Viral interleukin-10 expressed by human cytomegalovirus during the latent phase of infection modulates latently infected myeloid cell differentiation

The human cytomegalovirus UL111A gene is expressed during latent and productive infections, and it codes for homologs of interleukin-10 (IL-10). We examined whether viral IL-10 expressed during latency altered differentiation of latently infected myeloid progenitors. In comparison to infection with parental virus or mock infection, latent infection with a virus in which the gene encoding viral IL-10 has been deleted upregulated cytokines associated with dendritic cell (DC) formation and increased the proportion of myeloid DCs. These data demonstrate that viral IL-10 restricts the ability of latently infected myeloid progenitors to differentiate into DCs and identifies an immunomodulatory role for viral IL-10 which may limit the host's ability to clear latent virus.     

Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression

In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies.     

An immortalized human fibroblast cell line is permissive for human cytomegalovirus infection.

Human foreskin fibroblasts (HFF) were immortalized via retrovirus-mediated gene transfer of the E6 and E7 genes of human papillomavirus type 16. An immortalized fibroblast (IF) cell line which was morphologically akin to the parental cell line was isolated. The IF cell line was evaluated for permissiveness to human cytomegalovirus (HCMV) infection after the IF cell line surpassed the normal passage limitation of diploid fibroblasts. Western immunoblot analysis of representative HCMV-encoded immediate-early (72-kDa), early (gB), and late (gH) gene products demonstrated that the IF cell line produced these proteins analogous to those produced by the parental HFF cells. Similar quantities of infectious virus were produced in the IF and HFF cell lines as determined in one-step growth curve experiments. Compared with the HFF cells, morphologically identical plaques were produced in the IF cell line in approximately 10 to 12 days postinfection. These findings indicate that fibroblast cell lines immortalized with transforming genes of human papillomavirus retain complete permissiveness to HCMV infection and support plaque formation. The IF cell line will be useful for future genetic analysis of HCMV.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.     

控制人巨细胞病毒感染的策略

人巨细胞病毒（HCMV）感染对免疫受损人群危害极大,本综述了目前控制HCMV感染的策略,其中包括药物控制和免疫控制。阐述了使用药物控制HCMV感染的效果,疫苗在控制HCMV感染及引发疾病方面的作用及通过被动免疫控制HCMV感染的现状与进展。对这些策略的了解对控制HCMV感染具有重要意义。     

Permissive human cytomegalovirus infection of a first trimester extravillous cytotrophoblast cell line

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection in the United States and Europe. Despite the significant morbidity associated with prenatal HCMV infection, little is known about how the virus infects the fetus during pregnancy. To date, primary human cytotrophoblasts (CTBs) have been utilized to study placental HCMV infection and replication; however, the minimal mitotic potential of these cells restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. The aim of this study was to determine whether the human first trimester CTB cell line SGHPL-4 was permissive for HCMV infection and therefore could overcome such limitations. HCMV immediate early (IE) protein expression was detected as early as 3 hours post-infection in SGHPL-4 cells and progressively increased as a function of time. HCMV growth assays revealed the presence of infectious virus in both cell lysates and culture supernatants, indicating that viral replication and the release of progeny virus occurred. Compared to human fibroblasts, viral replication was delayed in CTBs, consistent with previous studies reporting delayed viral kinetics in HCMV-infected primary CTBs. These results indicate that SGHPL-4 cells are fully permissive for the complete HCMV replicative cycle. Our findings suggest that these cells may serve as useful tools for future mechanistic studies of HCMV pathogenesis during early pregnancy.     

Targeting the hemangioblast with a novel cell type-specific enhancer

Background

Hemangioblasts are known as the common precursors for primitive hematopoietic and endothelial lineages. Their existence has been supported mainly by the observation that both cell types develop in close proximity and by in vitro differentiation and genetic studies. However, more compelling evidence will arise from tracking their cell fates using a lineage-specific marker.

Results

We report the identification of a hemangioblast-specific enhancer (Hb) located in the cis-regulatory region of chick Cerberus gene (cCer) that is able to direct the expression of enhanced green fluorescent protein (eGFP) to the precursors of yolk sac blood and endothelial cells in electroporated chick embryos. Moreover, we present the Hb-eGFP reporter as a powerful live imaging tool for visualizing hemangioblast cell fate and blood island morphogenesis.

Conclusions

We hereby introduce the Hb enhancer as a valuable resource for genetically targeting the hemangioblast population as well as for studying the dynamics of vascular and blood cell development.     

A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus

Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 μl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that ～0.5% HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.     

Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line

The susceptibility of four different human cell lines (HUT 102, THP-1, MOLT-4, and HL-60) to infection by human CMV (HCMV) was studied. Only HUT 102 was susceptible and only immediate-early gene products were produced. However, THP-1, a monocytic cell line, could be infected by HCMV with a full cycle of replication after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which produced differentiation of the cell line into cells with characteristics of mature macrophages. Late (structural) Ag were demonstrated, as were infectious virions as detected by electron microscopy and infectious center assay. HL-60, a promyelocytic cell line, was not susceptible to HCMV infection after treatment with TPA despite differentiation into adherent cells with properties of macrophages, suggesting that cellular lineage was important. Treatment with TPA after infection resulted in a greatly reduced frequency of infected cells, suggesting that pretreatment was essential. Furthermore, continued presence of TPA was unnecessary after differentiation was induced. This study establishes the precedent of productive HCMV infection in human monocytic cells. The potential mechanism and relevance of enhanced replication induced by TPA are discussed.     

A novel mechanism for persistence of human cytomegalovirus in macrophages.

Human cytomegalovirus (HCMV) infection of monocyte-derived macrophages (MDM) results in delayed and nonlytic productive viral growth. During late stages of replication, infectious virus remains cell associated in cytoplasmic vacuoles. In order to understand HCMV survival and persistence in MDM, we examined mechanisms involved in the formation and trafficking of HCMV-containing vacuoles in these cells. Utilizing double-label immunofluorescence with antibodies to viral and cellular proteins, HCMV-containing vacuoles were associated with the Golgi apparatus marker mannosidase II but not with markers to early endosomes (transferrin receptor and rab5) or late endosomes and early lysosomes (LAMP-1 and -2). In addition, as late-stage viral infection progressed in MDM, the cells displayed increasing abnormalities in the Golgi apparatus. Analysis of structural features of infected cells revealed the disruption of the microtubule network. These observations suggest a novel mechanism by which HCMV is vacuolized in MDM, avoiding degradation and release from the cell.     

In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.     

Cytoskeletal disruption during human cytomegalovirus infection of human lung fibroblasts

Human cytomegalovirus (HCMV) infection causes a rapid, progressive disruption of the host cell cytoskeleton that correlates with actin depolymerization. Whole-mount (3D) electron microscopy was used to analyze the cytoskeleton of uninfected and HCMV-infected human lung fibroblast cells. Within 2 min of HCMV infection, localized areas of cytoskeletal disruption were observed. Disruption extended throughout the cytoplasm during the ensuing 45 to 90 min of infection and resulted in generalized cytoskeletal disorganization. Actin depolymerization occurred, as indicated by an increase in DNase I inhibition and alteration in the fluorescence pattern with rhodamine-conjugated phalloidin. Thus, actin appears to be the primary cytoskeletal target involved during HCMV infection. Fractionation of the virus seed inoculum showed that development of DNase I inhibitory activity in infected cells was associated only with the virus-containing fractions. Cytochalasin B treatment at early times of HCMV infection stimulated progeny virus production. This study demonstrates that rapid cytoskeletal disruption occurs during early periods of HCMV infection and indicates that actin depolymerization facilitates viral infectivity.     

Human cytomegalovirus expresses novel microRNAs during productive viral infection

MicroRNAs (miRNAs) are a large class of approximately 22-nucleotide non-coding RNAs that facilitate mRNA cleavage and translation repression through the RNA interference pathway. Until recently, miRNAs have been exclusively found in eukaryotic organisms. A non-immunogenic molecule requiring minimal genomic investment, these RNAs may offer an efficient means for viruses to modulate both their own and the host's gene expression during a productive viral infection. In this study we report that human cytomegalovirus (HCMV) expresses miRNAs during its productive lytic infection of four clinically relevant human cell types: fibroblast, endothelial, epithelial and astrocyte cells. The sequences of the miRNAs, expressed from the UL23 and US24 loci of the viral genome, were conserved among all HCMV strains examined and in chimpanzee cytomegalovirus. Furthermore, their expression was detected from both a laboratory-adapted strain and a clinical isolate of HCMV. The conservation of these miRNAs and their expression in different cell types suggests that they represent an evolutionarily primitive feature in the viral genome, and that virus-encoded miRNAs may be more common than previously believed.