Human cytomegalovirus proteins pp65 and immediate early protein 1 are common targets for CD8+ T cell responses in children with congenital or postnatal human cytomegalovirus infection

Recombinant modified vaccinia Ankara- and peptide-based IFN-gamma ELISPOT assays were used to detect and measure human CMV (HCMV)-specific CD8(+) T cell responses to the pp65 (UL83) and immediate early protein 1 (IE1; UL123) gene products in 16 HCMV-infected infants and children. Age at study ranged from birth to 2 years. HCMV-specific CD8(+) T cells were detected in 14 (88%) of 16 children at frequencies ranging from 60 to >2000 spots/million PBMC. Responses were detected as early as 1 day of age in infants with documented congenital infection. Nine children responded to both pp65 and IE1, whereas responses to pp65 or IE1 alone were detected in three and two children, respectively. Regardless of the specificity of initial responses, IE1-specific responses predominated by 1 year of age. Changes in HCMV epitopes targeted by the CD8(+) T cell responses were observed over time; epitopes commonly recognized by HLA-A2(+) adults with latent HCMV infection did not fully account for responses detected in early childhood. Finally, the detection of HCMV-specific CD8(+) T cell responses was temporally associated with a decrease in peripheral blood HCMV load. Taken altogether, these data demonstrate that the fetus and young infant can generate virus-specific CD8(+) T cell responses. Changes observed in the protein and epitope-specificity of HCMV-specific CD8(+) T cells over time are consistent with those observed after other primary viral infections. The temporal association between the detection of HCMV-specific CD8(+) T cell responses and the reduction in blood HCMV load supports the importance of CD8(+) T cells in controlling primary HCMV viremia.     

Characterization of the elements and proteins responsible for interferon-stimulated gene induction by human cytomegalovirus

Human cytomegalovirus (HCMV) infection of human fibroblast cells activates a large number of interferon-stimulated genes (ISGs) in a viral envelope-cell membrane fusion-dependent mechanism. In this study, we identified two interferon response elements, the interferon-stimulated response element (ISRE) and the gamma interferon-activated site (GAS), which act as HCMV response sites (VRS). Gel mobility shift assays showed that cellular proteins form specific and identical complexes with ISRE and GAS elements, and the binding of these complexes to ISRE and GAS is stimulated by HCMV infection. Point mutations in the consensus sequences of ISRE and GAS completely abolished their activities in response to HCMV-mediated transactivation, as well as their abilities to interact with HCMV-activated VRS-binding proteins. Interferon regulatory factor 3 does not appear to be present in the VRS-binding complexes or to be involved directly in HCMV-mediated ISG activation. Using ProteinChip technology, four potential proteins were identified, ranging from 20 to 42 kDa, in the VRS-binding complexes. The data suggest that HCMV infection activates VRS-binding proteins, which then bind to the VRS and stimulate ISG expression.     

Role of Milk Whey in the Transmission of Human Cytomegalovirus Infection by Breast Milk

Breast-fed infants are susceptible to human cytomegalovirus (HCMV) infection via breast milk. In our previous study, HCMV was isolated more frequently from breast milk at later than one month after delivery than from colostrum or early breast milk. To clarify the role of milk cells and whey in vertical infection by breast feeding, we separated breast milk into milk cells and whey and examined each fraction for the presence of HCMV. We collected breast milk from mothers who breast-fed their infants (aged from 3 days to 2 months). The breast milk was centrifuged and separated into the middle layer (layer of milk whey) and the pellet (containing milk cells). We attempted to isolate HCMV from whey and to detect HCMV immediate early (IE) DNA in both milk whey and cells. HCMV was isolated from 7 out of 35 (20.0%) whey samples and HCMV IE DNA was detected from 15 out of 35 (42.9%) whey and/or milk cells. Detection rates of HCMV IE DNA in the whey layer and milk cells were 39.1% (25 out of 64) and 17.2% (11 out of 64), respectively. HCMV IE DNA was not detected in colostrum, but was detected in breast milk samples one month after delivery. Therefore, cell-free HCMV shed into milk whey may have a more important role in vertical infection by breast milk than cell-associated HCMV in the milk.     

TLR9 -1486T/C and 2848C/T SNPs Are Associated with Human Cytomegalovirus Infection in Infants

Toll-like receptor 9 (TLR9) recognizes non-methylated viral CpG-containing DNA and serves as a pattern recognition receptor that signals the presence of human cytomegalovirus (HCMV). Here, we present the genotype distribution of single-nucleotide polymorphisms (SNPs) of the TLR9 gene in infants and the relationship between TLR9 polymorphisms and HCMV infection. Four polymorphisms (-1237T/C, rs5743836; -1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) in the TLR9 gene were genotyped in 72 infants with symptomatic HCMV infection and 70 healthy individuals. SNP genotyping was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digested fragments were separated and identified by capillary electrophoresis. The HCMV DNA copy number was measured by a quantitative real-time PCR assay. We found an increased frequency of heterozygous genotypes TLR9 -1486T/C and 2848C/T in infants with HCMV infection compared with uninfected cases. Heterozygous variants of these two SNPs increased the risk of HCMV disease in children (P = 0.044 and P = 0.029, respectively). In infants with a mutation present in at least one allele of -1486T/C and 2848C/T SNPs, a trend towards increased risk of cytomegaly was confirmed after Bonferroni’s correction for multiple testing (Pc = 0.063). The rs352139 GG genotype showed a significantly reduced relative risk for HCMV infection (Pc = 0.006). In contrast, the -1237T/C SNP was not related to viral infection. We found no evidence for linkage disequilibrium with the four examined TLR9 SNPs. The findings suggest that the TLR9 -1486T/C and 2848C/T polymorphisms could be a genetic risk factor for the development of HCMV disease.     

Cytokine response and polymerase chain reaction study of peripheral blood mononuclear cells in infants with human cytomegalovirus infection

Abstract We tried to detect human cytomegalovirus (HCMV) DNA in CD4 + and CD8 + T lymphocytes from fourteen infants with HCMV hepatitis using polymerase chain reaction (PCR) assay. HCMV was isolated from their urine and anti-HCMV IgM antibody was detected in their sera. One set of primers were designed from a region — a major immediate early (IE) gene. We detected HCMV IE DNA in the specimens obtained from six infants. HCMV IE DNA was detected from CD4 + cells in two cases and from CD8 + cells in one. In three cases, HCMV IE DNA was detected from both CD4 + and CD8 + cells. We also studied the relationship between HCMV infection and serum levels of cytokines. We determined serum levels of interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α) and soluble interleukin 2 receptor (sIL-2R) which were associated with the activation of T lymphocytes by enzyme immunoassay. In the acute phase of HCMV infection, titers of sIL-2R were correlated with serum levels of liver enzymes in some cases. IL-4 and TNF-α activities were not detected in sera. It is likely that expression of viral genome on T lymphocytes as well as activities of some cytokines are associated with active HCMV infection.     

Molecular analysis of the human cytomegalovirus strains isolated from infected infants.

Polymerase chain reaction (PCR) techniques were developed to facilitate the study of the molecular epidemiology of human cytomegalovirus (HCMV). In the present study analysis of HCMV DNA was applied for the determination of the reinfection frequency and genotypes of HCMV strains isolated from infected infants, treated with ganciclovir and non-treated. Urines from 92 infants, aged 1 to 5 months, were investigated. Isolates were analysed by PCR method using primers for a-seq and glycoprotein B (gB) HCMV genes. PCR products of gB gene were digested with RsaI and HinfI endonucleases (PCR-RFLP). A-seq gene amplified products were visualized on agarose gels and analysed by densitometry. Genotyping based on hypervariable a-seq region in comparison with restriction analysis of gB gene fragment allowed better differentiation and discrimination of particular HCMV strains. Analysis of the a-sequence PCR products allowed to distinguish 9 profile groups. The patterns obtained consisted of fragments with different size (100 bp to 350 bp), suggesting considerable diversity of HCMV strains. A-sequence analysis revealed that 5 (15.6%) of treated children and 14 (20.7%) of those non-treated, excreted virus of stable genotype. Twenty one (65.6%) of treated and 32 (52.5%) of non-treated children excreted HCMV with a-sequence product of different size, suggesting that in these cases reinfection was caused by genetically distinct strains. Results suggest that reinfection is more frequent in children treated with ganciclovir.     

Placental extravillous cytotrophoblasts persistently express class I major histocompatibility complex molecules after human cytomegalovirus infection

Human cytomegalovirus (HCMV) downregulates the class I major histocompatibility complexes (MHCs), HLA-A and -B, in infected fibroblasts to escape from antigen-specific cytotoxic T lymphocytes. The HCMV genes responsible for the downregulation of MHCs are US2, US3, US6, and US11, which encode type I membrane proteins working at the endoplasmic reticulum (ER). However, it is largely unknown whether HCMV downregulates the class I MHC molecules in placental extravillous cytotrophoblasts (EVT), which express HLA-C, -E, and -G to protect a semiallogenic fetus from maternal natural killer (NK) cells at the fetomaternal interface. Here, we report that differentiated EVT prepared from human first-trimester chorionic villi persistently express class I MHC molecules upon HCMV infection. When these US proteins were expressed in uninfected EVT, they were localized at the ER in the entire cytoplasm. However, subsequent HCMV infection resulted in dissociation of these US proteins from the ER, which relocated toward the cell membrane. In fibroblasts, these US proteins were localized at the ER before and after HCMV infection. These results suggest that the US gene products are not integrated into ER of HCMV-infected EVT and fail to downregulate class I MHC molecules.     

Fetal Human Cytomegalovirus Transmission Correlates with Delayed Maternal Antibodies to gH/gL/pUL128-130-131 Complex during Primary Infection

Primary human cytomegalovirus (HCMV) infections during pregnancy are associated with a high risk of virus transmission to the fetus. To identify correlates of intrauterine HCMV transmission, serial serum samples from HCMV transmitter and non-transmitter pregnant women with primary HCMV infection were analyzed for the presence of neutralizing antibodies against different glycoproteins and glycoprotein complexes, which are known to mediate entry into distinct types of host cells. Neutralizing activity was detected in the sera early after primary infection; absorption with a soluble pentameric complex formed by gH/gL/pUL128-131, but not with gH/gL dimer or with gB, abolished the capacity of sera to neutralize infection of epithelial cells. Importantly, an early, high antibody response to pentamer antigenic sites was associated with a significantly reduced risk of HCMV transmission to the fetus. This association is consistent with the high in vitro inhibition of HCMV infection of epithelial/endothelial cells as well as cell-to-cell spreading and virus transfer to leukocytes by anti-pentamer antibodies. Taken together, these findings indicate that the HCMV pentamer complex is a major target of the antibody-mediated maternal immunity.     

Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes.

Three monoclonal antibodies were characterized by examining their reactivity to human cytomegalovirus (HCMV) glycoproteins under reducing and nonreducing conditions and their reactivity to glycoproteins and disulfide-linked glycoprotein complexes isolated by ion-exchange high-performance liquid chromatography. One monoclonal antibody, 9E10, reacted with glycoprotein complexes which had molecular weights of 93,000 and 450,000 and eluted from the ion-exchange column at 0.3 and 0.9 M NaCl, respectively. All glycoproteins associated in these complexes could be immunoprecipitated under reducing conditions by 9E10, suggesting that they were related to one another. The most abundant glycoproteins immunoprecipitated by 9E10 had molecular weights of 50,000 to 52,000. In contrast to this antibody, two other monoclonal antibodies, 9B7 and 41C2, reacted with glycoprotein complexes which had molecular weights of 130,000 and greater than 200,000 and eluted from the ion-exchange column at 0.6 M NaCl. All glycoproteins associated in these complexes could be immunoprecipitated by 9B7 or 41C2 under reducing conditions, suggesting that they were also related to one another. The most abundant glycoprotein immunoprecipitated by 41C2 or 9B7 had a molecular weight of 93,000. In addition, it was also determined that a 93,000-molecular-weight glycoprotein which was not associated with other glycoproteins by disulfide bonds could not be precipitated by any of the three antibodies, suggesting that it was different from the other glycoproteins. The monoclonal antibodies were also examined for specificity and neutralizing activity. Monoclonal antibodies 41C2 and 9B7 were specific to HCMV as determined by immunofluorescent staining of skin fibroblast cells infected with several different viruses. However, 41C2 did not neutralize Towne strain HCMV, while 9B7 did. The neutralizing activity of 9B7 did require complement. These results suggested that 41C2 and 9B7 reacted with different antigenic sites on the same glycoproteins. Unlike 41C2 and 9B7, monoclonal antibody 9E10 was found to cross-react with adenovirus and herpes simplex virus as determined by immunofluorescent staining of infected skin fibroblast cells. Furthermore, 9E10 neutralized the Towne and Toledo strains of HCMV in the absence of complement.     

Identification of binary interactions between human cytomegalovirus virion proteins

Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis.     

Physical and functional interaction between the HCMV IE2 protein and the Wilms' tumor suppressor WT1

Human cytomegalovirus (HCMV) is a major renal pathogen in congenitally infected infants and renal allograft recipients. It has been shown that human kidney cells of glomerular, tubular, and vascular origin were all infected by HCMV in vitro. It has previously been demonstrated that the IE2 protein of HCMV directly associates with the zinc finger domain of Egr-1. The zinc finger region of WT1 is a sequence-specific DNA-binding domain which also recognizes the consensus DNA binding site (5'-CGCCCCCGC-3') of Egr-1, thus suggesting a possible interaction between WT1 and IE2. Here we demonstrate that HCMV IE2 binds to the C-terminal region of WT1 containing zinc finger domain in vivo as well as in vitro and that WT1 can inhibit IE2-driven transactivation of the responsive promoter. Our results suggest that WT1 may be able to regulate the functional activity of HCMV IE2. Furthermore, these data may provide new insights into the possible involvement of HCMV in WT1-related pathogeneses.     

Human Cytomegalovirus UL50 and UL53 Recruit Viral Protein Kinase UL97, Not Protein Kinase C,for Disruption of Nuclear Lamina and Nuclear Egress in Infected Cells

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.     

Glycoprotein H-related complexes of human cytomegalovirus: identification of a third protein in the gCIII complex.

Previous studies have described three disulfide-bonded glycoprotein complexes within the envelope of human cytomegalovirus (HCMV). These have been designated gCI, gCII, and gCIII. Although gCI has been identified as homodimeric glycoprotein B (gB, gpUL55), the compositions of gCII and gCIII remain incompletely defined. Earlier studies suggested that gCIII was composed of glycoprotein H (gH, gpUL75) complexed with a second glycoprotein, the gL homolog of HCMV. We characterized the gCIII complex of HCMV using recombinant vaccinia virus-expressed gH and gL. Our results indicated that authentic gCIII was not reconstituted by coexpression of gH and gL. The presence of a third, structurally and antigenically unique glycoprotein with an estimated molecular mass of 125,000 Da in virion-derived gCIII complexes suggested that at least three proteins were necessary for formation of this envelope glycoprotein complex. This third glycoprotein, gp125, contained both simple and complex N-linked carbohydrates and had an estimated deglycosylated mass of 64,000 Da. Furthermore, we demonstrated that mature gH existed as both a covalently complexed and noncovalently associated component of the gCIII complex within the envelope of infectious extracellular virions. These findings provide further evidence for the structural complexity of the envelope of HCMV and emphasize the uncertainties associated with the previous assignment of specific functions to envelope proteins of HCMV.     

Characterization of a novel third member of the human cytomegalovirus glycoprotein H-glycoprotein L complex.

A prerequisite for understanding the molecular function of the human cytomegalovirus (HCMV) gH (UL75)-gL (UL115) complex is a detailed knowledge of the structure of this complex in its functional form, as it is present in mature virions. The gH protein is known to be a component of a 240-kDa envelope complex designated as gCIII (D. R. Gretch, B. Kari, L. Rasmussen, R. C. Gehrz, and M. F. Stinski, J. Virol. 62:875-881, 1988). However, the exact composition of the gCIII complex remains unknown. In this report, we attempted reconstitution of the gCIII complex by coexpression of gH and gL in the baculovirus expression system. Formation of recombinant gH-gL complexes of approximately 115 kDa was demonstrated; however, no higher-molecular-mass (approximately 240-kDa) recombinant gH-gL complexes were detected, suggesting that the presence of gH and gL alone is not sufficient for reconstitution of the gCIII complex. To identify other mammalian and/or HCMV factors which may be necessary for gCIII formation, immunoprecipitates of gH and gL from HCMV-infected fibroblasts and purified HCMV virions were examined. This analysis did reveal a number of coprecipitating proteins which associate either transiently or integrally with gH and gL. One coprecipitating protein of 145 kDa was shown to be an integral component of gCIII, along with gH and gL. Characterization of the 145-kDa protein demonstrates that it is structurally and antigenically unrelated to gH and gL and that it appears to be virally encoded. Together, these data indicate that the 145-kDa protein is a third novel component of the mature HCMV gH-gL complex.     

Allelic MHC class I chain related B (MICB) molecules affect the binding to the human cytomegalovirus (HCMV) unique long 16 (UL16) protein: Implications for immune surveillance

Unique long 16 (UL16) is a viral glycoprotein produced in a host cell infected with human cytomegalovirus (HCMV). It down regulates surface expression of MICB, one of the NKG2D ligands, by forming stable intracellular complexes and retained in the endoplasmic reticulum. Down expression of MICB renders cells less susceptible to NK cell lysis via the NKG2D receptor. Diverse UL16 sequences were identified from different strains of HCMV. MICB is known to be polymorphic. It is not known whether these polymorphisms affect the interactions between these molecules leading to alteration of the immune surveillance of HCMV. The soluble Fc fusion variant UL16 proteins from four laboratory and clinical isolates (AD169, Toledo, PH, and TR) were produced. Four allelic MICB alleles (008, 003, 004, and 00502) were cloned and stable cell lines expressing these MICB alleles were produced. The binding activities of variant UL16 to allelic MICB proteins were determined by flow cytometry. The variants of UL16 proteins did not affect the binding activities to allelic MICB proteins. However, diverse MICB alleles differentially bound UL16. We found that MICB*008 which contains methionine and asparagine at the amino acid positions 98 and 113, respectively, in the alpha 2 domain showed decreased binding activities to UL16 when compared to MICB*003, 004, and MICB*00502 containing isoleucine and aspartic acid, respectively. This finding may imply that MICB*008 is a protective allele and involved in the immune surveillance of HCMV infected patients.     

Recent clinical isolates of cytomegalovirus suppress human cytomegalovirus-specific human leukocyte antigen-restricted cytotoxic T-lymphocyte activity.

Peripheral blood mononuclear cells harvested from healthy adults seropositive for human cytomegalovirus (HCMV) and cultured with laboratory strain AD-169 demonstrated human leukocyte antigen-restricted and HCMV-specific killing on target cells infected with either HCMV laboratory strain AD-169 or recent low-passage HCMV isolates. These results indicated that the determinants recognized by cytotoxic T lymphocytes (CTLs) are shared among different strains of HCMV. However, when low-passage isolates, rather than high-passage AD-169 virions, were used to stimulate CTL activity, the lytic response was significantly lower against all targets. Mixing of AD-169 and low-passage HCMV isolates induced low CTL activity. Collectively, the findings suggest that low-passage HCMV isolates have dual effects--antigenic stimulation and immunosuppression--whereas laboratory strain AD-169 is primarily immunogenic. The study of several recent isolates indicated that they varied in their ratio of immunostimulation to suppression, that infectious virus was necessary to produce suppression, and that suppressive isolates did not have to be present at the initiation of culture to exert their suppressive effects.     

The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms, the HCMV protein US9, counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically downregulates MICA*008. UL147A primarily induces MICA*008 maturation arrest, and additionally targets it to proteasomal degradation, acting additively with US9 during HCMV infection. Thus, UL147A hinders NKG2D-mediated elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines.