMV display MHC I allotype specificity, complementarity, and cooperativity.

Suppression of major histocompatibility complex (MHC) class I-mediated presentation of human cytomegalovirus (HCMV)peptides is an important mechanism to avoid CD8 T lymphocyte recognition and killing of infected cells. Of particular interest is how MHC class I presentation of essential regulatory immediate early (IE) proteins of HCMV can be effectively compromised at times when known viral immunoevasins are not abundantly expressed. The tegument protein pp71 had been suggested to be involved in MHC class I downregulation. Intriguingly, this polypeptide is also critically engaged in the initial derepression of the major IE gene locus, leading to enhanced expression of IE proteins IE1-pp72 and IE2-pp86. Using a set of viral mutants, we addressed the role of pp71 in MHC class I presentation of IE1-pp72-derived peptides. We show that the amount of "incoming" pp71 positively correlates with IE1-pp72 protein levels and with the presentation of IE1-derived peptides. This indicates that the amount of the IE1 protein, induced by pp71, rather than a putative immunoevasive function of the tegument protein, determines MHC class I antigen presentation of IE1-derived peptides. This process proved to be independent of the presence of pp65, which had been reported to interfere with IE1 presentation. It may thus be beneficial for the success of HCMV replication to limit the level of pp71 delivered from infecting particles in order to avoid critical levels of MHC class I presentation of IE protein-derived peptides.

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.