In Vivo Replication, Latency, and Immunogenicity of Murine Cytomegalovirus Mutants with Deletions in the M83 and M84 Genes, the Putative Homologs of Human Cytomegalovirus pp65 (UL83)

We previously identified two open reading frames (ORFs) of murine cytomegalovirus (MCMV), M83 and M84, which are putative homologs of the human cytomegalovirus (HCMV) UL83 tegument phosphoprotein pp65 (L. D. Cranmer, C. L. Clark, C. S. Morello, H. E. Farrell, W. D. Rawlinson, and D. H. Spector, J. Virol. 70:7929-7939, 1996). In this report, we show that unlike the M83 gene product, the M84 protein is expressed at early times in the infection and cannot be detected in the virion. To elucidate the functional differences between the two pp65 homologs in acute and latent MCMV infections, we constructed two MCMV K181 mutants in which either the M83 or M84 ORF was deleted. The resultant viruses, designated DeltaM83 and DeltaM84, respectively, were found to replicate in NIH 3T3 cells with kinetics identical to those of the parent strain. Western blot analysis demonstrated that except for the absence of M83 or M84 protein expression in the respective mutants, no global perturbations of protein expression were detected. When DeltaM83 and DeltaM84 were inoculated intraperitoneally (i.p.) into BALB/c mice, both viruses showed similar attenuated growth in the spleen, liver, and kidney. However, only DeltaM83 was severely growth restricted in the salivary glands, a phenotype that was abolished upon restoration of the M83 ORF. DeltaM83's growth was similarly restricted in the salivary glands of the resistant C3H/HeN or highly sensitive 129/J strain, as well as in the lungs of all three strains following intranasal inoculation. Using a nested-PCR assay, we found that both DeltaM83 and DeltaM84 established latency in BALB/c mice, with slightly decreased levels of DeltaM83 and DeltaM84 genomic DNAs, relative to K181, observed in the salivary glands and lungs. Immunization of BALB/c mice with 10(5) PFU of K181, DeltaM83, or DeltaM84 i.p. provided similar levels of protection against lethal challenge. Although immunization with 200 PFU of DeltaM83 also provided complete protection, this dose allowed both the immunizing and challenge viruses to establish latency in the spleen. Our results show that the two MCMV pp65 homologs differ in their expression kinetics, virion association, and influence on viral tropism and/or dissemination.     

Localization of Human Cytomegalovirus Structural Proteins to the Nuclear Matrix of Infected Human Fibroblasts

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.     

Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation

The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant (DeltaUL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with DeltaUL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented DeltaUL32-BAC replication but murine CMV M32 failed to complement, consistent with evolutionary divergence of rodent and primate cytomegaloviruses. Infection with DeltaUL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.     

Two antigenic peptides from genes m123 and m164 of murine cytomegalovirus quantitatively dominate CD8 T-cell memory in the H-2d haplotype.

The importance of CD8 T cells for the control of cytomegalovirus (CMV) infection has raised interest in the identification of immunogenic viral proteins as candidates for vaccination and cytoimmunotherapy. The final aim is to determine the viral "immunome" for any major histocompatibility complex class I molecule by antigenicity screening of proteome-derived peptides. For human CMV, there is a limitation to this approach: the T cells used as responder cells for peptide screening are usually memory cells that have undergone in vivo selection. On this basis, pUL83 (pp65) and pUL123 (IE1 or pp68 to -72) were classified as immunodominant proteins. It is an open question whether this limited "memory immunome" really reflects the immunogenic potential of the human CMV proteome. Here we document an analogous focus of the memory repertoire on two proteins of murine CMV. Specifically, ca. 80% of all memory CD8 T cells in the spleen as well as in persisting pulmonary infiltrates were found to be specific for the known IE1 peptide 168YPHFMPTNL176 and for the peptide 257AGPPRYSRI265, newly defined here, derived from open reading frame m164. Notably, CD8 T-cell lines of both specificities protected against acute infection upon adoptive transfer. In contrast, the natural immune response to acute infection in draining lymph nodes and in the lungs indicated a somewhat broader specificity repertoire. We conclude that the low number of antigenic peptides identified so far for CMVs reflects a focused memory repertoire, and we predict that more antigenic peptides will be disclosed by analysis of the acute immune response.     

Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response

We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration. Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response. The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3). In contrast, NF-kappaB is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus. pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells. pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state. Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response. By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response.     

Highly protective in vivo function of cytomegalovirus IE1 epitope-specific memory CD8 T cells purified by T-cell receptor-based cell sorting

Reconstitution of antiviral CD8 T cells is essential for controlling cytomegalovirus (CMV) infection after bone marrow transplantation. Accordingly, polyclonal CD8 T cells derived from BALB/c mice infected with murine CMV protect immunocompromised adoptive transfer recipients against CMV disease. The protective population comprises CD8 T cells with T-cell receptors (TCRs) specific for defined and for as-yet-unknown viral epitopes, as well as a majority of nonprotective cells with unrelated specificities. Defined epitopes include IE1/m123 and m164, which are immunodominant in terms of the magnitude of the CD8 T-cell response, and a panel of subordinate epitopes (m04, m18, M45, M83, and M84). While cytolytic T-lymphocyte lines (CTLLs) were shown to be protective regardless of the immunodominance of the respective epitope, the individual contributions of in vivo resident epitope-specific CD8 T cells to the antiviral control awaited investigation. The IE1 peptide 168-YPHFMPTNL-176 is generated from the immediate-early protein 1 (IE1) (pp89/76) of murine CMV and is presented by the major histocompatibility complex class I (MHC-I) molecule Ld. To quantitate its contribution to the protective potential of a CD8-T memory (CD8-TM) cell population, IE1-TCR+ and IE1-TCR- CD8-TM cells were purified by epitope-specific cell sorting with IE1 peptide-loaded MHC-immunoglobulin G1 dimers as ligands of cognate TCRs. Of relevance for clinical approaches to an adoptive cellular immunotherapy, sorted IE1 epitope-specific CD8-TM cells were found to be exceedingly protective upon adoptive transfer. Compared with CTLLs specific for the same epitope and of comparable avidity and TCR beta-chain variable region (Vbeta)-defined polyclonality, sorted CD8-TM cells proved to be superior by more than 2 orders of magnitude.     

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.     

Development of a vaccine against murine cytomegalovirus (MCMV), consisting of plasmid DNA and formalin-inactivated MCMV, that provides long-term, complete protection against viral replication

We previously demonstrated that immunization of mice with plasmid DNAs (pDNAs) expressing the murine cytomegalovirus (MCMV) genes IE1-pp89 and M84 provided synergistic protection against sublethal viral challenge, while immunization with plasmids expressing putative virion proteins provided no or inconsistent protection. In this report, we sought to augment protection by increasing the breadth of the immune response. We identified another MCMV gene (m04 encoding gp34) that provided strong and consistent protection against viral replication in the spleen. We also found that immunization with a DNA pool containing 10 MCMV genes that individually were nonprotective elicited reproducible protection against low to intermediate doses of challenge virus. Moreover, inclusion of these plasmids into a mixture with gp34, pp89, and M84 DNAs provided even greater protection than did coimmunization with pp89 and M84. The highest level of protection was achieved by immunization of mice with the pool of 13 pDNAs, followed by formalin-inactivated MCMV (FI-MCMV). Immunization with FI-MCMV elicited neutralizing antibodies against salivary gland-derived MCMV, and of greatest importance, mice immunized with both the combined pDNA pool and FI-MCMV had undetectable levels of virus in the spleen and salivary glands after challenge. Intracellular cytokine staining of splenocytes from pDNA- and FI-MCMV-immunized mice showed that pDNA immunization elicited high levels of pp89- and M83-specific CD8(+) T cells, whereas both pDNA and FI-MCMV immunizations generated strong CD8(+)-T-cell responses against virion-associated antigens. Taken together, these results show that immunization with pDNA and inactivated virus provides strong antibody and cell-mediated immunity against CMV infection.     

The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture.

The phosphoprotein pp65 (ppUL83) of human cytomegalovirus (HCMV) is abundantly synthesized during lytic infection in cultured fibroblasts. As a major constituent of extracellular particles, it gains entry to infected cells immediately after adsorption and subsequently translocates to the cell nucleus. This efficient transport is mediated by unique nuclear localization signals. To study the function of pp65, a viral deletion mutant was constructed by replacing the pp65 gene with the bacterial neomycin phosphotransferase gene, driven by the simian virus 40 early promoter. The resulting virus, RVAd65, could be grown and selected on human fibroblasts without complementation. The deletion of the pp65 gene in RVAd65 was verified by using Southern blot and PCR analyses. The lack of expression from the gene was investigated by immunoblotting with pp65-specific monoclonal antibodies. Single-cycle growth analyses showed that RVAd65 grew to levels of infectivity comparable to those of the wild-type virus. Therefore, pp65 is nonessential for the growth of HCMV in human fibroblasts. Electron microscopy revealed no differences in the processes of virion morphogenesis, although the maturation appeared to be delayed. However, the kinetics of expression of the immediate-early genes UL122 and UL123, the early gene UL44, and the late gene UL32 were the same in RVAd65-infected cells as in wild-type virus-infected cells in immunoblot analyses. In vitro phosphorylation assays showed that some of the virion proteins were labelled to a markedly reduced extent by virion-associated kinases in RVAd65 compared with wild-type virus. We therefore conclude that although deletion of the pp65 gene does not abolish replication of HCMV, a recombinant virus lacking pp65 displays phenotypic alterations compared with wild-type virus during growth in cultured fibroblasts.     

Strong CD8 T-cell responses following coimmunization with plasmids expressing the dominant pp89 and subdominant M84 antigens of murine cytomegalovirus correlate with long-term protection against subsequent viral challenge

We previously showed that intradermal immunization with plasmids expressing the murine cytomegalovirus (MCMV) protein IE1-pp89 or M84 protects against viral challenge and that coimmunization has a synergistic protective effect (C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 74:3696-3708, 2000). Using an intracellular gamma interferon cytokine staining assay, we have now characterized the CD8+ T-cell response after DNA immunization with pp89, M84, or pp89 plus M84. The pp89- and M84-specific CD8+ T-cell responses peaked rapidly after three immunizations. DNA immunization and MCMV infection generated similar levels of pp89-specific CD8+ T cells. In contrast, a significantly higher level of M84-specific CD8+ T cells was elicited by DNA immunization than by MCMV infection. Fusion of ubiquitin to pp89 enhanced the CD8+ T-cell response only under conditions where vaccination was suboptimal. Three immunizations with either pp89, M84, or pp89 plus M84 DNA also provided significant protection against MCMV infection for at least 6 months, with the best protection produced by coimmunization. A substantial percentage of antigen-specific CD8+ T cells remained detectable, and they responded rapidly to the MCMV challenge. These results underscore the importance of considering antigens that do not appear to be highly immunogenic during infection as DNA vaccine candidates.     

Murine Cytomegalovirus Capsid Assembly Is Dependent on US22 Family Gene M140 in Infected Macrophages

Macrophages are an important target cell for infection with cytomegalovirus (CMV). A number of viral genes that either are expressed specifically in this cell type or function to optimize CMV replication in this host cell have now been identified. Among these is the murine CMV (MCMV) US22 gene family member M140, a nonessential early gene whose deletion (RVΔ140) leads to significant impairment in virus replication in differentiated macrophages. We have now determined that the defect in replication is at the stage of viral DNA encapsidation. Although the rate of RVΔ140 genome replication and extent of DNA cleavage were comparable to those for revertant virus, deletion of M140 resulted in a significant reduction in the number of viral capsids in the nucleus, and the viral DNA remained sensitive to DNase treatment. These data are indicative of incomplete virion assembly. Steady-state levels of both the major capsid protein (M86) and tegument protein M25 were reduced in the absence of the M140 protein (pM140). This effect may be related to the localization of pM140 to an aggresome-like, microtubule organizing center-associated structure that is known to target misfolded and overexpressed proteins for degradation. It appears, therefore, that pM140 indirectly influences MCMV capsid formation in differentiated macrophages by regulating the stability of viral structural proteins.An important feature of cytomegalovirus (CMV) pathogenesis is dissemination of virus to target organs by infected monocytes that harbor CMV DNA (14, 36). Differentiation of these cells into macrophages as they extravasate into tissues triggers production of infectious virus. Infection with human CMV (HCMV) drives monocytes toward a proinflammatory macrophage phenotype with an extended life span (7, 36). This cellular differentiation process is accompanied by numerous physiological changes that could, in theory, negatively affect virus replication. These include increases in reactive oxygen intermediates, phagocytosis, lysosomal compartments, mitochondrial activities, secretory enzymes, and antiviral cytokines, as well as numerous receptors that render these cells highly interactive with the extracellular matrix and immune modulators (7, 14). It is likely, therefore, that CMV encodes products to counteract factors that are adverse to replication or to subvert cellular events to enhance replication. The mouse model has been utilized to identify genes within murine CMV (MCMV) that facilitate replication within differentiated macrophages.To date, four MCMV genes or gene regions have been identified as determinants of macrophage tropism in that deletion of or insertion within these genes has no impact on virus replication in fibroblasts but significantly depresses replication in macrophages (reviewed in reference 14). Gene M78 encodes a G protein-coupled receptor that facilitates accumulation of immediate-early RNA at low multiplicities (29). The product of gene M45 is virion associated and conveys antiapoptotic functions by binding to RIP1 (4, 21). The US22 gene family member M36 encodes a virion-associated protein that has antiapoptotic function by binding to caspase 8 (24). Finally, the US22 gene family members M139, M140, and M141 optimize MCMV replication in fully differentiated tissue macrophages both in vitro and in vivo (16, 17, 24). Two additional MCMV genes of unknown function are transcribed in infected macrophages but not fibroblasts (38), indicating additional adaptations specific to this target cell.The M139, M140, and M141 early gene products form at least three stable complexes that colocalize to a perinuclear cis-Golgi region in infected macrophages (18). Sequence analysis of the three genes does not reveal any consensus functional domains, with the exception of a putative nuclear localization sequence within M139 and M141 (15). Deletion of one or more of the three genes does not influence apoptosis (24) or tumor necrosis factor alpha or nitric oxide activities (unpublished data). Therefore, the function of these genes remains unknown. It is apparent that M140 is fundamental to the function(s) of these genes for several reasons. First, the degree of impairment in replication of RVΔ140 is equal to that of a mutant deleted of all three genes (16). Second, pM140 is a component of all three complexes identified: a pM140-pM141 complex and two pM139-pM140-pM141 complexes that differ in size and/or mass (18). Third, pM140 localizes to the nucleus but is redistributed to the cytoplasm when expressed with pM141 (18).In this study, we took a broad approach to assessing the function of M140 in optimizing MCMV replication in macrophages. The data indicate that impairment in replication of MCMV deleted of M140 is after genome replication but prior to virion assembly. Both the copy numbers and rates of viral genome replication for revertant and mutant virus in macrophages were comparable. However, in the absence of M140, capsids were rarely detected by electron microscopy in the nucleus or cytoplasm. Mutant viral DNA was highly sensitive to DNase, although cleavage of DNA was not compromised. Because cleavage of viral DNA is linked to encapsidation, these data suggest that procapsid stability, rather than formation, was altered by the M140 deletion. In support of this, steady-state levels of the major capsid protein (MCP) and M25 tegument protein in mutant virus-infected cells failed to accumulate to wild-type (WT) levels. The fact that pM140 localized to an aggresome-like structure, which is typically important in regulating viral and cellular protein degradation, implies that pM140 may function to regulate viral protein degradation. These studies indicate that pM140 indirectly regulates virus capsid assembly by regulating the stability of viral structural proteins.     

Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65)

The cytotoxic T-lymphocyte (CTL) response against the murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) 89-kDa phosphoprotein pp89 plays a major role in protecting BALB/c mice against the lethal effects of the viral infection. CTL populations specific to MCMV early-phase and structural antigens are also generated during infection, but the identities of these antigens and their relative contributions to overall immunity against MCMV are not known. We previously demonstrated that DNA vaccination with a pp89-expressing plasmid effectively generated a CTL response and conferred protection against infection (J. C. Gonzalez Armas, C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 70:7921-7928, 1996). In this report, we have sought (i) to identify other viral antigens that contribute to immunity against MCMV and (ii) to determine whether the protective response is haplotype specific. DNA immunization was used to test the protective efficacies of plasmids encoding MCMV homologs of human cytomegalovirus (HCMV) tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86), and nonstructural antigens (IE1-pp89 and M84). BALB/c (H-2(d)) and C3H/HeN (H-2(k)) mice were immunized by intradermal injection of either single plasmids or cocktails of up to four expression plasmids and then challenged with sublethal doses of virulent MCMV administered intraperitoneally. In this way, we identified a new viral gene product, M84, that conferred protection against viral replication in the spleens of BALB/c mice. M84 is expressed early in the infection and encodes a nonstructural protein that shares significant amino acid homology with the HCMV UL83-pp65 tegument protein, a major target of protective CTLs in humans. Specificity of the immune response to the M84 protein was confirmed by showing that immunization with pp89 DNA, but not M84 DNA, protected mice against subsequent infection with an MCMV deletion mutant lacking the M84 gene. The other MCMV genes tested did not generate a protective response even when mice were immunized with vaccinia viruses expressing the viral proteins. However, the M84 plasmid was protective when injected in combination with nonprotective plasmids, and coimmunization of BALB/c mice with pp89 and M84 provided a synergistic level of protection in the spleen. Viral titers in the salivary glands were also reduced, but not to the same extent as observed in the spleen, and the decrease was seen only when the BALB/c mice were immunized with pp89 plus M84 or with pp89 alone. The experiments with the C3H/HeN mice showed that the immunity conferred by DNA vaccination was haplotype dependent. In this strain of mice, only pp89 elicited a protective response as measured by a reduction in spleen titer. These results suggest that DNA immunization with the appropriate combination of CMV genes may provide a strategy for improving vaccine efficacy.     

Cytomegalovirus infection in Gambian infants leads to profound CD8 T-cell differentiation

Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85% by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection. CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28(-) CD62L(-) CD95(+) perforin(+) granzyme A(+) Bcl-2(low)). Strikingly, the overall CD8 T-cell population developed a similar phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12 months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptide-specific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly deleterious effect.     

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.     

Preemptive CD8 T-Cell Immunotherapy of Acute Cytomegalovirus Infection Prevents Lethal Disease,Limits the Burden of Latent Viral Genomes,and Reduces the Risk of Virus Recurrence

In the immunocompetent host, primary cytomegalovirus (CMV) infection is resolved by the immune response without causing overt disease. The viral genome, however, is not cleared but is maintained in a latent state that entails a risk of virus recurrence and consequent organ disease. By using murine CMV as a model, we have shown previously that multiple organs harbor latent CMV and that reactivation occurs with an incidence that is determined by the viral DNA load in the respective organ (M. J. Reddehase, M. Balthesen, M. Rapp, S. Jonjic, I. Pavic, and U. H. Koszinowski. J. Exp. Med. 179:185–193, 1994). This predicts that a therapeutic intervention capable of limiting the load of latent viral genome should also reduce the risk of virus recurrence. Here we demonstrate the benefits and the limits of a preemptive CD8 T-cell immunotherapy of CMV infection in the immunocompromised bone marrow transplantation recipient. Antiviral CD8 T cells prevented CMV disease and accelerated the resolution of productive infection. The therapy also resulted in a lower load of latent CMV DNA in organs and consequently reduced the incidence of recurrence. The data thus provide a further supporting argument for clinical trials of preemptive cytoimmunotherapy of human CMV disease with CD8 T cells. However, CD8 T cells failed to clear the viral DNA. The therapy-susceptible portion of the DNA load differed between organs and was highest in the lungs. The existence of an invariant, therapy-resistant load suggests a role for immune system evasion mechanisms in the establishment of CMV latency.Recurrence of productive infection by reactivation of latent viral genome in the immunocompromised host is a feature common to the members of the herpesvirus family (39; reviewed in reference 38). Specifically, in the case of human cytomegalovirus (CMV), the human herpesvirus type 5, primary as well as recurrent infection during the temporal immunodeficiency early after bone marrow (BM) transplantation (BMT) entails a risk of graft failure and severe organ manifestations of CMV disease (8, 44). Early findings by Quinnan et al. (24) have suggested a correlation between efficient reconstitution of the cellular immune response and the control of post-BMT CMV infection, and more recent clinical data have attributed this control to the reconstituted CD8 T cells (35). Accordingly, restoration of antiviral immunity in the critical phase before the reconstitution by BMT becomes effective should diminish the risk of CMV disease. Experimental research with the model of murine CMV infection has positively demonstrated the antiviral and protective efficacy of adoptively transferred acutely sensitized (31, 34) or memory (28) CD8 T cells recovered from immune donors as well as of short-term CD8 T-cell lines propagated in culture (32). These studies have been pivotal for clinical trials of a preemptive CD8 T-cell immunotherapy of post-BMT human CMV infection in patients (37, 43).Infection of the BMT recipient can accidentally result from the transmission of infectious virus, however, productive infection is more commonly initiated by reactivation of latent CMV in either the transplant or the recipient’s own organs or, occasionally, both (11). For the murine model system, we have previously demonstrated the existence of multiple organ sites of CMV latency at which the latent viral DNA is retained after the resolution of productive primary infection and after clearance of the viral genome from hematopoietic leukocytic cells in BM and blood (27). In accordance with the wide distribution of the latent viral DNA, recurrence was found to occur focally in any of the organs, which led us to propose the concept of multifocal CMV latency and recurrence (27). Most importantly, the incidence of recurrence was found to correlate with the load of latent viral DNA in the respective tissue. Specifically, low virus dissemination and rapid control of infection in immunocompetent adult mice resulted in a low load and was associated with a low risk of recurrence, whereas the delayed control of infection in neonatal mice resulted in a high load and was associated with a high risk. Furthermore, there were also organ-specific differences. In accordance with the high incidence of interstitial CMV pneumonia after BMT, the lungs were identified as having a high load of latent CMV (2, 17).It is apparent that antiviral CD8 T cells generated during primary infection as well as memory cells present during latency do not eradicate latently infected cells under physiological conditions, since latency would not exist if they did. However, it has been open to question whether adoptive transfer of antiviral CD8 effector cells could prevent the escape of virus into latency. We will show here that modulation of primary infection by experimental CD8 T-cell immunotherapy has indeed had an effect on the load of latent viral DNA in tissues. The effect of the therapy is of relevance, since the load of latent viral DNA can be kept below the threshold required for effective recurrence. Our data thus provide a further supporting argument for clinical trials of cytoimmunotherapy. Interestingly, however, the data also predict that no dosage of CD8 T cells will prevent the establishment of latency.     

Mast Cells Expedite Control of Pulmonary Murine Cytomegalovirus Infection by Enhancing the Recruitment of Protective CD8 T Cells to the Lungs

The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV infection as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic infection triggered degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by infection of MC-deficient Kit^W-sh/W-sh “sash” mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of “sash” mice restoring both, efficient CD8 T-cell recruitment and infection control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease.     

Acyclovir Therapy Reduces the CD4+ T Cell Response against the Immunodominant pp65 Protein from Cytomegalovirus in Immune Competent Individuals

Cytomegalovirus (CMV) infects the majority of the global population and leads to the development of a strong virus-specific immune response. The CMV-specific CD4+ and CD8+ T cell immune response can comprise between 10 and 50% of the T cell pool within peripheral blood and there is concern that this may impair immunity to other pathogens. Elderly individuals with the highest magnitude of CMV-specific immune response have been demonstrated to be at increased risk of mortality and there is increasing interest in interventions that may serve to moderate this. Acyclovir is an anti-viral drug with activity against a range of herpes viruses and is used as long term treatment to suppress reactivation of herpes simplex virus. We studied the immune response to CMV in patients who were taking acyclovir to assess if therapy could be used to suppress the CMV-specific immune response. The T cell reactivity against the immunodominant late viral protein pp65 was reduced by 53% in people who were taking acyclovir. This effect was seen within one year of therapy and was observed primarily within the CD4+ response. Acyclovir treatment only modestly influenced the immune response to the IE-1 target protein. These data show that low dose acyclovir treatment has the potential to modulate components of the T cell response to CMV antigen proteins and indicate that anti-viral drugs should be further investigated as a means to reduce the magnitude of CMV-specific immune response and potentially improve overall immune function.     

Long-term stable expanded human CD4+ T cell clones specific for human cytomegalovirus are distributed in both CD45RAhigh and CD45ROhigh populations

T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4+ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4+ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4+ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN-gamma secretion. We analyzed the clonal composition of CD4+ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4+ clones and sequencing the TCR beta-chain. In a given carrier, most of the CD4+ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4+ clones in whole PBMC and in purified subpopulations of CD45RAhighCD45ROlow and CD45RAlowCD45ROhigh cells. Individual CD4+ T cell clones could be large (0.3-1.5% of all CD4+ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RAhigh and CD45ROhigh subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28-CD45RAhigh CD4+ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4+ T cell clone, which resembles that seen in the CD8+ T cell response against HCMV pp65.