Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys

Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a full-length UL/b' region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b' region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b' region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one na?ve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.     

Human cytomegalovirus UL145 gene is highly conserved among clinical strains

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects in newborns. A region (referred to as UL/b') present in the Toledo strain of HCMV and low-passage clinical isolates) contains 22 additional genes,which are absent in the highly passaged laboratory strain AD169. One of these genes,UL145 open reading frame (ORF), is located between the highly variable genes UL144 and UL146. To assess the structure of the UL145 gene,the UL145 ORF was amplified by PCR and sequenced from 16 low-passage clinical isolates and 15 non-passage strains from suspected congenitally infected infants. Nine UL145 sequences previously published in the GenBank were used for sequence comparison. The identities of the gene and the similarities of its putative protein among all strains were 95.9 -100% and 96.6-100%, respectively. The post-translational modification motifs of the UL145 putative protein in clinical strains were conserved,comprising the protein kinase C phosphorylation motif (PKC)and casein kinase II phosphorylation site (CK-II). We conclude that the structure of the UL145 gene and its putative protein are relatively conserved among clinical strains, irrespective of whether the strains come from patients with different manifestations, from different areas of the world, or were passaged or not in human embryonic lung fibroblast (HELF) cells.     

Genetic variability of human cytomegalovirus UL132 gene in strains from infected infants

Human cytomegalovirus (HCMV) displays genetic polymorphisms. HCMV infects a number of organs and cell types, leading to the hypothesis that HCMV disease and tissue tropism may be related to specific sequence variability. A gene in UL/b' of HCMV, UL132 open reading frame (ORF), encodes glycoprotein (gpUL132) which is identified as a low-abundance structural component of HCMV. In this study, the sequence variability of the UL132 gene was studied in 30 clinical strains. The results showed that a large number of nucleotide non-synonymous substitutions occurred in the UL132 ORF, particularly in the 5' half, in comparison to the UL132 of reference strain, Toledo. The UL132 variants of the clinical strains were clustered clearly into three major groups in the phylogenetic tree: G1(10/30), G2(9/30), and G3(11/30). The precise definition of UL132 genotypes and their putative functions would be helpful in a better understanding of the HCMV.     

Human cytomegalovirus regulates surface expression of the viral protein UL18 by means of two motifs present in the cytoplasmic tail

UL18 is a trans-membrane viral protein expressed on human cytomegalovirus (HCMV)-infected cells, and its surface expression determines the interaction of infected cells with lymphocytes expressing the CD85j (LIR-1/ILT2) receptor. We previously showed that the UL18-CD85j interaction elicits activation of T lymphocytes. However, in in vitro cell models UL18 displays mostly undetectable surface expression. Thus, we asked how surface expression of UL18 is regulated. Domain-swapping experiments and construction of specific mutants demonstrated that two motifs on its cytoplasmic tail, homologous to YXXPhi and KKXX consensus sequences, respectively, are responsible for impairing UL18 surface expression. However, the presence of the whole HCMV genome, granted by HCMV infection of human fibroblasts, restored surface expression of either UL18 or chimeric proteins carrying the UL18 cytoplasmic tail, starting from the third day after infection. It is of note that the two motifs responsible for cytoplasmic retention are identical in all 17 HCMV strains examined. We disclosed a control mechanism used by the HCMV to regulate the availability of UL18 on the infected-cell surface to allow interaction with its ligand on T and NK cells.     

Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.     

Human cytomegalovirus UL144 open reading frame: sequence hypervariability in low-passage clinical isolates

Human cytomegalovirus (HCMV) infects a number of organs and cell types in vivo, leading to the hypothesis that HCMV disease and tissue tropism may be related to specific sequence variants. A potential component of HCMV variant strains is the UL144 open reading frame (ORF), which encodes a homologue of the herpesvirus entry mediator, HveA, a member of the tumor necrosis factor receptor superfamily. Sequence analysis of the UL144 ORF in 45 low-passage clinical isolates demonstrated significant strain-specific variability. In individual isolates, nucleotide substitutions occur at up to 21% of the 531 positions, resulting in approximately the same percentage of substitutions in the predicted 176-amino-acid sequence. Phylogenetic analysis indicated that the nucleotide and amino acid sequences diverge into three major groups. For genotypic comparison, the known hypervariable region encompassing the proteolytic cleavage site of the glycoprotein B (gB) gene was also sequenced. All of the isolates could be typed according to the four known gB groups; however, the gB and UL144 sequence groups appeared to be phylogenetically unlinked. The predicted UL144 product homology with tumor necrosis factor receptor family members, along with the unexpectedly high level of sequence variability of the UL144 ORF, suggests that the predicted product may play a role in HCMV infectivity and subsequent host disease.     

The UL97 gene product of human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase.

The temporal expression of the UL97 gene product during human cytomegalovirus (HCMV) infection of human foreskin fibroblasts (HFF) and subcellular localization of this protein were analyzed by using a polyclonal antiserum raised against a truncated UL97 protein of 47 kDa. The UL97 protein was detectable 16 h after infection by Western blot (immunoblot) analysis. Since only reduced UL97 expression occurred in the presence of two inhibitors of DNA replication, phosphonoacetic acid and ganciclovir, we conclude that UL97 is an early-late gene, requiring DNA replication for maximum expression. By indirect immunofluorescence, the protein could be visualized in the nuclei of virus-infected HFF 22 h after infection. Nuclear localization of the UL97 protein was also detected in thymidine kinase-deficient 143B cells infected with a recombinant vaccinia virus containing the entire UL97 open reading frame (ORF), as well as in HFF transiently expressing the entire UL97 ORF under the control of HCMV major immediate-early promoter. However, transiently expressed 5'-terminal deletion mutants of the UL97 ORF in addition showed a cytoplasmic localization of the UL97 protein, confirming the presence of a nuclear localization site in the N-terminal region of the protein. Our high-pressure liquid chromatography analyses confirmed the ganciclovir phosphorylation by the UL97 protein, but no specific phosphorylation of natural nucleosides was observed, indicating that the UL97 protein is not a nucleoside kinase. During plaque purification of recombinant UL97-deficient HCMV, this virus was growth defective; hence, we presume that UL97 may be essential for the viral life cycle.     

Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus.

The UL144 open reading frame found in clinical isolates of human CMV (HCMV) encodes a structural homologue of the herpesvirus entry mediator, a member of the TNFR superfamily. UL144 is a type I transmembrane glycoprotein that is expressed early after infection of fibroblasts; however, it is retained intracellularly. A YXXZ motif in the highly conserved cytoplasmic tail contributes to UL144 subcellular distribution. The finding that no known ligand of the TNF family binds UL144 suggests that its mechanism of action is distinct from other known viral immune evasion genes. Specific Abs to UL144 can be detected in the serum of a subset of HCMV seropositive individuals infected with HIV. This work establishes a novel molecular link between the TNF superfamily and herpesvirus that may contribute to the ability of HCMV to escape immune clearance.     

Selective induction of Th2-attracting chemokines CCL17 and CCL22 in human B cells by latent membrane protein 1 of Epstein-Barr virus

Chemokines are likely to play important roles in the pathophysiology of diseases associated with Epstein-Barr virus (EBV). Here, we have analyzed the repertoire of chemokines expressed by EBV-infected B cells. EBV infection of B cells induced expression of TARC/CCL17 and MDC/CCL22, which are known to attract Th2 cells and regulatory T cells via CCR4, and also upregulated constitutive expression of MIP-1 alpha/CCL3, MIP-1 beta/CCL4, and RANTES/CCL5, which are known to attract Th1 cells and cytotoxic T cells via CCR5. Accordingly, EBV-immortalized B cells secreted these chemokines, especially CCL3, CCL4, and CCL22, in large quantities. EBV infection or stable expression of LMP1 also induced CCL17 and CCL22 in a B-cell line, BJAB. The inhibitors of the TRAF/NF-kappa B pathway (BAY11-7082) and the p38/ATF2 pathway (SB202190) selectively suppressed the expression of CCL17 and CCL22 in EBV-immortalized B cells and BJAB-LMP1. Consistently, transient-transfection assays using CCL22 promoter-reporter constructs demonstrated that two NF-kappa B sites and a single AP-1 site were involved in the activation of the CCL22 promoter by LMP1. Finally, serum CCL22 levels were significantly elevated in infectious mononucleosis. Collectively, LMP1 induces CCL17 and CCL22 in EBV-infected B cells via activation of NF-kappa B and probably ATF2. Production of CCL17 and CCL22, which attract Th2 and regulatory T cells, may help EBV-infected B cells evade immune surveillance by Th1 cells. However, the concomitant production of CCL3, CCL4, and CCL5 by EBV-infected B cells may eventually attract Th1 cells and cytotoxic T cells, leading to elimination of EBV-infected B cells at latency III and to selection of those with limited expression of latent genes.     

UL146 variability among clinical isolates of human cytomegalovirus from Japan

Human cytomegalovirus (HCMV) is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb' is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb' region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR) in only 17/56 strains (30%), while the success rate for UL145/UL147 gene was 18/56 strains (32%). After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1% to 52.9% at the DNA level and from 34.5% to 67% at the amino acid level. Seven groups had the interleukin-8 (IL-8) motif ERL (Glu-Leu-Arg) CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hyper-variable in clinical strains from Japan suggesting the possibility of a different function in each sequence group.     

ESR and NMR studies provide evidence that phosphatidyl glycerol specifically interacts with poxvirus membranes

Background

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and treatment strategies. Antisense molecules are promising gene-targeting agents for specific regulation of gene expression. External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. The UL49-deletion BAC of HCMV was significantly defective in growth in human foreskin fibroblasts. Therefore, UL49 gene may serve as a potential target for novel drug development to combat HCMV infection. In this study, DNA-based EGS molecules were synthesized to target the UL49 mRNA of human cytomegalovirus (HCMV).

Results

By cleavage activity assessing in vitro, the EGS aimed to the cleavage site 324 nt downstream from the translational initiation codon of UL49 mRNA (i.e. EGS324) was confirmed be efficient to direct human RNase P to cleave the target mRNA sequence. When EGS324 was exogenously administered into HCMV-infected human foreskin fibroblasts (HFFs), a significant reduction of ~76% in the mRNA and ~80% in the protein expression of UL49 gene, comparing with the cells transfected with control EGSs. Furthermore, a reduction of about 330-fold in HCMV growth were observed in HCMV-infected HFFs treated with the EGS.

Conclusions

These results indicated that UL49 gene was essential for replication of HCMV. Moreover, our study provides evidence that exogenous administration of a DNA-based EGS can be used as a potential therapeutic approach for inhibiting gene expression and replication of a human virus.     

Human cytomegalovirus <Emphasis Type="Italic">UL145</Emphasis> gene is highly conserved among clinical strains

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects in newborns. A region (referred to as UL/b′) present in the Toledo strain of HCMV and low-passage clinical isolates) contains 22 additional genes, which are absent in the highly passaged laboratory strain AD169. One of these genes, UL145 open reading frame (ORF), is located between the highly variable genes UL144 and UL146. To assess the structure of the UL145 gene, the UL145 ORF was amplified by PCR and sequenced from 16 low-passage clinical isolates and 15 non-passage strains from suspected congenitally infected infants. Nine UL145 sequences previously published in the GenBank were used for sequence comparison. The identities of the gene and the similarities of its putative protein among all strains were 95.9–100% and 96.6–100%, respectively. The post-translational modification motifs of the UL145 putative protein in clinical strains were conserved, comprising the protein kinase C phosphorylation motif (PKC) and casein kinase II phosphorylation site (CK-II). We conclude that the structure of the UL145 gene and its putative protein are relatively conserved among clinical strains, irrespective of whether the strains come from patients with different manifestations, from different areas of the world, or were passaged or not in human embryonic lung fibroblast (HELF) cells.